Articles in next issue
June – July, 2019, vol. 8, no. 6
Nitrates cause the most serious problems when dispersed in water since they cause the depletion of aquifers and the eutrophication of rivers. Sources of nitrate comprise natural cycle and human activities, mainly from uncontrolled land discharges of treated or raw domestic and industrial waste waters, landfills, and animal wastes predominantly from animal farms (Vitousek et al., 1997 and Galloway et al., 2008). Therefore, several studies focused on the nitrate removal from wastewater in order to achieve an acceptable concentration in treated waters to be discharged into the environment. Denitrifying microorganisms are ubiquitous in nature and they have been isolated from different ecological sources. Nitrogen containing ions such as nitrate and nitrite occur widely in a variety of process streams, such as those coming from the extensive use of fertilizers. These species can have serious consequences when released in the environment, due to the possible health effects for many organisms including humans (Matsuzaka et al., 2003). Various researchers have isolated denitrifying bacteria from diverse environments such as agricultural soils, deep sea sediments, wastewater treatment plants, biofilms of long term aerobic/anoxic denitrifying reactor and have isolated potential denitrifiers from various nitrate enriched regions such as petrochemical industry effluent, greenhouse soil of agricultural land (Zumfit, 1997; Rezaee et al., 2010; Wang et al., 2013; Zhou et al., 2014, Kong et al., 2018). In this study, the isolated denitrifying bacteria from municipal waste water with high denitrification potential were identified and characterized, using their morphological and biochemical properties, and 16S rRNA analyses. 16s rDNA gene sequence was used for the identification of isolated strain. The effect of isolate on nitrate reduction with different process parameters was studied. The study also analyzed with general factorial design and model variation trends for three parameters of pH, Temperature and C: N ratio. Response surface methodology (RSM) has an important application in the process design and optimization as well as the improvement of existing design (Box and Draper, 1987). This methodology is more practical than other methods of approaches arise from an experimental methodology which includes interactive effects among the variables and, eventually, it depicts the overall effects of the parameters on the process (Bas and Boyaci, 2007). In the last few years, RSM has been applied to optimize and evaluate the interactive effects of independent factors in numerous chemical and biochemical processes (Yang and Hwang, 2003; Ahmadi et al., 2005; Aghamohammadi et al., 2007; Zinatizadeh et al., 2009).
MATERIAL AND METHODS
Composition of Nitrate rich (NR) medium was NH4Cl-0.3g/L, KH2PO4-1.5g/L, Na2HPO4.7H2O- 7.9g/L, KNO3-2 g/L, disodium succinate-27g/L, MgSO4.7H2O-5 mL/L (20g/L).
Composition of trace element solution : 5 mL/L (composition – EDTA, 50.0 g/L; ZnSO4, 2.2 g/L; CaCl2, 5.5 g/L; MnCl2.4H2O, 5.06 g/L; FeSO4.7H2O, 5.0 g/L; (NH4)6Mo7O24.4H2O, 1.1 g/L; CuSO4.5H2O, 1.57 g/L; and CoCl2.6H2O, 1.61 g/L; pH 7.2) and Na2SO3– 100 mg/L .
Composition of bromothymol blue (BTB) medium: L-asparagine, 1 g/L; KNO3,1g/L; KH2PO4, 1 g/L; FeCl2, 0.05 g/L; CaCl2, 0.2 g/L; MgSO4.7H2O, 1 g/L; BTB reagent, 1 mL/L (1% in ethanol); and agar, 20 g/L (pH 7.3).
Isolation and screening of denitrifier by enrichment methods
The isolation of microorganisms was from nitrate enriched (66mg/L) municipal waste water was carried out by enrichment technique. 5 mL or 5g of collected samples were transferred separately into NR medium and incubated in a shaker at 30°C and at 150 rpm for 2days, and the flasks were maintained under aerobic and anoxic conditions separately. After two days, 5 mL of the sample was withdrawn from each, and transferred to fresh NR media and incubated under the same conditions. This procedure was repeated for two more times. After 6 days of incubation period, the samples were diluted from 10-3 to 10-7since they were more turbid. The resulting bacterial suspensions were plated onto bromothymol blue (BTB) medium plates using spread plate technique and incubated at 30°C for 1–3 days to screen denitrifiers. The colonies formed on each plate were counted using a colony counter and the Colony Forming Units (CFU) per ml or g was calculated by a standard plate count method. The blue color colonies and /or halo forming colonies on the BTB medium which indicated the presence of denitrifying microorganisms of the cultures were subjected to further screening. The colonies showing halo on the selective BTB medium were further screened by inoculating into NR medium and incubated under aerobic (Erlenmeyer flasks) and anoxic (BOD bottles) conditions separately. 10 mL of samples were withdrawn after 48 hours and then centrifuged at 1000 rpm for 10 min, and the supernatant was analyzed for residual nitrate to select the efficient denitrifier.
Culture maintenance and storage
Nutrient Agar (NA) medium was used for the growth and maintenance of bacteria. Nutrient agar medium containing the components-peptone 5.0 g/L, sodium chloride 5.0 g/L, yeast extract 2.0 g/L, beef extract 1.0 g/L and agar 15.0 g/L was used and the initial pH of the medium was maintained at 7.2. The medium components were suspended in distilled water before autoclaving at 121ºC for 20 min at 15 psi. The medium was then cooled prior to transferring into sterile petri dishes. The molten agar was left to cool and solidify at room temperature. The bacteria were streaked on the agar medium and incubated at 30ºC for 1 to 2 days and after observing growth they were stored at 4ºC. The pure cultures of bacteria were sub-cultured at regular intervals.
Identification of isolate
Based on the results of various screening tests, the bacterial isolate, designated as ASA was identified as a potential denitrifier and it was selected for further studies. The bacterial isolate was sent to Cyxton Biosolutions Pvt. Ltd., Hubballi, Karnataka, India for partial 16s RNA sequencing for its identification. The purity of stains, DNA isolation, purity and quantification, 1.5kb PCR run, sequencing, blast and gene bank run, and phylogenetic tree were analyzed.
Denitrification studies of a potential Isolate
Nitrate rich (NR) medium inoculated with isolate was used for denitrification to study the effect of various physico-chemical parameters on denitrification. The experiments were conducted to study the effect of parameters like initial pH of the medium, incubation temperature, agitation speed, carbon sources, and carbon to nitrogen ratio on denitrification. In each case, sample cultures were withdrawn for every 6h of incubation for biomass, and nitrate analysis.
The statistical method of factorial design of experiments (DOEs) eliminates systematic errors with an estimate of the experimental error and minimizes the number of experiments. In this study, the central composite design (CCD) was applied to design the experimental conditions using Design-Expert software (version 9.0). The experimental design consisted of 2k factorial points augmented by 2k axial points and a center point, where k is the number of variables (Lim and Lee, 2013; Rastegar et al., 2011). The behavior of the system was explained by the quadratic equation
Where Y represents the response, βo is the interception coefficient, βi is coefficient of the linear effect, βii is the coefficient of quadratic effect and βij is the coefficient of the interaction effect. Xi and Xj represent the coded independent variables. From the initial screening experiments by one factor at a time method, the factors such as temperature (A), pH (B) and C: N ratio (C) was found to have a significant effect on percentage denitrification. Hence, these variables were chosen as the independent variables. The model obtained from regression analysis was employed to generate response surface and contour plots. The quality of the fitting of the polynomial model equation was expressed via the coefficient of determination, R2, and its statistical significance was analyzed through an F-test in the same program. The significance of the regression coefficient was tested via a t-test (Rastegar et al., 2011; Yue et al., 2007). Numerical optimization of the process was performed through the model developed in the software according to different limitations placed on the primary variables.
Growth of the bacterium was monitored by measuring the optical density at 610 nm using UV-Visible Spectrophotometer (Genesys 10S). The culture samples were centrifuged at 10,000 rpm for 10 min (Make Laby, T-60) and supernatant was used for nitrate analysis by UV-Visible Spectrophotometer (Genesys 10S) (Dhamole et al., 2007; Joshi et al., 2014). This study was done using APHA 2012 Section 4500 NO3-B method and materials used were HCl, nitrite ion standard (1000 mg NO3– /L), and Reverse Osmosis (RO) water samples. Sample analysis was carried out using UV-Visible Spectrophotometer (Genesys 10S) and followed 220 nm and 275 nm wavelength.The nitrate removal percentage was estimated by taking the difference with initial and final concentration of nitrate and divided by the initial concentration.
RESULTS AND DISCUSSION
Isolation of microorganism
Aerobic denitrifying bacteria were isolated from municipal waste water plant nearby the Dharwad city, Karnataka, India. Four strains were obtained as denitrifier from the initial solid agar screening techniques. Here the colonies formed blue color and/or halos zones on BTB agar medium due to an increase in pH (Kim et al., 2008). These strains were individually separated and further employed in liquid cultures for confirming the denitrification process. Among the four strains, ASA3 showed 98% nitrate reduction and it was further sent for identification.
Identification of microorganism
The bacterial isolate was further identified by 16S rRNA partial genome sequencing method. The culture sample was processed for isolation and purification of genomic RNA. The isolated RNA was amplified using PCR with a combination of primers FDD2 – RPP2 as shown in Fig.1.a. After the amplification process, the 16S rRNA genomic sequence was identified using the Basic Local Alignment Search Tool (BLAST). The genomic sequence is presented in Fig. 1.b. Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The first step in a PCR cycle is the denaturation step, where the hydrogen bonds holding the complementary strands of DNA together are broken. The second step in a PCR cycle is the annealing step, in which the primers anneal, or attach, to the DNA template. A phylogenetic tree was constructed by the neighbor joining method as shown in Fig. 1.c. (Kim et al., 2008; Saitou et al., 1987). With closest phylogenetic identification, the isolate was identified as Enterobacter sp. NCCP-29.
Figure 1 (a) Amplification profile of the strain, (b) 16S rRNA gene sequence of the bacterial isolate (c) Phylogenetic tree based on a comparison of the 16S rRNA gene sequence
Effect of physical and chemical parameters on the denitrification efficiency
The one factor at a time (OFAT) approach was adopted to select the significant physical parameters and the levels for the denitrification process using isolated microorganism are given in Table 1.
Table 1 Parameters and their range for OFAT studies
|Agitation speed (rpm)||0–250|
Effect of initial medium pH on denitrification
The effect of pH on denitrification was studied by growing the isolated organism in NR media of different initial medium pH with a known initial nitrate concentration. The isolate was grown for 48h and the residual nitrate concentration and biomass quantity were estimated thereafter. The results of the effect of initial medium pH on denitrification are presented in Fig. 2. It is evident from the Fig. 2 that at pH 6.5, the maximum nitrate removal of 97 % was achieved and the corresponding biomass quantity observed was 0.89 g/L. The results indicate that the isolate was found to be more effective in nitrate removal at pH ranging from 6-7.
The decomposition of carbonate ions and carbon dioxide stripping in the acidic pH range, resulting in carbon source deficiency, may be one of the reasons for less growth which further leads to less nitrate removal. It is known that, pH can affect directly the bacterial growth and its enzymatic activities (Campos and Flotats, 2003). At alkaline pH, when the reaction proceeds, the alkalinity of the reaction mixture increases which may reduce the activity of the microorganisms. Wang et al., (1995) reported that, because many enzymes are involved in the denitrification process and that enzyme kinetics are pH dependent and hence, denitrification is also pH dependent. The control of pH is important in the completion of entire denitrification without accumulation of intermediates.
Figure 2 Effect of initial medium pH on percentage of nitrate removal by Enterobacter sp. NCCP-29
Effect of incubation temperature on percentage denitrification
The effect of incubation temperature on denitrification by isolated organism was studied by incubating NR media at different temperatures such as 20, 25, 30, 35 and 40°C. The NR media with the initial nitrate supplement of 300 ppm was inoculated with isolated strain and incubated in an incubator shaker at the above-mentioned temperatures. Fig. 3 presents the results of the effect of incubation temperature on nitrate removal by the isolate. This organism showed its maximum growth and nitrate removal efficiency at an incubation temperature of 30°C. At this temperature, biomass yield was 0.91g/L. At lower temperatures i.e., 20 and 25°C, the biomass yield obtained were 0.21 g/L and 0.23 g/L, respectively. The corresponding percentages of nitrate removals were found to be 47%, and 54%, respectively. At higher temperatures, i.e., 35°C and for 40°C, the percentage denitrification observed was 71% and 65% with corresponding biomass yield of 0.45 g/L and 0.52 g/L, respectively.
Figure 3 Effect of incubation temperature on percentage nitrate removal by an Enterobacter sp. NCCP-29
It was observed from the above study that the temperature is one of the important parameters for the growth of microorganism and nitrate removal. Temperature variation affects the folding of enzyme structure, which further alters the enzyme kinetics. At a certain temperature, the arrangement of the proper catalytic site will be formed and at this particular temperature, enzyme activity is maximum. The activity of the enzyme is affected by the temperature of the growth process. Wang et al., (1995) reported that at 30°C, the culture reduced nitrate optimally and Arrhenius-type expressions were also used in describing the effect of temperature on each of the parameter. Suet al., (2001) reported that at 30°C after 20 h, the concentration of nitrate decreased rapidly in the presence of Pseudomonas stutzeri.
Effect of agitation speed on denitrification
The effect of mixing on denitrification was studied by varying the agitation speed of the flasks containing the NR medium with an initial nitrate supplement of 300 mg/L. The flasks were inoculated with isolate culture and incubated at 30ºC at different agitation speeds at 0 rpm (static culture), 50 rpm, 100 rpm, 150 rpm, 200 rpm, and 250 rpm. The results on the effect of agitation speed on nitrate removal are presented in Fig. 4. It is evident from Fig.4 that with an increase in agitation speed, no significant change in nitrate removal until 200 rpm was observed. In the present study, a maximum of 98% nitrate removal was achieved in the culture kept at an agitation speed of 150 rpm in an incubator shaker. The corresponding biomass quantity produced was 0.98 g/L. At agitation speed of 250 rpm, a decrease in nitrate removal, as well as biomass growth, was observed. In this condition, the observed percentage denitrification was 56% and the amount of biomass produced was 0.57 g/L. At agitation speed of 250 rpm, the reduction in nitrate removal may be because of the death of cells due to rupturing at high agitation speed.
Figure 4 Effect of agitation speed on percentage nitrate removal by Enterobacter sp. NCCP-29
In the literature, the effect of agitation speed is discussed with reference to DO level in denitrification process. With the increase in agitation speed, there was a corresponding increase in oxygen transfer into the media, which enhances the dissolved oxygen (DO) level of the medium (Taylor et al., 2009; Liang et al., 2011). Liang et al., (2011) reported that 420 mg/L NO3– N was completely removed within 30 h at 160 rpm and a further increase in agitation speed has not much effect in the efficiency in nitrate removal.
Effect of different carbon sources on denitrification
Growth rate and metabolic process of a bacterium mainly depend on the carbon consumption and the type of carbon source. In the present study, different carbon sources were selected to study their effect on the denitrification efficiency of the isolate. The carbon sources included in this study were dextrose, ethanol, sodium acetate, sodium succinate and methanol. Fig. 5 presents the results of the effect of different carbon sources on denitrification by isolated strain. The maximum nitrate removal of 95% with the corresponding biomass yield of 0.98 g/L was obtained in the medium supplemented with sodium succinate as a carbon source. In the case of other carbon sources like ethanol and sodium acetate, a satisfactory nitrate removal was observed. The nitrate removal efficiency was considerably less when dextrose and methanol were used as carbon sources. In these cases, nitrate removal was 72% and 39%, respectively. The corresponding biomass quantities produced were 0.33 g/L and 0.19 g/L, respectively.
It was observed that maximum nitrate removal could be achieved in the cases of medium supplemented with sodium succinate, sodium acetate, and ethanol as carbon sources. Sodium succinate and sodium acetate are the intermediate molecules of the TCA cycle in the metabolic activity and hence, they can easily be utilized by bacteria as electron donors. The presence of these compounds enhances the microbial growth, which in turn resulted in more nitrate removal. Choice of the carbon source is very important to avoid this incomplete denitrification because of the toxicity for many bacteria at higher levels of nitrite accumulation. Acetate donates electrons closer to nitrate reductase, in the upstream region of the respiratory chain to either ubiquinone or cytochrome (Van Rijn et al., 1996), but acetate as carbon source stimulates denitrification in activated sludge samples (Eilersen et al., 1995). Methanol donates electrons closer to nitrite reductase (Van Rijn et al., 1996).
Figure 5 Effect of different carbon sources on nitrate removal by Enterobacter sp. NCCP-29
Effect of carbon to nitrogen ratio on denitrification
The optimization of carbon source concentration is very important in denitrification process. Denitrification rate will be reduced if the concentration of the carbon concentration becomes too low or high. In the present study, carbon to nitrogen ratios 3:1, 2:1 and 1:1 for the carbon sources of ethanol (a), sodium acetate (b) and for sodium succinate (c) were considered to prepare NR media, for inoculation with isolate. While preparing NR media, carbon concentration was only altered, and nitrate concentration was kept constant.
The results of the effect of carbon to nitrogen ratios on denitrification are presented in Fig. 6. It is evident from Fig. 8that, for all individual carbon sources, 3:1 ratio was found to be the optimum ratio for nitrate removal. At this ratio, the maximum nitrate removal of 98% was achieved for the medium containing sodium succinate. The corresponding biomass yield obtained was 1.29 g/L.
Figure 6 Effect of different carbon sources and carbon to nitrogen ratio on percentage nitrate removal by Enterobacter sp. NCCP-29
Nitrate removal is strongly dependent on the amount of carbon available (Elefsiniotis et al., 2004). Liang et al., (2011) reported at an insufficient carbon concentration, the electron flow is too low to provide enough energy for cell growth and causes accumulation of intermediate such as nitrite. When excess carbon substrates are added, it inhibits the growth of the bacteria, which may, in turn, delay the denitrification process. Wang et al., (2007) reported that the optimal C/N ratio was 5.5 – 6.0 for nearly complete denitrification by Pseudomonas sp.
Response Surface Methodology (RSM) studies
Central Composite Design (CCD) was used to optimize the concentration of three independent variables temperature, pH and C: N ratio (Table 2). The behavior of the system was explained with the quadratic equation
Where Y represents the response, βo is the interception coefficient, βi is coefficient of the linear effect, βii is the coefficient of quadratic effect and βij is the coefficient of interaction effect. Xi and Xj represent the coded independent variables. From the initial screening experiments by one factor at a time method, the factors such as temperature (A), pH (B) and C: N ratios (C) were found to have significant effect on percentage denitrification. Hence, these variables were chosen as the independent variables. To maximize the percentage denitrification, these variables were optimized using response surface methodology. Central Composite Design (CCD) was used to optimize the three independent variables. A 23 factorial design augmented by 6 axial points (α = 2) was implemented in 20 experiments where in the effect of each compound on denitrification rate was taken as a response. A total number of 20 experiments were conducted in duplicates to establish the relationship between independent variables (pH, Temperature and C:N ratio) and dependent variable/response (percentage denitrification). Details of the design have been mentioned in Table 3.
Table 2 Coded significant parameters and their actual levels for optimization by CCD
Table 3 Full factorial central composite design matrix and their observed response
|pH||Temperature||C:N Ratio||Y=% of removal (Actual)||Y=% of removal (Predicted)|
Experiments were carried out according to the design as given in Table 3. Experiments were conducted in random run order and tabulated in standard order. Actual and predicted values were compared. Actual values were the response obtained from the particular experimental run and predicted response were values determined by approximating functions employed by the model and are presented in Fig 7.
Figure 7 Comparison of experimental and predicted values for nitrate removal
Adequacy of the model is tested by determining the significant variables with ANOVA. ANOVA consists of statistical result which was tested by means of specified classification difference. It consists of classified and cross-classified statistical results analyzed by Fisher’s statistical test (F-test). F-value is defined as the ratio of the mean square of regression to the mean square residual or error (Anupam et al., 2011; Nejad et al., 2011). The coefficient of determination (R2) and the adjusted R2wasevaluated to test the global fit of the model. The value of R2 was found to be 93.8 % and hence the model did not explain only 6.2% of the total variability. The value of R2 (adj) was 88.35% deviates only by 6.2 % from the R2 value. This indicates that the model is highly significant and there is only a meager chance to include any insignificant terms in the model. The significance of individual coefficients and its interactions are determined by students T-test and P-value. T-value gives the ratio of estimated parameter effect to the estimated parameter standard deviation. If P-value is less than 0.05, then it is statistically significant at a confidence level of 98% (Anupam et al., 2011). P-value is used as a tool to check the significance of coefficients, a small P-value (<0.05) and a larger regression and T-value indicates the significant effect of the variables on the response variable (Nejad et al., 2011; Anupam et al., 2011). Square (P<0.001) and interaction (P<0.001) effects of all the three variables temperature, pH and C:N ratio had a significant influence on the percentage denitrification. Regression analysis of the data resulted in the polynomial equation which is given below. Regression equation in uncoded units
The positive coefficients of variables indicate that they have a synergistic effect on rate of denitrification whereas the negative coefficients of variables indicate an antagonistic effect on the rate of denitrification. The results of the ANOVA are given in Table 4. P-value of pH (A) is less than 0.05, also the P-value of AC is also less than 0.05. These are said to play a significant role in denitrification. These are said to be interacting, so the Response surface plots obtained are elliptical in nature (Fig.9 ) while other response surface plots obtained were circular in nature (Fig. 8 & 10).
Table 4 ANOVA test for response function of nitrate removal
|Source||Sum of squares||Df||Mean square||F Value||P Value|
|R2= 93.87% Adj R2=88.35% Predicted R2=49.86%|
Figure 8 Response surface plot for temperature and pH on percentage denitrification
Figure 9 Response surface plot for the effect C:N ratio and pH on percentage denitrification
Figure 10 Response surface plot for the effect C: N and temperature on percentage denitrification
Fig 8, 9, 10 represent the three dimensional response and contour plots for the parameters (pH, temperature and C:N ratio) which affect the denitrification efficiency. It can be seen from the figures that, the percentage denitrification decreased beyond the neutral range of pH, at very low and very high temperatures and at higher C:N ratio. Maximum percentage denitrification was obtained at a temperature of 30°C, pH 6.5 and C: N ratio of 3:1. These optimized values were validated by conducting experiments using these conditions, the percentage denitrification was found to be 98%. These results are in good agreement with the predicted results and confirmed adequacy of the model.
In this study, a potential novel denitrifying bacterial isolate was isolated from municipal waste water and identified as Enterobacter sp. NCCP-29. The bacterium showed the highest nitrate removal capability of 98% without nitrite accumulation. The bacterium, Enterobacter sp. NCCP-29 isolated in the present study was found be an efficient denitrifier in aerobic condition, 98 % of initial 300 ppm of nitrate was denitrified within 48hunder aerobic condition. It also showed significant nitrate removal at pH 6.5, 150 rpm agitation speed, the temperature of 30°C, and for sodium succinate as a carbon source and 3:1 as C:N ratio. Moreover, the denitrification activity of the bacterium was not much affected by the increase in agitation speed up to 200 rpm, which indicated the aerobic denitrification process of the bacterium. These results suggest that Enterobacter sp. NCCP-29may be a prospective candidate for aerobic wastewater treatment. The developed models with high correlation based on the experimental results of the CCD and RSM were useful to understand the direct effect of nitrate concentrations on the performance of the denitrification process. The optimum operational conditions in order to have a maximum denitrification rate with more than 98% removal of nitrate was achieved with pH 6.5, temperature 30°C and C:N of 3:1
ACKNOWLEDGMENT: The authors wish to thank, VGST (GRD 478), Government of Karnataka, Bangalore for funding the project and Management, Principal and Department of Chemical Engineering, SDMCET for the support and encouragement to carry out the work at the department. We also thank Department of Biotechnology Engineering, NMAMIT, Nitte for the kind support.
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Fish is the main animal protein source for the populations (Okpanachi et al., 2018). While Benin’s population is growing (10,000,000 inhabitants in 2014), the cover of animal protein needs is in deficit. Facing this situation, in 2015, Benin imported 81,327 tons of frozen fish, dominated by Horse mackerel, Atlantic mackerel and Sardinella (FAO, 2017). Thus, in order to ensure the quality integrity of these fish, conservation by continuous cold is indispensable. The conservation methods used are freezing, refrigeration and ice preservation (Gandrota et al., 2012; Tolstorebrov et al., 2017; Lanlan et al., 2017). These methods preserve the freshness state, nutritional qualities and taste properties of fish and aim to extend their shelf life (Nalan and Pilar, 2015; Varghese and Mathew, 2016).However, when fish are poorly preserved or undergo break in the cold chain, they are likely to favor pathogenic micro-organisms development or toxins production (ANSES, 2010; Simoes, 2016) and provoke inconveniences, in particular to the food industry sector. The prolonged storage of cold-stored fish can degrade cell walls, cause weight loss and promote proteins denaturation. The freshness state, vitamins and nutritional value of these fish are also altered. These various deteriorations are the cause of three major mechanisms: cellular autolysis, microbial growth and oxidative reactions (Ghaly et al., 2010; Sharifian et al., 2014). Thus, the quality of imported frozen sea-fish (Scomber scombrus, Trachurus trachurus), which already undergo relatively long storage before being discharged into local markets, is questioned (Abubakar and Uzairu, 2015; Kabamba, 2016). This depends on the storage temperature and storage duration as well as on the freezing technique used (Popelka et al., 2012). In addition to the dubious quality of these fish, in Benin, the surveys carried out by Bocodaho (2015) and Gnimavo (2015) on frozen fish imported in Benin revealed that the storage temperature is not constant during storage in fish-shops of Cotonou (98%) and Abomey Calavi (78%). This break in the cold chain is provoked for several reasons including the power cut due to generators failure and the consumer electricity high price. According to Moons (2016), any liquid having undergone this break in the cold chain is likely to present risks to the consumer health. In general, changes that occur during the storage limit these fish use as a raw material for the processing sector or as a food for the consumer (Bou m’handi et al., 2015).In order to improve these fish quality, several studies were carried out on their microbiological quality during smoking (Degnon et al., 2012, 2013, Chabi et al., 2014; Kpodekon et al., 2014) and preservation (Wabi, 2010). Apart from the microbiological quality, no study was carried out on the impact of break in the cold chain on the technological and organoleptic qualities of frozen fish, despite the frequent power cut registered In Benin. The objective of the study is to evaluate the impact of break in the cold chain on the technological and organoleptic qualities of Atlantic mackerel (Scomber scombrus) and Horse mackerel (Trachurus trachurus) in south Benin. These two fish species were chosen because of their high frequency in households’ consumption in south Benin. MATERIAL AND METHODSStudy areaThe data collection on the impact of break in the cold chain on the technological and organoleptic qualities of Atlantic mackerel (Scomber scombrus) and Horse mackerel (Trachurus trachurus) in south Benin was carried out from June to December 2016 in the Township of Abomey Calavi.This Township is located at 12 meters of altitude between 6° 26′ North latitude and 2° 21′ East longitude. It is in the Atlantic Department, South of Benin and bounded to the North by the Township of Zè, to the South by the Atlantic Ocean, to the East by the Township of Sô-Ava and Cotonou, and to the West by the Township of Tori-Bossito and Ouidah. The climate is of the subequatorial type characterized by two rainy seasons and two dry seasons. The Township has two bodies of water, Nokoué Lake and Cotonou Lagoon, a sea front juxtaposed to Cotonou Lagoon, marshes, streams and swamps. These bodies of water offer the Township a very lively artisanal fishing activity. The township has also several local markets (Kpota, Glodjigbé, Akassato, Zinvié and Zè). The Kpota market has an embankment where caught fish in the Nokoué Lake are regularly sold. In this market fish are marketed whole or processed. Fish processing is of artisanal type. Once the cold chain break tests performed, the data on the technological and organoleptic parameters were collected and analyzed at the Laboratory of Animal Biotechnology and Meat Technology of the Department of Animal Production and Health of the Polytechnic School of Abomey-Calavi of the University of Abomey-Calavi. Experimental procedure and samples A total of 120 Atlantic mackerel (Scomber scombrus) and 120 Horse mackerel (Trachurus trachurus) were sampled for the assessment of the effect of break in cold chain on these fish quality at the laboratory. These fish were sampled at the Food Products Distribution Common Agency in Cotonou, at the supply place of wholesalers and retailers. They were then transported to the laboratory in a cooler at 4° C in accordance with the ISO 7218: 2007 standard. Two preservation methods were tested: freezing (-18° C) and refrigeration (+4° C).Several fish batches were formed: the batch1: intended for refrigeration didn’t undergo power cut during the experimentation, it is the control batch1; the batch 2: intended for freezing didn’t undergo power cut during the experimentation, it is the control batch 2; The batches 3, 4 and 5: intended for refrigeration, underwent respectively power cuts of 3 hours, 6 hours and 12 hours per day for 3 days each; The batches 6, 7 and 8: intended for freezing, respectively underwent power cuts of 3 hours, 6 hours and 12 hours per day for 3 days each. For each batch, according to the preservation mode and to the power cuts, ten (10) fish were considered and this by species. The control batches that didn’t experience power cut, received each 10 fish per species for each preservation mode.The technological parameters measures were taken. The pH at 1h, 3h and 6h after each break in the cold chain were taken over 3 days and this by batch. For the 3h of break, only pH at 1h and 3h were taken.The water content of fish fillets was determined according to the Codex Stan 167-1989 standard. The fish yield (eviscerated fish weight*100/ total weight of whole fish) and filleting yield (fillet weight*100/total weight) were calculated. Concerning the organoleptic parameters, the fish freshness state was assessed according to the regulation 790/2005/EC, the color was taken according to Mathis (2003) using a colorimeter (Konica Minolta CR-400 INC) where the red index (a*), the yellow index (b*), the lightness (L*) were measured and the chroma was calculated by the following formula: C* = (a* ² + b* ²)1/2. Statistical analysisThe Proc means procedure of SAS (2013) was used to calculate the averages of the different technological and organoleptic variables. The General Linear Model (GLM) procedure was used for the variance analysis and the variation sources taken into account were fish species, power cut techniques, power cut duration, and the interaction between species and power cuts. The F test was used to determine the significance of each effect of the variance analysis model and averages were calculated and compared by the student t test.
Technological characteristics of fish
Effect of species, preservation technique and break duration on the technological characteristics of fish
The whole fish weight and the various organs/parts weight (eviscerated fish, fillets, viscera) of Atlantic mackerel were significantly higher than those of Horse mackerel (P<0.001). By contrast, Horse mackerel showed a higher yield than Atlantic mackerel (93.75% vs 92.42%, P<0.001) (Table 1). The whole fish weight, eviscerated fish weight, net weight, viscera weight and the remains were respectively 405.99g, 373.63g, 183.84g, 30.35g and 222.1g in Atlantic mackerel against 178.38g, 167.56g, 73.13g, 10.82g and 105.25g in Horse mackerel. The fillet yield was significantly higher (P<0.05) in Atlantic mackerel (45.85%) than in Horse mackerel (41.17%) (Table 1). The water content was significantly higher (P<0.01) in Horse mackerel (71.38%) than in Atlantic mackerel (65.55%). This water content differs from one species to another (P<0.01) (Table 1). The pH values in Atlantic mackerel ranged from 6.13 to 6.24 and those in Horse mackerel from 6.27 to 6.3. The pH of Atlantic mackerel decreased from 3h to 6h of break in cold chain (6.24 to 6.13). By contrast, the one of Horse mackerel from 1h to 3h of break (6.3 to 6,27) and increased at 6h of break (6,29). No influence of the preservation technique was observed on the whole fish weight, eviscerated fish weight, viscera weight, remains, and water content. On the contrary, the refrigerated experimental batch showed the best fish yield and the lowest fillet yield. The different pH values were influenced by the preservation technique (freezing and refrigeration). The highest pH values were found in frozen fish and the lowest in refrigerated fish. Apart from pH6, which was higher (P<0.05) in the refrigerated control batch than in the refrigerated experimental batch, no difference was found between pH values in the same preservation technique (Table 1).
The break duration influenced significantly the different weights (whole fish, eviscerated fish, viscera, fillet and the remains). Thus, the whole fish weight, the eviscerated fish weight and the remains weight were significantly higher (P<0.05) for the 6 hours of break than for the 3 hours and 12 hours of breaks. The viscera weight was significantly higher for fish with 6 hours and 12 hours of break than with 3 hours of break. The fillet weight at 6 hours (140.40 g) of preservation break was significantly higher than the weight at 3 hours (112 g) of preservation break. By contrast, no influence of the break in the cold chain was observed on fish yield, fillet yield, water content and pH (Table 2).
Organoleptic characteristics of fish
Variation of color according to the day, the species and the break duration
The color variability between day, species, and break duration is shown in the Table 3. The lightness, the yellow index and the red index didn’t vary with the break day. On the contrary, the chroma was higher on the days 2 and 3 than on the day 1 (P<0.05).The red index of Atlantic mackerel (Scomber scombrus) was higher (P<0.01) than that of Horse mackerel (Trachurus trachurus). The same trend was obtained for the yellow index and the chroma.The lightness, yellow index and chroma didn’t vary with the break in cold chain duration. However, the red index was higher (P<0.05) for the 6 hours of break than for the 12 hours of break. Whatever the day, the lightness, red index, yellow index and chroma didn’t significantly vary from one species to another. The same trends were obtained for these parameters according to the sex on the day 1 and to the break duration on the day 3 (Table 4). By contrast, fish were lighter for 3 hours of breaks than for 12 hours of breaks on the day 1. The red index, yellow index, and chroma didn’t significantly vary with the break duration on the day 1. On the day 2, the lightness and red index didn’t vary with sex, while the yellow index and chroma were significantly higher in the female than in the male. On the same day 2, the lightness, the red index and the chroma didn’t significantly vary by the break duration whereas the yellow index was lower for the 3 hours of breaks than for the breaks of 6 hours and 12 hours. On the day 3, females were lighter than males while the red index, yellow index, and chroma were higher in males than in females (Table 4).
Assessment of the freshness state of fish
The preservation technique had no influence on the freshness state of the skin, the eye, the gills and the abdominal cavity. By contrast, it highly influenced (P<0.001) the freshness state of the back muscle, abdominal wall, spine and the organs color. The back muscle of frozen fish and of refrigerated control batch fish was firm, rigid and elastic, whereas the refrigerated experimental batch fish had a dorsal muscle that decreased in elasticity. The abdominal wall was intact in the frozen fish (control and experimental batches) and in the refrigerated control batch fish, whereas it was less soft in the refrigerated experimental batch fish (Table 5). The break duration had no effect on the state of gills and abdominal cavity but it has significantly influenced the freshness state of the skin, eye, back muscle, abdominal wall, spine and of the organ color (P<0.001) (Table 6). With the breaks of 3 hours and 12 hours, the fish skin was light without luster with slightly unclear mucus and odor neither seaweed nor bad, while with a break of 6 hours, this skin was light, shimmering without discoloration with aqueous and transparent mucous and a seaweed odor. For the breaks of 3 hours and 6 hours, the dorsal muscle was firm, rigid and elastic and became less elastic when the rupture lasted 12 hours. The abdominal wall was intact for the breaks of 3 hours and 12 hours but less soft for the break of 6 hours. The spine was slightly pink with adhesions for the breaks of 6 hours and 12 hours while it was colorless and broke down instead of being removed for the breaks of 3 hours. The organs color was red masts for the breaks of 6 hours but bright red for the breaks of 3 hours and 12 hours.
Table 1 Effect of the species and preservation technique on the technological characteristics of fish
|Variation Source||Species||Techniques||Significance test|
|Scomber scombrus||Trachurus trachurus||TBF||CBF||CBR||TBR|
|Whole fish weight (g)||405.99||104.37||178.38||45.5||304.5a||60.70||277.2a||74.70||285.3a||145.30||299.7a||157.8||***||NS|
|Eviscerated fish weight (g)||375.63||98.42||167.56||44.64||279.9a||48.40||255.1a||68.50||264.2a||133.50||280.7a||147.4||***||NS|
|Fillets weight (g)||183.84||40.85||73.13||18.12||133.1a||34.60||125.3a||29.20||130.8a||69.40||125.9a||69.5||***||NS|
|Viscera weight (g)||30.35||9.19||10.82||2.15||24.6a||14.24||22.05a||12.92||21.12a||12.45||18.97a||10.9||***||NS|
|Fish yield (%)||92.42||1.9||93.75||1.29||92.36b||3.44||92.23b||3.63||92.78b||1.28||93.72a||0.88||***||***|
|Water content (%)||65.55||6.45||71.38||10.86||67.67a||14.28||72a||13.56||70.2a||5.59||66.2a||10.23||**||NS|
TBF : Tested Batch Freezing ; CBF : Control Batch Freezing ; CBR : Control Batch Refrigeration ; TBR : Tested Batch refrigeration ; SD : Standard Deviation ; pH : Hydrogen potential. NS: P>0.05; *: P <0.05; **: P<0.01; ***: P<0.001; The averages of the same line followed by different letters differ significantly at the threshold of 5%;
Table 2 Effect of cold chain break on the technological characteristics of fish
|Variation source||Break||Significance test|
|Whole fish weight (g)||264b||102||341.7a||197.2||295.7b||121.4||*|
|Eviscerated fish weight (g)||247.7b||94.5||318.4a||184.8||275.5b||111.8||*|
|Fillets weight (g)||112b||53.4||140.4a||82.8||128.9ab||56.3||*|
|Viscera weight (g)||16.26b||9.52||23.29a||13.9||20.17a||10.44||*|
|Fish yield (%)||94.03a||1.56||93.11a||1.99||93.33a||1.16||NS|
|Water content (%)||67.5a||15.4||64a||7.72||67.83a||7.93||NS|
SD : Standard Deviation ; pH : Hydrogen potential. NS: P>0.05; *: P <0.05; ***: P<0.001; The averages of the same line followed by different letters differ significantly at the threshold of 5%;
Table 3 Color variability between days, species and break duration
|Break duration||3 hours||41.71a||6.72||8.85ab||3.45||14.66a||6.73||174.40a||97.90|
L*: lightness; a*: red index; b*: yellow index; J: Day; SD: Standard deviation; NS: P>0.05; *: P<0.05; **: P<0.01; the intra-class means of the same column followed by different letters differ significantly at the threshold of 5%;
Table 4 Color variability of fish fillets by species, sex and cold break duration per day
|Variation source||Day 1|
|Break duration||3 hours||46.53ab||6.23||10.2a||4.88||7.42a||3.01||94.7a||81.90|
|Break duration||3 hours||38.68||5.55||8.33||2.26||17.73b||3.03||198.7||64.00|
|Break duration||3 hours||39.92||5.92||8.01||2.42||18.81||6.3||230||93.4|
L*: lightness; a*: red index; b*: yellow index; SD: Standard deviation; NS: P>0.05; *: P<0.05; **: P<0.01; ***: P<0.001; the intra-class means of the same column followed by different letters differ significantly at the threshold of 5%;
Table 5 The technique effect on the freshness state of fish
|Abdominal cavity||2.91a||0.28||3a||0||3a||0||3 a||0||NS|
TBF : Tested Batch Freezing ; CBF : Control Batch Freezing ; CBR : Control Batch Refrigeration ; TBR : Tested Batch refrigeration ; SD : Standard Deviation ; NS: P>0.05; ***: P<0.001 ; The averages of the same line followed by different letters differ significantly at the threshold of 5%;
Table 6 The break effect on the freshness state of fish
SD : Standard Deviation ; NS: P>0.05; ***: P<0.001 ; The averages of the same line followed by different letters differ significantly at the threshold of 5%;
Effect of species, preservation technique and break duration on the technological characteristics of fish
The difference between the whole fish weight, the various organs/parts weight (eviscerated fish, fillets and viscera), the yields and the water content of Atlantic mackerel and Horse mackerel is due to the genetic type.Atlantic mackerel (Scomber scombrus) are generally large and fatty fish (Alinasabhematabadi, 2015) and Horse mackerel (Trachurus trachurus) have a fairly compressed elongated body with a wide head (FAO, 2018). The noted differences in the various organs and yields are due to the whole weights difference between the two species. This weight is higher in S. scombrus than in T. trachurus. The remains of both species are very important in relation with the whole weight and are a limiting factor for high fillet weights obtaining. Thus, S. scombrus has a higher fillet weight than T. trachurus, which explains the higher filleting yield of S. scombrus compared to T. trachurus.In addition, morphometric parameters such as standard length, total length, body perimeter, condition factor, and body shape influence different yields (eviscerated, beheaded, and carcass). Studies on the carcass characteristics of Labeo rohita (Sahu et al., 2013) and on those of Hypselobarbus pulchellus (Raghunath et al., 2016) have shown that the fish body perimeter is a good indicator to assess yields (carcass and filleting). In the current study, the round body shape of Atlantic mackerel influenced the fillet yield compared to T. trachurus of which the body shape is flat. Even the Toubiana (2016) studies on the yield prediction of Dicentrarchus labrax reveal that fish with a small head have a better fillet yield. In this study, S. scombrus have a small pointed head and show a better yield than T. trachurus which have a wide head. Besides, the differences in fillet yield may also be related to lower trimming losses (Bugeon et al., 2008).Alfaro et al. (2013) report for the same species a water content of 61-63% close to 71.38 ± 10% found in the current study. In Atlantic mackerel, weights ranging from 318 to 556 g were reported by Fjermetad et al. (2000) and water contents ranging from 65 to 78%, by Aubourg et al. (2005). The values reported in this study are within the ranges reported by these authors. In addition, the frozen fish showed a better yield and a higher water content than the refrigerated fish; this is due to the storage temperature in both techniques because the break duration has no influence on these two variables. According to Magnussen et al. (2008), the lower is the storage temperature, the better is the yield.The highest pH values were found in frozen fish because the freezing temperature is lower (-18° C) than that of the refrigeration (+ 4° C) and allows fish to better maintain their freshness state. Contrary to our study, Obemeata and Christopher (2012) didn’t show this preservation technique effect (freezing and refrigeration) on the pH until the 2nd week of preservation in Tilapia guineensis. Even, Akter et al. (2014) observed a pH increase in frozen Pangasianodon hypophthalmus at -20° C after 100 days. Alfaro et al. (2013) reported pH values ranging from 6.2 to 6.9 in Atlantic mackerel (Scomber scombrus) kept at temperatures ranging from 2 to 10° C and present no correlation with the bacterial load growth. In addition, Aubourg et al. (2001) reported that pH is not a good freshness state indicator. The influence of the break duration on the whole fish weight, the eviscerated fish weight and the remains weight is due to changes in the fish freshness state. Thus, 3 hours after the cold break, fish preserve better their states, 6 hours after, they lose water (preservation ice water in particular) and consequently lose weight; then 12 hours after the preservation, fish degradation begins and several elements are already in decomposition (denaturation) and gases take form, which increases the fish weight. In addition, during freezing, 95% of the fish composition water freezes and the ice crystals formation damages the texture and is factor favoring water and nutrients losses during freezing (FAO and OMS, 2012; Liu and al., 2013; Sampels, 2014). Water losses are obtained in Epinephelus coioides fillets stored at +4° C (Sharifian et al., 2014) and in Chinese carp fillets (Ctenopharyngodon idella) stored at -3° C (Liu and al., 2013). Also, weight losses were registered in salmon (Salmo salar) eviscerated and refrigerated at +4.5° C on the day 0 and after two days, the weight loss stabilized at about 2% (Erikson and al., 2011). These water losses lead to pathogenic microorganisms’ development or toxin formation (Moons, 2016). The break in the cold chain aggravates the water losses in the current study. However, according to Erikson and al. (2011) the weight increase of preserved fish is unacceptable from a commercial point of view.Moreover, no influence of the break duration was noted on the pH which confirms the hypothesis of Aubourg and al. (2001) according to which pH is not a good freshness state indicator.
Effect of break in the cold chain on color and freshness state
The day of the cold break has no influence on the lightness, red index, yellow index and chroma of fish (Horse mackerel and Atlantic mackerel). The works of Hong and al. (1996) on Atlantic mackerel (Scomber scombrus) show no variation in lightness, red index, yellow index and chroma during the first experimentation week but from the 14th day, the flesh of these Atlantic mackerels kept at -2° C was lighter, redder and yellower. Similarly, no difference in color (L*; a*; b*) was observed in salmon fillets (Salmo salar) stored at +4.5° C on the experimentation day 4, but the difference in color appears on the 11th day (Erikson and al., 2011). The current study lasted only 3 days and doesn’t show this influence.The red index decreases with the break duration and this is characteristic of the progressive deterioration of the freshness state. Thus, fish were lighter for 3 hours of breaks than for 12 hours of breaks on the day 1. Similarly, Knockaert and al. (2007) claim that usually, a raw fresh of fish fillet has a translucent appearance. The lightness difference observed during a refrigerated storage makes the flesh to become progressively milky and opaque. This phenomenon is probably due to proteins denaturation. This denaturation has already been reported in most of the preserved fish (Bratt, 2010) and in ling-cod fillet (Ophiodon elongates) refrigerated at 2° C and frozen at 20° C (Duan and al. 2010). The freshness state of the various organs didn’t vary from one species to another, and this shows that the fish were subjected to the same experimental conditions.
The dorsal muscle and abdominal wall of the frozen fish and the control batch of refrigerated fish showed a better freshness state than that of the refrigerated experimental batch and this is justified by the storage temperature of each technique. Indeed with refrigeration, the fish are kept at +4°C of temperature whereas with freezing, they are kept at -18° C of temperature and so, they are better preserved. This also justifies the difference in the abdominal wall that was soft in refrigerated Atlantic mackerel while intact in frozen Atlantic mackerel. The influence of preservation temperature on fish freshness state in general has been reported by Duan and al. (2010) and particularly on Trachurus trachurus by Alfaro and al. (2013). During refrigeration, the cold stops or slows down the cellular activity, the enzymatic reactions and induces during the cold break water losses. The muscular structures don’t toughen but soften when the temperature increases with the cold break. The temperature variation at the cold break explains the differences in the abdominal wall for each of the preservation techniques. Obemeata and Christopher (2012) also reported this effect of storage temperature on the freshness state of Tilapia guineensis. Indeed, the high temperatures accelerate the fish degradation which is on the contrary slowed down at low temperature. As a result, if the fresh fish is kept at a low temperature, the quality loss is slow. The faster the low temperature is reached during fish refrigeration, the more effectively the deterioration phenomenon is inhibited (Shawyer and Medina Pizzali, 2005). Margeirsson et al. (2010a) and Mai and al. (2012) confirm that at a temperature close to 0° C a high quality of freshness state is obtained. The break duration has no effect on the gills and abdominal cavity state because these organs are not superficial. In addition, the experimentation duration is relatively short (12 hours of break at the maximum) and didn’t reveal the freshness state changes of these organs. By contrast, it influences the freshness state of the skin, eye, dorsal muscle, abdominal wall and spine which are superficial organs and therefore immediately subjected to the environment influence. Thus, immediately after death, the fish muscle is completely relaxed and the elastic and flexible texture usually lasts a few hours, after which the muscle contracts (Bouazaoui, 2011). When it toughens, the body stiffens up and the fish is then in rigor mortis state (Bouazaoui, 2011). ConclusionThe impact of break in the cold chain on the technological qualities of Scomber scombrus and Trachurus trachurus shows that the weight, yield and water content of fish vary between the two species. The cold chain maintenance during freezing guarantees a better fish freshness state. Preservation by refrigeration deteriorates the fish freshness state and modifies the internal organs state (abdominal wall, spine, and organs color). The red index and the yellow index of S. scombrus are more affected than those of T. trachurus. The cold chain interruption influences the fish quality. In general beyond the 3 hours of break, the fish quality deteriorates. After 6 hours of the cold break, fish are more and redder. On the day 2 of cold break and after 6 hours of time, fish lose their sensory quality because of the flesh yellow index which is more affected. However, fish evisceration prior preservation and continuous maintenance of the cold chain would guarantee a high quality of the preserved and consumed fish in Benin.
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Perylene (PRL) (C10H12), as a representative of polycyclic aromatic hydrocarbons (PAHs) has been reported as hazardous pollutant (Donaldson et al., 1953). It is an extensively determined 5-ring PAH, derives from varied sediment environments such as marine sediments (Slater et al., 2013), fresh water and river sediments and peats (Hu et al., 2014). PRL has been identified in marine and terrestrial sediments ad well as in brown coals, crude oils and sedimentary rocks (Marynowski et al., 2015). The occurrence of PRL is mainly associated with the terrestrial organic matter (Stefanova et al., 2013). The terrestrial soil holds the precursors of PRL which are being transported by rivers to the coasts (Varnosfaderany et at., 2014). Although PRL has been the object of several environmental studies, report is very less on its toxicity. Respiratory tract lesions including tumors in respiratory tract have been reported as toxic effects of PRL (Kephalopoulos et al., 2014). The carcinogenic effects of PRL in human and animals exposed orally or by inhalation have also been reported (US EPA, 2007). The coke oven workers were found to be affected by high concentration of PRL which reduced their levels of serum immunoglobins (Irwin et al., 1997). Cunha et al. (2006) reported the toxicity of PRL on benthic bacteria and macro-fauna. The predominance of PRL over other PAHs has been found in marine sediments throughout the world at concentrations that are different from those of other PAHs (Itoh and Hanari, 2010).
There are many physico-chemical methods for remediation of PAHs which include chemical oxidation, photolysis, incineration, landfilling, volatilization and adsorption. But applications of these methods are limited due to certain drawbacks such as high operating cost, formation of toxic products etc. (Gan et al., 2009). Microbial remediation is one of the most significant natural processes that can influence the fate of pollutants in both aquatic and terrestrial environment. Biodegradation is a promising method to remediate PAHs as it is inexpensive, environmental friendly and able to convert toxic substances into harmless products compared to conventional methods that need high cost and may produce hazardous by-products which can affect the environment (Qin et al., 2017).
Biosurfactants have become one of the most versatile chemicals appropriate to be used for various industrial and environmental purposes. There are reports on bacteria and yeasts producing biosurfactants which are having lot of applications in various fields including remediation of number of pollutants(Raza et al., 2007).
Recently, nanoparticles have been used as reductants or catalysts to improve various reactions due to their high surface areas and other characteristics (Nezahat et al., 2009). In addition, effect of nanoparticles on microorganisms has additionally created great interests. Nanoparticles are able to assist microbial activities (Shin and Cha, 2008). But extremely limited studies have been conducted on bioremediation of pollutants in presence of nanoparticles and produced biosurfactants in the growth medium using microbes (El-Sheshtawy and Ahmed, 2017).
Remediation of PRL through biological method is receiving attention now-a- days. There are relatively few publications on successful microbial remediation of PRL (Hesham et al., 2006; Silva et al., 2009; Mandal and Das, 2018) . In addition, the use of microbial consortia is considered to be more stable and effective than using single organism because of their diversity and synergistic effect of metabolic activity that occur in microbial consortia (Hesham et al., 2006; Mishra et al., 2014; Mandal and Das, 2018). So far, no report is available on nanobioremediation of PRL in the presence of nanoparticles and produced biosurfactant in the medium using microbial consortium.
In the present study, an attempt has been made to optimize the remediation process using essential variables that enhance the biodegradability of PRL using 3-level Box- Behnken design which can provide a mathematical model showing the influence of each variable and their interactions. Experiments were conducted to study the biodegradation of PRL using yeast consortium in presence of ZnO nanoparticles and produced biosurfactant in the growth medium. To the best of our knowledge, this is the first study in which process optimization has been done towards nanobioremediation of PRL using yeast consortium in presence of ZnO nanoparticles and produced biosurfactant in medium.
MATERIALS AND METHODS
In our previous study, the yeast consortium YC02 was already reported as potential degrader of PRL (Mandal and Das, 2018) which consist of three yeast isolates viz. Hanseniaspora opuntiae NS02, Debaryomyces hansenii NS03 and Hanseniaspora valbyensis NS04. So, YC02 was selected for the present study.
Synthesis and characterization of ZnO nanoparticles
The synthesis of ZnO nanoparticles was done following the standard method (Litt and Almquist, 2009). The ZnO nanoparticles were prepared by the precipitation technique using zinc chloride [ZnCl2.9H2O] (Sigma-Aldrich). In practical, 2.6 g of ZnCl2 was dissolved in 1000 ml distilled water and precipitated using 33% dilute aqueous ammonium hydroxide solution (Sigma-Aldrich) at room temperature until the pH reached a value of 13. The slurry was then agitated for 30 min. The resultant precipitates were filtered and washed carefully, and the sample was dried at 100ºC for 12 h. The characterization of ZnO nanoparticles was done using UV spectroscopy, X ray diffraction (XRD), Fourier transform Infrared spectroscopy (FTIR) analysis, TEM and EDX analysis.
Production of biosurfactant in culture media
For biosurfactant production, a mineral salt medium (MSM) was prepared. Trace element solution containing (g L-1): 0.116 of FeSO4.7H2O, 0.232 of H3BO3, 0.41 of CoCl2.6H2O, 0.008 of CuSO4.5H2O, 0.008 of MnSO4.H2O, 0.02 of (NH4)6Mo7O24 and 0.174 of ZnSO4 were added to MSM (Haghighat et al., 2008). The yeast consortium YC02 was inoculated in 500 mL Erlenmeyer flasks containing 150 mL MSM of initial pH 7.0 and incubated at 30ºC for 3 days under shaking condition of 130 rpm.
Biosurfactant production by YC02 in MSM was confirmed by various tests viz. drop collapsing test (Bodour and Miller-Maier, 1998), methylene blue agar plate method (Satpute et al., 2008) and emulsification test ( E24) (Bodour et al., 2004) following the standard procedures.
Biodegradation of PRL in presence of ZnO nanoparticles and produced biosurfactant
The biodegradation experiments were conducted in sterilized 500 mL Erlenmeyer flask containing 100 mL of sterilized mineral salt medium supplemented with PRL (50 mg L-1) under different set of conditions as follows : (i) PRL + YC02 (ii) PRL + YC02+ produced biosurfactant (iii) PRL + YC02+ ZnO nanoparticles (0.5 g L-1) (iv) PRL +YC02 + produced biosurfactant + ZnO nanoparticles (0.5 g L-1). Flasks without inoculation were maintained as control. The residual PRL was extracted from the different set of conditions and biodegradation percentage was calculated.
GC-MS analysis was done to determine the residual PRL and the degraded products in the culture broths (Arulazhagan et al., 2010). Flasks from different set of conditions were withdrawn after 6 days of incubation. The degraded products were extracted using ethyl acetate. The solvent was removed under vacuum by rotary evaporation (SuperfitTM Rotary vacuum Digital bath) prior to analysis. Aliquots of 2-5 µL were injected directly for GC–MS analysis, (JEOL GC MATEII) using silica as stationary phase. The inlet temperature was 220ºC; oven temperature was increased from 50 to 250ºC at 10ºC rev min-1; the GC interface temperature was 250ºC; carrier gas was nitrogen at a flow rate of 1.0 mL rev min-1. Mass spectrum conditions had the ionization energy 70 eV, ion chamber temperature was maintained at 250ºC with tungsten filament which was used for the ionization of molecules. The concentration of PRL was calculated by comparing the peak areas of each treated sample with that of the peak area of the abiotic control. For identification of the degraded products, the mass spectra of the products formed were compared with respective mass spectra of authentic compounds and also with the mass profile of the same compound available in the National Institute of Standard Technology (NIST) library, USA.
The FTIR spectra of PRL and degraded products were used to determine the vibrational frequency changes in functional groups. The extended degraded products dissolved in ethyl acetate, were mixed with KBr and made in the form of pellets (13 mm in diameter and 1 mm thickness). IR spectroscopy was investigated with the IR affinity-1 FT-IR spectrophotometer (Shimadzu). The scanning wavenumber ranged from 4,000 to 400 cm‑1 and the spectral resolution was 4 cm-1.
Response surface methodology (RSM) using Box-Behnken design (BBD) was used for optimization of parameters and to determine the significant single factors, interactions and quadratic terms. Each factor was varied at three different levels -1, 0 and +1 signifying low, medium and high values. The minimum and maximum ranges of pH, temperature, shaking speed, inoculum dosage and zinc oxide nanoparticle concentrations were determined as shown in Table 1. A design of 46 experiments were formulated and the experiments were carried out in 250 mL Erlenmeyer flasks containing 100 mL of production medium (PRL 50 mg L-1). The experiments were performed twice to optimize the levels of the selected variables viz., pH (A), temperature (B), shaking speed (C), inoculum dosage (D) and zinc oxide nanoparticle concentrations (E). The range of the variables was chosen based on preliminary experiments. The 3D contour plots were prepared to evaluate the optimized parameters, which influence the response. The respective responses were analyzed by using a second order polynomial equation, and the data were fitted to the equation by multiple regression procedures. Later, an experiment was conducted in triplicates using the optimum values for variables given by response surface optimization to validate the predicted value and the observed value of the responses. The results of the experimental design were analyzed and interpreted using Design-Expert version 11.0 (Stat-Ease Inc. Minneapolis, MN, USA) statistical software (Sahoo and Gupta, 2012).
Table 1 Independent factors and its level used in response surface design for PRL biodegradation
Factors Name Level (0) Low level(-1) High level(+1)
A pH 7 5 9
B Temperature (°C) 30 10 50
C Shaking speed (rpm) 130 110 150
D Inoculum dosages (%) 3 1 5
E ZnO nanoparticle concentration (g L-1) 2 1 3
Degradation kinetics was performed in triplicates. The zero order (Wang et al., 2002), first order (Agarry et al., 2013) and second order (Capellos and Bielski, 1972) kinetic models were used to define the degradation of PRL in mineral medium.
RESULTS AND DISCUSSION
Characterization of ZnO nanoparticles
UV-Visible Spectroscopy analysis of ZnO nanoparticles
The optical characterization of the sample was recorded on UV-Vis absorption spectrophotometer showed in Figure 1a. The UV-Visible absorption spectroscopy of ZnO nanoparticles in deionized water exhibited maximum absorption at two different wavelengths of 240 nm and 390 nm which clearly indicated the gradual formation of ZnO nanoparticles. Similar result was reported by Kulkarni and Shirsat (2015).
XRD analysis of ZnO nanoparticles
The XRD pattern of ZnO nanoparticle exhibited well-defined peaks at 2θ values of 13.13, 15.28, 26.13, 26.90, 33.31, 38.94 and 59.69 which correspond to the 010, 011, 113,104, 213, 123 and 401 planes respectively (Figure 1b). The intensity of ZnO nanoparticle peaks at 2θ values of 15.28, 26.90, 33.31 and 59.69 reflected high degree of crystallinity in nanoparticles. Similar result was demonstrated by Kumar and Rani (2013).
FTIR analysis of ZnO nanoparticles
The FTIR spectrum of dispersed ZnO nanoparticles in deionized water was shown in Figure 1c. Infrared studies were carried out in order to ascertain the purity and nature of the zinc oxide nanoparticles. Metal oxides generally give absorption bands in fingerprint region i.e. below 1000 cm-1 arising from inter-atomic vibrations. The peaks at observed at 3257.77, 3165.19, 3101.54 cm-1 were due to O-H stretching vibration arising from hydroxyl groups from the water on nanoparticles. The absorption peaks at 1548.84, 1498.69, 1361.03, 1354.03, 1039.63 and 947.05 cm-1 were due to de-ionized water used as solvent. The absorption peaks at 690.52, 514.99, 480.29 cm-1 were corresponding to the Zn-O bond stretching and deformation vibration, respectively. Similar FTIR spectra were observed in case of zinc oxide nanoparticles (Parthasarathi and Thilagavathi, 2011; Kumar and Rani, 2013).
TEM and EDX analysis of ZnO nanoparticles
The morphological and structural properties of the prepared nanoparticle are illustrated in the TEM images (Figure 1d). The ZnO nanoparticle particles are highly agglomerated with roughly the average particle size of 45 mm. The elemental analysis of ZnO nanoparticle was performed using energy dispersive X-ray (EDX) analysis (Figure 1e). The EDX spectrum of ZnO nanoparticle indicated the presence of Zn and O as present on ZnO nanoparticle. The peak of Zn and O were noted on EDX which confirmed ZnO nanoparticle.
Figure 1 Characterization of ZnO nanoparticle: (a)UV-Visible Spectroscopy analysis of ZnO nanoparticle, (b) XRD analysis of synthesized ZnO nanoparticles, (c) FTIR analysis of synthesized ZnO nanoparticle, (d) TEM micrographs of synthesized ZnO nanoparticle and (e) EDX of synthesized ZnO nanoparticle.
Biosurfactant produced by yeast consortium (YC02) in the MS medium was tested by methylene blue agar test, drop collapsing test and emulsification index (%). The positive results of all the tests confirmed the biosurfactant producing ability of YC02. The flat drop appearance in micro titer plate confirmed the positive results for drop collapse test. Dark blue halo zone in the methylene blue agar plate supplemented with CTAB confirmed the presence of anionic biosurfactant (Figure not shown). The emulsification index was found to be 61.7%.
GC-MS analysis for PRL biodegradation
The degradation of PRL (50 mg L-1) by yeast consortium YC02 in MSM was found to be 67.0% after 6 days of incubation. Improvement in degradation (68.3%) was noted when MSM was supplemented with ZnO nanoparticles (0.5 g L-1). Maximum degradation of PRL (70.0%) was observed in presence of ZnO nanoparticles (0.5 g L-1) and produced biosurfactant in MSM (Figure 2a). This implementation proved that ZnO nanoparticles were capable of assisting the microbial activities which agree with other studies (Shin and Cha, 2008). Hesham et al. (2006) reported the degradation of PRL (6.03 mg Kg-1) by a mixture of five yeast strains which was found to be 70% after a period of 42 days. The soil fungi viz. Aspergillus sp. and Achremonium sp. were also reported to be capable of degrading PRL 37.0% and 20.5% respectively after a period of 30 days at the concentration 0.05 (w v-1) (Silva et al., 2009). Therefore, yeast consortium YC02 used in the present study was found to be more efficient in degrading PRL at much higher concentration (50 mg L-1) within a short period (6 days) compared to the earlier reports. According to the Figure 2a, it can be concluded that the presence of ZnO nanoparticles could enhance the ability of yeast consortium YC02 in terms of improving the biosurfactant properties which followed by enhancing the biodegradation process. So far, extremely limited studies have been reported on combined effect of nanoparticles and biosurfactant towards degradation of environmental pollutants (Zhang et al., 2011).
FTIR spectra of control perylene (Figure 2b) showed the characteristic absorption peaks at 3047 cm-1 (=CH stretch in aromatic rings), 2850.79-2953.02 cm-1 (C-H stretching in cyclic ring), 1741.72-1921.1 cm-1 (weak overtone and combination bands in aromatic compounds), 1490.97-1587.42 (variable aromatic ring stretching), 759.75-885.33 cm-1 (strong out of plane CH deformations in aromatic compounds) and 462.92-540.07 cm-1 (ring deformations in aromatic compounds). The second spectra illustrated, degraded perylene products by yeast consortium, YC02 showed a presence of 2970.38-2796.78 cm-1 representing H-bonded OH stretch in carboxylic acid. Sharp absorption peak of 1739.79 cm-1 (overtone and combination bonds in aromatic compounds), 1637.56 cm-1 (C=O stretching, enol form in β-kentone esters ), 1537.27 cm-1 (aromatic ring stretching), 1427.32 cm-1 (in plane OH bonding in carboxylic acids), 1369.46 cm-1 (medium CH3 deformations in isopropyl groups), 1215.15 cm-1 (C-O-C antisym stretch in vinyl ethers), 1004.91 cm-1 (ring breathing mode of carbon ring in cyclic compounds), 954.76 cm-1 (=CH out of plane deformation in vinyl compounds), 615.29 cm-1 (C-OH out of plane deformations in alcohols) and 572.86-534.28 cm-1 (ring deformations in aromatic compounds). These results suggest that the parental compound has undergone significant changes after degradation.
A statistical tool, three level 5 variables Box-Behnken Design was implemented to enhance the biodegradation of PRL using yeast consortium (YC02). The factors were optimized by BBD with six central points, the response PLR biodegradation (%) was studied and the second-order polynomial equation was given below:
Y= 74.2200 + 0.8750A – 0.25B – 0.125C + 0.3125D – 0.5625 E + 3.25AB + 3.00AC -1.50 AD + 3.25AE – 0.25BC + 1.25 BD + 0.75 BE + 0.25 CD – 2.5 CE + 1.25 DE – 5.19 A2 – 5.52 B2 -7.35 C2 – 4.44D2 – 3.44 E2
Where, Y was representing PRL biodegradation (%) as response and A, B, C, D and E were coded terms for the five test variables viz. pH, temperature, shaking speed, inoculum dosages and ZnO nanoparticle concentration respectively. The lack of fit analysis was found to be not significant which considered that the model is fit.
Figure 2 (a) GC-MS analysis of PRL degradation under different sets of condition after 6 days. (b) FT-IR spectrum of PRL before degradation and after degradation in presence of ZnO nanoparticle and biosurfactant.
The total determination coefficient R-Squared value was found to be 0.9790, indicating a realistic fit of the model to the experimental data (Table 2). This also indicates that 97% variation of response can be elucidated effectively and approves that 3% of the variations occur while performing the experiments. The ratio of 28.59 indicated an adequate signal, thus this model can be used to navigate the design space (Table 2). In this case, A, E, AB, AC, AD, AE, BD, CE, DE A2, B2, C2, D2 and E2 were found to be significant model terms (p< 0.05) as tabulated in Table 2. When considered in linear terms, variables namely pH and ZnO nanoparticle concentration had shown the highest influence on PRL biodegradation, as compared to temperature, shaking speed and inoculum dosages. When considered with respect to squared terms, all the variables had positive significance on PRL biodegradation as tabulated in Table 2. The interactive effect of variables, AB, AC, AD, AE, BD, CE and DE were found to be the most significant in response as shown in Figure 3. In Figure 3a, the response plot AB (pH vs temperature) indicated a significant reduction from acidic to alkaline pH and from lower (10°C) to higher temperature on PRL biodegradation. The maximum PRL biodegradation was observed at pH 7 and at temperature of 30°C. The response plots of AD (pH vs inoculum dosages) and BD (temperature vs inoculum dosages) were found to be significant and maximum degradation was observed at 3% inoculum as shown in Figure 3c, e. The response plots of AC (pH vs shaking speed) was found to be significant and maximum degradation was observed at 120 rpm as shown in Figure 3b. The response plot of AE (pH vs ZnO Nps), CE (shaking speed vs ZnO Nps) and DE (inoculum dosage vs ZnO Nps) were found to be significant and maximum PRL biodegradation was observed at 2.0 gL-1 ZnO nanoparticle as shown in Figure 3d, f, g. The supplementation of ZnO nanoparticle in the production medium had showed significant impact on PRL biodegradation (%) using the yeast consortium. However, a significant reduction in the PRL degradation was observed at higher concentration of ZnO nanoparticles (3 g L-1). This could be because of the addition of high amount of ZnO Nps which become toxic to yeast cells. Similar trend by El-Sheshtawy and Ahmed (2017) was reported where crude oil was degraded using Bacillus licheniformis in the presence of different concentration nanoparticles and produced biosurfactant.
A statistical model was validated by executing point prediction tool of BBD from an optimum value of all the 5 variables A, B, C, D and E. The actual PRL biodegradation (74.00 ± 0.01%) was in close agreement with the predicted value (74.00 ± 0.8%) indicating the validity of the model (Table 3). Maximum biodegradation of PRL was found to be 74.0 ± 0.01 (%) at central values of all the factors viz., pH (7.0), temperature (30ºC), shaking speed (130 rpm), ZnO nanoparticle (2 g L-1) and inoculum dosages (3%). The normal plot for residuals and predicted vs actual plots were represented (Figure 3h-i) respectively. Thus, the biodegradation of PRL by YC02 was found to be increased from 70.0% to 74.0% in aqueous medium under optimized condition using BBD.
The kinetic study was performed applying the predicted optimum conditions. The kinetic data on degradation of PRL (50 mg L-1) was best fitted with the first order kinetic model in case of all set of conditions. The highest regression coefficient (R2) values of (0.9938) of PRL degradation was noted in case of biosurfactant producing YC02 in presence of ZnO nanoparticles as shown in Table 4. The calculated degradation rate constant (K) of PRL is 0.206 d-1 and the theoretical half-life of PRL is 3.364 days implied that the removal of PRL by yeast consortium was time dependent process and degradation rate was directly proportional to substrate concentration (Jin et al., 2017).
To conclude, anionic biosurfactant was produced by yeast consortium YC02 which could enhance the degradation of perylene (PRL) in the growth medium. However, the best improvement on PRL degradation evaluated to 70.0% which was recorded in the presence of biosurfactant and ZnO nanoparticles both. In addition, statistical optimization of growth parameters remarkably enhanced the PRL degradation (74 %) by YC02 in presence of ZnO nanoparticles and produced biosurfactant which establishes the novelty of our work. Results suggested the potential applicability of the yeast consortium YC02 for the bioremediation of PRL contaminated sites using biosurfactant and specific concentration of ZnO nanoparticles. Kinetic studies suggested that degradation of PRL by yeast consortium was time dependent process and degradation rate was directly proportional to substrate concentration. According to the best of our knowledge, this is the first report on nanobioremediation of PRL, a high molecular weight PAH using yeast consortium. Further work on application of YC02 in nanoremediation of PRL from the real-world contaminated environment is underway in order to ascertain its relevance in pollution control.
Table 2. ANOVA for Response Surface Quadratic Model (Response: PRL biodegradation %)
|Source||Sum of Squares||df||Mean Square||F-value||p-value|
|Lack of Fit||16.33||20||0.8167||2.04||0.2201||not significant|
Figure 3 3-D interactions between the different variables for response (PRL biodegradation %) where, (a) pH vs. Temperature (AB) (b) pH vs. Shaking speed (AC) (c) pH vs. Dosage (AD) (d) pH vs. ZnO nanoparticles (AE) (e) Temperature vs. Dosage (BD) (f) Shaking speed vs. ZnO nanoparticles (CE) (g) Dosage vs. ZnO nanoparticles (DE) (h) Normal plot of residuals (i) Predicted vs. actual plot.
Table 3 Actual versus predicted value for Response: PRL biodegradation (%)
C:Shaking speed (rpm)
D: Dosage (%)
E:ZnO nanoparticle (g L-1)
|Response: PRL biodegradation (%)|
|Actual value||Predicted values|
Table 4. Kinetic parameters for the degradation of perylene (PRL) using yeast consortium YC02
Set of conditions Kinetics equation of degradation Rate constants of degradation
________________________ R2 K(d-1) T1/2 (days)
PRL + YC02 Ct = -5.520t + 48.91 0.9935 5.520 4.529
PRL + YC02 + biosurfactant Ct = -5.565t + 48.77 0.9918 5.565 4.492
PRL + YC02 + Ct = -5.885t + 48.43 0.9735 5.885 4.428
ZnO nanoparticles (0.5 g L-1)
PRL + YC02 + biosurfactant Ct = -5.960t + 48.38 0.9741 5.960 4.195
+ ZnO nanoparticle (0.5 g L-1)
PRL + YC02 lnCt = -0.1822t + 3.9433 0.9891 0.1822 3.804
PRL + YC02 + biosurfactant lnCt = -0.1847t + 3.9401 0.9920 0.1847 3.752
PRL + YC02 + lnCt = -0.2008t + 3.9303 0.9929 0.2008 3.451
ZnO nanoparticles (0.5 g L-1)
PRL + YC02 + biosurfactant lnCt = -0.2060t + 3.9321 0.9938 0.2060 3.364
+ ZnO nanoparticle (0.5 g L-1)
PRL + YC02 1/Ct = 0.0066t + 0.0166 0.9316 0.0066 3.030
PRL + YC02 + biosurfactant 1/Ct = 0.0067t + 0.0167 0.9379 0.0067 2.985
PRL + YC02 + 1/Ct = 0.0076t + 0.0168 0.9720 0.0076 2.632
ZnO nanoparticles (0.5 g L-1)
PRL + YC02 + biosurfactant 1/Ct = 0.0079t + 0.0166 0.9697 0.0079 2.532 + ZnO nanoparticles (0.5 g L-1)
ACKNOWLEDGEMENT: The authors are grateful to VIT, Vellore for providing necessary laboratory facilities.
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Microorganisms populate in almost every possible region on the earth from those providing favorable circumstances for their survival and reproduction to those offering the severe or extreme conditions for their growth. The microorganisms that can survive in such extreme conditions are widely known as extremophiles. Among the extremophiles, thermophiles have received a huge attention in recent years because of their capability to survive at a very high temperature; even, they are active at an elevated temperature (Beg et al., 2000; Bharadwaj et al., 2010; Akmar et al., 2011; Huang et al., 2011).
Thermophilic microbes are found in various biotopes such as thermal springs, geothermal sediments and marine solfataras (Rothschild and Manicineli, 2001; Pathak and Rathod, 2014). Thermal springs are having very high water temperature or in other words we can say that the temperature is much more than the surroundings (Sen et al., 2010).
Thermal springs are the proofs of geological activity that indicate very high temperature and widely available in the Himalayan region (Kumar et al., 2004). Thermal springs are the affluent source of thermophilic microorganisms which can be tapped for various applications in different fields. Thermophilic microbes are containing enzymes that are stable at a very high temperature, which make them useful for the pharmaceutical, biotechnological, food processing and chemical industries (Tekere et al., 2015). Physico-chemical properties are also playing an important role in the density and diversity of microbes in the thermal springs.
Though a lot of work has been done on various aspects of thermal springs that includes the works of Kumar et al., (2004) on soil microbial diversity from two different thermal springs of Uttarakhand, Sharma et al., (2008) on characterization and identification of various strains of Geobacillus spp. enumerated from Saldhar thermal spring; Akmar et al., (2011) on isolation of new thermophilic bacteria from hot spring; Bhusare and Wakte, (2011) on hot water spring of Unkeshwar; Ghati et al., (2013) on Esterolytic thermophilic bacteria from an Indian hot spring; Pathak and Rathod, (2014) on culturable diversity of bacteria in the thermal spring of Unkeshwar, India; Rawat, (2015) on bacterial diversity of a sulphur spring in Uttarakhand and Tekere et al., (2015) on bacterial diversity in some African thermal springs. But no work has been done so far on the microbial diversity and physico-chemical attributes in the water of Ringigad and Saldhar thermal springs. Therefore, a maiden attempt has been made to provide basic data on microbial diversity and physico-chemical characteristics of Ringigad and Saldhar hot water spring for further studies.
MATERIALS AND METHODS
Ringigad is a thermal spring located at 17 Km from Joshimath towards Suraithota (Figure 1) between latitude 30033’14” N and longitude 79040’0.06” E at an altitude of 1,850 m above mean sea level in the Chamoli district of Uttarakhand. The approximate area covered by this hot water spring is 45 m2. The maximum in-situ temperature of Ringigad hot water spring is 890C.
However, Saldhar is also a thermal spring located at 19 Km from Joshimath towards Suraithota (Figure 2) just near to the road on the right side. It is situated between latitude 39029’25” N and 79039’29” E at an altitude of 1,900 m above mean sea level in the Chamoli district of Uttarakhand. The approximate area that is covered by this hot water spring was 70 m2. The maximum in-situ temperature of Saldhar is 920C.
Figure 1 Study area (Ringigad, a thermal spring near Joshimath)
Figure 2 Study area (Saldhar, a thermal spring near Joshimath)
Water of both the thermal springs was sampled during the year 2014 and 2015 in two sampling operations each year. In both the years, the water sample was collected in the month of June and August to observe any possibility of change among the physico-chemical parameters during the dry and rainy months. The main objective of current study is to explore the microbial diversity in two hot water springs. Water samples were collected from the origin place of both the hot water springs in autoclaved thermoflask. However, water samples collected for the isolation and identification of
culturable microbial diversity was placed in an ice box filled with freezed ice packs and analyzed within 24 hours. Few of the water quality parameters like pH, water temperature, dissolved oxygen and free carbon dioxide were measured at the sampling site whereas, for the remaining physico-chemical characteristics, the water samples were transferred to the Laboratory of Environmental Microbiology and Biotechnology, Department of Environmental Sciences, H.N.B. Garhwal University, Srinagar-Garhwal, Uttarakhand, India at its earliest possible. All the physico-chemical characteristics were analyzed by following the standard protocols available in APHA, (2005) and microbial diversity by following the standard methods outlined in Harley and Prescott, (2002); Morello et al., (2003).
Water samples were analyzed for a predefined set of physical and chemical characteristics. Water temperature was measured by dipping the digital thermometer 10 cm in the hot water of the spring carefully and noted down the readings. The temperature range of digital thermometer was (-50 0C to +300 0C)
pH was measured both at the site by using portable pH meter of Electronics India (Model No. 7011) and in the laboratory by using the Toshcon bench top multiparameter analyzer (Model No. TPC-17). Dissolved oxygen was measured by using the modified Winkler method at the sampling site. Conductivity, salinity and total dissolved solids (TDS) were measured by using the Toshcon
Bench Top Multiparameter analyzer (Model No. TPC-17). Free CO2, total alkalinity, chlorides, total hardness, Calcium and Magnesium were measured by following the protocols outlined in APHA, (2005). Nitrates, Sulphates and Phosphates were measured by Spectrophotometric method by using Systronic UV-VIS Spectrophotometer (model No. 117).
Microbal isolation and enumeration
Nutrient Agar media (HiMEDIA) was used for the estimation of colony forming units (CFUs) of bacteria. Media pH for microbial isolation was adjusted according to the pH of sampling sites. SDA media was used for isolation of fungal species. It was supplemented with 50 mg/l of each (Streptomycin and Ampicillin) to prevent the bacterial contamination. AIA media was used for the isolation of actinomycetes. After the isolation of microbial colonies, each unique colony was streaked on separate media plate to get the pure culture of each microbial colony (Clesceri et al., 1998).
To study the morphological characteristics, the purified selected microbial isolates were observed by naked eyes and under the Phase Contrast Microscope (Nikon Eclipse TS100). Morphology of selected bacterial isolates that was observed in the laboratory is given in Table 1.
Table 1 Morphological and Biochemical characterization of bacterial isolates identified from hot springs of the Garhwal Himalaya
|Bacillus cibi||Bacillus subtilis|
|Size||2 mm||2-5 mm||2-3 mm||1 mm||3-4 mm||2-3 mm|
|Color||Yellowish grey||Grayish yellow||Cream||Translucent||Orange yellow||White|
|Methyl Red (MR)||–||–||–||–||–||–|
|Voges Proskauer (VP)||–||+||–||–||–||+|
Abbreviations: +: positive; -: negative; v: variable
Moreover, detailed biochemical characterizations were carried out to identify the bacterial and archeal isolates from the hot water spring, up to possible genus or species level. The result of biochemical tests for selected bacterial isolates recorded in the laboratory is given in Table 1. Identification of all the fungal isolates were made by microscopic analysis by using the taxonomic keys and standard procedures. To confirm the identification of the microbes done in the laboratory, the pure culture of each isolate was collected and then sent for further identification and confirmation to National Centre for Microbial Resources
(NCMR), National Centre for Cell Sciences (NCCS), Pune by using the MALDI-TOF MS.
Statistical treatment (minimum; maximum; mean; standard deviation) of the physico-chemical parameters of water was conducted.
RESULTS AND DISCUSSION
Data of all the fifteen physico-chemical characteristics obtained under two sampling operations each year during a period of two years (2014-2015) from the Ringigad and Saldhar hot water springs of the Garhwal Himalaya. The data for physico-chemical parameters of Ringigad and Saldhar hot water springs has been presented in Table 2 & 3.
The minimum water temperature of Ringigad thermal spring was recorded 820C as minimum and 890C as maximum at the site. The pH of water of Ringigad thermal spring varied from 6.7 to 6.9, indicated the slightly acidic nature of water. Similar range of pH was also recorded by Kumar et al. (2013) for hot springs of Kullu district in Himachal Pradesh; Singh et al. (2015) for hot springs of Jharkhand and West Bengal region; Ghilamicael et al. (2017) for hot springs in Eritrea.
The value of dissolved oxygen (DO) varied from 0.8 mg/l to 1.2 mg/l. Dissolved oxygen concentration is inversely proportional to the temperature. As the temperature of water goes up the concentration of dissolved oxygen in the water goes down (Rana et al. 2018). Fazlzadeh et al. (2017) recorded the DO concentration within a range of 3.25 mg/l to 3.57 mg/l for thermal springs in Iran. The electrical conductivity (EC) was ranged from 4.48 mS/cm to 4.68 mS/cm in the water of hot spring. Haki and Gezmu (2012) recorded the similar range of electrical conductivity for the hyperthermal springs of Ethiopia. Salinity of the samples ranged from 2.1SAL to 2.3SAL throughout the sampling period. Similar range of salinity was also recorded by Hamzah et al. (2013) for thermal springs of Malaysia.
The concentration of total dissolved solids (TDS) was ranged between 2.47 mg/l to 2.53 mg/l in the water of Ringigad thermal spring. High range of total dissolved solids was recorded by Hamzah et al. (2013) for thermal springs of Malaysia. The concentration of free CO2 was ranged between 35.2 mg/l and 48.4 mg/l during the study period.
The concentration of total hardness varied from 228 mg/l to 234 mg/l. High range of alkalinity (196 mg/l) was recorded by Kumar et al. (2013) for thermal springs of Kullu district in Himachal Pradesh. The concentration of Calcium varied from 63.2 mg/l to 69.6 mg/l. Magnesium concentration varied from 14.20 mg/l to 17.13 mg/l. A similar range (12.60 mg/l to 15.62 mg/l) of magnesium was recorded by Singh et al. (2015) for thermal springs of Jharkhand and West Bengal. The concentration of chlorides varied from 11.36 mg/l to 14.2 mg/l concentration observed in the sample. Extreme high range of alkalinity (197.38 mg/l) was recorded by Kumar et al. (2013) for thermal springs of Kullu district in Himachal Pradesh.
Alkalinity of the water ranged from 270.0 mg/l to 300 mg/l during the study period. However, a very high range (165.2 mg/l) of alkalinity was recorded by Kumar et al. (2013) for thermal springs of Kullu district in Himachal Pradesh. The concentration of nitrates present in the water sample was ranged from 0.239mg/l to 0.256 mg/l. Sulphates concentration were also found within a range of 0.276 mg/l to 0.289 mg/l. The concentration of phosphates in the water
sample was ranged from 0.026 mg/l to 0.028 mg/l. A very high range of sulphates and nitrates were recorded by Sherpa et al. (2013) for thermal springs of Sikkim in India.
The minimum water temperature of Saldhar thermal spring was recorded 870C as minimum and 920C as maximum at the site. The pH of water of Saldhar thermal spring varied from 9.1 to 9.3, indicating that the water is alkaline in nature. This high pH may be due to the presence of cyanobacteria present at the site in the form of algal mat. The cyanobacteria use the carbonates and bicarbonates that increase the pH of the water at a high level. Similar range of pH (6.9-9.5) was recorded by Singh et al. (2015) for the water samples of thermal springs in Jharkhand and West Bengal of India; Guzman et al. (2004) also recorded the similar range of pH (7.76 to 9.98) for thermal springs in the pacific coast of Guerrero, Mexico.
The values of dissolved oxygen (DO) varied from 0.4 mg/l to 0.6 mg/l. Fazlzadeh et al. (2017) recorded the DO concentration within a range of 3.25 mg/l to 3.57 mg/l for thermal springs in Iran and Kumar et al. (2013) recorded 2.52 mg/l of dissolved oxygen for hot springs of Kullu district in Himachal Pradesh. The electrical conductivity (EC) was ranged from 6.04 mS/cm to 6.08 mS/cm in the water of hot spring. Homma and Tsukahara (2008) recorded the similar range of conductivity for the Northernmost area of the Itoigawa Shizuoka Tectonic Line. Salinity of the samples ranged from 3.0SAL to 3.28SAL throughout the sampling period.
The concentration of total dissolved solids (TDS) was ranged between 3.10 mg/l to 3.34 mg/l in the water of Saldhar thermal spring. The concentration of free CO2 was recorded between 30.8 mg/l and 35.2 mg/l during the study period. High range of total dissolved solids was recorded by Hamzah et al. (2013) for thermal springs of Malaysia. The concentration of free CO2 was ranged between 35.2 mg/l and 48.4 mg/l during the study period.
The concentration of total hardness varied from 228 mg/l to 234 mg/l. High range of alkalinity (196 mg/l) was recorded by Kumar et al. (2013) for thermal springs of Kullu district in Himachal Pradesh. The concentration of Calcium varied from 63.2 mg/l to 69.6 mg/l. Magnesium concentration varied from 14.20 mg/l to 17.13 mg/l. A similar range (12.60 mg/l to 15.62 mg/l) of magnesium was recorded by Singh et al. (2015) for thermal springs of Jharkhand and West Bengal.
The concentration of total hardness varied from 248 mg/l to 256 mg/l. The concentration of Calcium varied from 52.20 mg/l to 56.11 mg/l. Magnesium concentration varied from 28.28 mg/l to 28.83 mg/l. The concentration of chlorides varied from 9.94 mg/l to 14.20 mg/l observed in the water sample.
Alkalinity of the water ranged from 290 mg/l to 320 mg/l during the study period. The concentration of nitrates present in the water sample was ranged from 0.089 mg/l to 0.096 mg/l. Sulphates concentration were also found within a range of 0.287 mg/l to 0.311 mg/l. The concentration of phosphates in the water sample was ranged from 0.021 mg/l to 0.24 mg/l.
Table 2 Physico-chemical parameters for water of Ringigad thermal spring in the Garhwal Himalaya
Mean ± SD
|Water Temperature (0C)||82.0||89.0||83.0||87.0||82.0||89.0||85.25±3.30|
|Dissolved oxygen (mg/l)||1.2||0.8||1.2||0.8||0.8||1.2||1.0±0.03|
|Free CO2 (mg/l)||35.2||44.0||35.2||48.4||35.2||48.4||40.7±6.6|
|Total Alkalinity (mg/l)||270.0||300.0||275.0||290.0||270.0||300.0||283.75±13.77|
|Total Hardness (mg/l)||228.0||232.0||228.0||234.0||228.0||234.0||230.5±3.0|
Table 3 Physico-chemical parameters for water of Saldhar thermal spring in the Garhwal Himalaya
Mean ± SD
|Water Temperature (0C)||89.0||92.0||87.0||90.0||87.0||92.0||89.5±2.08|
|Dissolved oxygen (mg/l)||0.6||0.4||0.6||0.4||0.4||0.6||0.5±0.12|
|Free CO2 (mg/l)||30.8||35.2||30.8||30.8||30.8||35.2||31.9±2.20|
|Total Alkalinity (mg/l)||295.0||320.0||290.0||310.0||290.0||320.0||303.75±13.77|
|Total Hardness (mg/l)||248.0||256.0||252.0||256.0||248.0||256.0||253.0±3.83|
In Ringigad, an overall, seven species of bacteria (Brevibacillus borstelensis, Aeromonas veronii, Paenibacillus dendritiformis, Bacillus cerus, Bacillus cibi, Streptococcus pyogenes and Strenotrophomonas maltophila) and four species of actinomycetes (Streptomyces albus, Streptomyces canescens, Thermoactinomyces candidus and Thermoactinomyces thalopophilum) were recorded. However, three species of fungi (Sclerotium rolfsii, Fusarium oxysporum and Sclerotinia sclerotiorum) were also recorded. The α-diversity of microbes in the Ringigad thermal spring was found to be fourteen during the study period (Table 4). In Saldhar, an overall, six species of bacteria (Bacillus cerus, Streptococcus pyogenes, Bacillus subtilis, Brevibacillus parabrevis, Brevibacillus reuszeri and Geobacillus stearothermophilus) and three species of actinomycetes (Streptomyces albus, Thermoactinomyces candidus and Thermoactinomyces thalopophilum) were recorded. However, three species of fungi (Aspergillus tubingensis, Trichoderma harzianum and
Sclerotinia sclerotiorum) were also recorded. The α-diversity of microbes in Saldhar spring was found to be twelve during the study period (Table 4). The microbes that were found in both the thermal springs were typical to other thermal springs. All the microbial species are hyperthermophiles and can be identified only in the thermal springs having such an extreme temperature.
Keeping in view, it has been concluded that both the thermal springs (Ringigad and Saldhar) are having a very high temperature. The concentration of dissolved oxygen is very low in which only the thermophilic microbes can survive. The pH of both the springs revealed the alkaline nature of the water of the hot springs. Apart of the high temperature, the α-diversity of microbes in Ringigad is 14 and in Saldhar it is 12.
Table 4 Microbial diversity of Ringigad and Saldhar hot water springs of Garhwal Himalaya (Abbreviations: +: present; -: absent)
Acknowledgement: One of the authors (Rahul Kumar) is thankfully acknowledge for the fellowship given by the University Grant Commission, New Delhi through Hemvati Nandan Bahuguna Garhwal University (A Central University), Srinagar-Garhwal, Uttarakhand, India for undertaking the present work.
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Sweetpotato is a highly nutritious vegetable and its consumption has been increased in various parts of the world in recent years (Sato, 2016). A USDA survey reported that sweetpotato consumption in the U.S. increased from 1.9 kg to 3.4 kg per capita annually between 2000 and 2014 (Johnson et al., 2015). There are several cultivars of sweetpotatoes that vary in their flesh colour, sugar composition and percentage dry matter (La Bonte et al., 2000). Although, traditionally, the fresh produce market prefers orange-fleshed roots (Coolong et al., 2012) and in the US, orange-fleshed cultivars generally occupy over 90% of sweetpotato production area (Carpena, 2009). According to the North Carolina Sweetpotato Commission (2015), the more consumer-recognized orange-flesh sweetpotato cultivars are Beauregard, Hernandez, Jewel, Carolina Ruby, Porto Rico, Cordner and Covington. Sweetpotatoes are known to be a good source of energy, protein, fibre, and minerals including potassium, vitamin A, carotenoids and phenolic compounds (Sajeev et al., 2012; Ellong et al., 2014; Laurie et al., 2012; Button, 2015). They are rich in starch, which represents more than 50% of the carbohydrate components (Ellong et al., 2014). Sweetpotatoes are majorly consumed cooked, baked or fried. Ovens and pressure cookers are currently present in a lot of homes. Sometimes, sweetpotatoes may be pureed or candied to improve shelf life (Padmaja, 2012).
Sweetpotato cultivars react differently when cooked (either a soft or firm texture or colour changes after cooking). Degras (1998) reported that changes may occur in the nutrient and chemical composition of sweetpotatoes while cooking. These changes can alter the starch, dextrins, sugar, carotene and anthocyanin contents (Magness et al., 1971; Messiaen, 1975; Duke, 1983; Susheelamma, 1992). Reddy and Sistrunk (1990) discovered that baking or microwaved cooked sweet potatoes contained high reducing sugars, total sugars and pectins than steamed ones. Martin (1986) reported that the percentage of starch that is converted to maltose in moist sweetpotato cultivars was 63-69% and about 54% for dry cultivars. Starch digestion has been said to increase with cooking and cooked sweetpotato starch was more prone to enzymatic breakdown compared to uncooked starch. Bradbury et al. (1985) observed a significant rise in the amount of dietary fibre in boiled and steamed sweetpotatoes possibly due to conversion of part of the starch to resistant starch. In addition, it has been shown that by diluting anthocyanins in cooking water, it could cause a fade colour (Ellong et al., 2014). The white to orange flesh stains more and the cream flesh may change to yellow or greenish or even grey (Ellong et al., 2014). This change was reportedly caused by the carotenoids degradation (Ellong et al., 2014). Furthermore, enzymatic browning can occur through polyphenol oxidase (PPO) reactions. PPO catalyzes the process of oxidation of mono, di, and poly phenols to o-quinones (Lourenco et al., 1992). It is highly likely that the method of cooking the sweetpotato could alter the dry matter content (Leighton et al., 2010). It has been previously shown that water loss due to evaporation during steaming process can increase the dry matter content of cooked samples (Truong et al., 1997).
Sweetpotato offers great possibility for usage in the food industry for the production of commercial products owing to the fact that sweetpotato is highly rich in starch content (Woolfe, 1992). It becomes imperative to have comprehensive understanding of the functional properties of the different sweetpotato cultivars in order to identify the most appropriate use for food processing (Agnes et al., 2012). Texture (dry matter content) is one of the most crucial parameters directly linked to product quality (Bhattiprolu, 2004). Texture analysis is a measure of food properties relating to how food sample feels in the mouth (Bhattiprolu, 2004). Textural quality can be assessed by use of instruments or by analysis of important constituents (Bach, 2012). According to Truong et al. (1997), parameters provided by an instrument can be good predictors of cooked sweetpotato texture. These parameters include certain characteristics such as mechanical (e.g. mealiness), geometrical (e.g. graininess), compositional (e,g. wateriness) (Szczesniak, 1963), adhesiveness (work required to overcome the force of attraction holding food samples) and chewiness (length of time required to chew a sample) (Bhattiprolu, 2004). Fluctuating levels of firmness that emerge from various cooking treatment could be the motivation to measure the differences among varying cooking methods, for example, 29% diminishing in hardness for baked samples, 44% for pressure cooked and 96% for open cooked specimens when compared with raw samples (Bernad, 2013). Generally, sweetpotato is mostly cooked at home; however, since home preparation is usually lengthy (80-90 min at 204oC for baked sweetpotato), many interested consumers may not use the product due to the length of time required for cooking (Truong and Walter, 1994). Sometimes, it is more reliable to adopt instrumental methods for assessing food texture rather than sensory methods. This is because they can be carried out under more controlled conditions. It also offers advantage of saving time and reducing costs, as well as providing more consistent results that are not subjective (Bhattiprolu, 2004).
The major marketable form of sweetpotato is fresh root (Truong and Walter, 1994). The quality of fresh market sweetpotato can vary due to differences in cultivar, conditions of cultivation, and post-harvest handling (Walter, 1987). Several studies have examined the reasons for textural distinction among cultivars and sweetpotato products and to decipher the effect on buyer preference and acceptance (Truong et al., 1997; Tomlins et al., 2004). Sensory characteristics of boiled or baked sweetpotato and preferences of consumers on different types of sweetpotato cultivars have been investigated (Laurie et al., 2013; Leksrisompong, 2012). Nevertheless, to the best of our knowledge, no study has been done to evaluate instrumentally the effect of different cooking methods on the textural properties of sweetpotato cultivars produced in an organic management system. The main goal of this study was to determine textural differences among six cultivars (Hernandez, Japanese Purple, Murasaki, Orleans, Old Yellow and O’Henry) prepared using open cooking, baking and pressure cooking methods. At the end of this study we will come to know the cultivars with the most desirable texture characteristics that can be grown locally which will be valuable for agricultural producers.
MATERIALS AND METHODS
Cultivar field production and harvest
Six sweetpotato cultivars of various flesh and texture attributes were gathered toward the conclusion of the 2016 cultivation season from the Tennessee State University, Nashville TN certified organic farm. Production practices applied were done following the regulations of the National Organic Program. The cultivars include Orleans, Old Yellow, Murasaki, O’Henry and Japanese Purple which have been grown in limited amounts for fresh root markets and processing industry and Hernandez, a moist type sweetpotato and major commercial cultivar well liked in the southern part of the U.S. Sweetpotato slips were purchased in June from Jones Family Farms, Bailey, N.C., Slade Farms, Surrey, V.A., Barefoot farms, TN, USA and planted immediately. It took four months for the slips to mature to vines and the sweetpotato was harvested. After harvest, root curing was done at 13-16 °C and 80-90% humid conditions for 5-7 days and set aside for eight weeks before conducting the experiment. Sweetpotato roots were graded according to USDA grading standards. As sweetpotatoes differ in size, only samples that had similar magnitude and shape were chosen for the examination. For each cultivar, three sweetpotato roots were selected randomly, sorted, washed, peeled, and diced into cubes. Roots of average diameter measurement of 1.96 inches, length of 5.06 inches and weight of 5.12 oz. were selected for experimental use.
Sweetpotato cooking methods and experimental design
The open/non-conventional cooking technique, pressure cooking and baking were the three cooking techniques applied in this experiment. Distilled water was utilized as a part of cooking to keep ions from affecting the firm structure of the sweetpotato cultivars.
Open cooking was performed specifically on high heat with a 2-L stainless steel pot containing 20 oz. to 38 oz. of bubbling water and they were cooked without peeling their skins (Leighton et al., 2010). No top cover was utilized for pots in the open cooking technique. A fixed time of 20 minutes cooking was employed.
Pressure cooking was done in a 2-L stainless steel pot, with 20-38 ounces of water and they were cooked without peeling their skins (Leighton et al., 2010). Cooking pots were secured with the customary top for pressure cooking. In pressure cooking, the temperature used was 100 °C and with similar specific time of 20 minutes.
Baked samples were heated in aluminum container at 204 °C for 90 min within an oven. Cooking duration was chosen with the assistance of sensory tests (Leksrisompong et al., 2012).
Instrumental texture profile analysis
After cooking, all sweetpotato were left to cool at room temperature (30±2°C), then peeled, diced into one-inch cube square samples and put away in independently sealed and labeled polythene bags to prevent loss of moisture before completing instrumental examination. Texture Profile Analysis (TPA) was done utilizing a texture analyzer TA HD Plus (Texture Technologies) with a level plate of 40 mm in breadth. The samples were packed to 75% of their unique stature by two continuous compressions. The crosshead speed was set at 1.66 mm/sec. Configured height was at 50 mm. Pre-test speed was set at 1.00 mm/sec while post-test speed was 5.00 mm/sec. Testing compression was done as follows. The plate approaches the specimen (one each squared sweetpotato cube) from the calibrated height (50 mm) with the pre-test speed; packed it to half of the original height with test speed; plate goes back to the original position using post-test speed. Once the test is finished, the pulverized example was expelled, and the stage surface was cleaned to evacuate the extracted dampness or water. At that point, the next specimen was set underneath the plate. Three samples for each treatment were tested. Care was taken to guarantee the removal of the specimen from the plate when the plate finished the second compression cycle and came back to its original position. The sample was compressed twice in order to mimic the mastication process. Six test parameters resulted from the analysis of a force versus time curve (Figure 1) was obtained during the compression test.
Figure 1 Typical texture (TPA) profile curve showing measurement of texture parameters
As described by Bourne (1978), we assessed the hardness, chewiness, springiness, cohesiveness, gumminess, and resilience. These terms were defined as follows (Szczesniak, 1975): Hardness: force required to cause a deformation, Chewiness: time required to chew a food sample to a state suitable for swallowing, Springiness: the rate at which a deformed material goes back to its intact state after deformation, Cohesiveness: extent to which a sample can be deformed before rupturing, Adhesiveness: work necessary to overcome the force of attraction between the food surface and other materials in contact with the food, Gumminess: energy required to breakdown a semi-solid food to a suitable state for swallowing, Resilience: a product of a low degree of hardness and a high degree of cohesiveness.
Data collection and calculation were accomplished using exponent software of the texture analyzer. Instrumental texture parameters from the force versus time curves were recorded. Three sweetpotatoes per cultivar were analyzed in each treatment. Data from the texture profile analysis were combined for analysis of variance (ANOVA) using PROC GLM in SAS (Ver. 9.4, SAS, Inc., Cary, N.C.) to determine significant influences of primary parameters – cultivar and cooking methods on the secondary parameters (hardness, springiness, cohesiveness, gumminess, chewiness, and resilience). If interactions of cultivar and cooking methods were significant, they were used to explain the results. When the main effect was significant, Fisher’s least significant difference (lsd) test was used for multiple comparisons between mean values of the variables (cultivar and cooking methods).
RESULTS AND DISCUSSION
The ANOVA results indicated that the texture profile parameters were significantly affected by the thermal treatments. Cultivar and cooking method affected the instrumental texture parameters of the sweetpotatoes (Table 1).
Table 1 ANOVA results (F values) showing effects of cultivar, cooking methods and their interactions on instrumental texture parameters.
|Sources||Degree of Freedom||F-Value||P-Value|
|Cultivar * Cooking method||10||295.39||<0.0001|
|Cooking method||2||31.19||< 0.0001|
|Cultivar * Cooking method||10||37.74||< 0.0001|
|Cooking method||2||28.18||< 0.0001|
|Cultivar * Cooking method||10||6.25||< 0.0001|
|Cultivar * Cooking method||10||106.52||< 0.0001|
|Cultivar * Cooking method||10||86.96||< 0.0001|
|Cultivar * Cooking method||10||16.38||< 0.0001|
Effect of Cultivars on textural characteristics of sweetpotato
Old Yellow differed from other cultivars, having the highest values for cohesion, gumminess and chewiness (Table 2). In the chewing process, the cell wall experiences twisting or breaking based on the characteristics of the cell wall (Waldron et al., 1997). As for springiness, Old Yellow also held the highest value and did not present significant difference with the Japanese Purple cultivar. Hernandez held the highest value for hardness although it did not differ significantly from Japanese Purple (Table 2). Dry matter content has been reported to be connected to some degree with the texture of potatoes, although this reality is not really clear (Van Marle et al., 1997). According to Truong et al. (2011), such sweetpotato cultivars with high dry matter content have firm and mealy texture after cooking while those with low dry matter content have soggy texture after cooking. As for resilience, Hernandez also held the highest value, however, it did not vary significantly from Japanese Purple and Old yellow cultivars (Table 2). O’Henry produced the lowest values for gumminess and hardness. O’ Henry also produced the lowest values for chewiness and resilience, however, it was not significantly different from Murasaki and Orleans. Walter et al. (1997) reported that the product processed from soft-sweet type sweetpotato was softer, moister, had fewer particles, more mass cohesion, was easier to swallow, and had an oily mouthfeel. Orleans produced the lowest parameters for springiness and cohesiveness, however, it did not vary significantly from Murasaki (and O’ Henry in the case of cohesiveness) (Table 2).
Table 2 Sweetpotato cultivars and their instrumental texture parameters.
|Hernandez||Japanese Purple||Murasaki||Orleans||Old Yellow||O’Henry|
|2.47 dc||2.43 d||2.62 bcd||2.66 bcd||263 bcd||2.61 bcd|
|231.50 a||221.76 ab||197.56 bc||197.71 bc||182.02 c||89.39 d|
|0.64 c||0.73 ab||0.55 d||0.53 d||0.75 a||0.69 bc|
|0.10 bc||0.10 b||0.08 bcd||0.07 d||0.13 a||0.08 cd|
|19.30 b||17.83 b||16.95 b||15.07 b||28.54 a||7.97 c|
|12.58 b||12.33 b||10.40 bc||9.28 bc||26.54 a||5.30 c|
|0.44 a||0.33 a||0.04 b||0.07 b||0.32 a||0.03 b|
* Mean values in a row with different letters differ significantly at P<0.05 by LSD. Ranking by high to low values among the cultivars.
Texture, or mouth-feel, is a major attribute in deciding overall consumer acceptance of sweetpotato cultivars. A mixture of sensory attributes of the sweetpotato root can impact consumer taste and overall acceptability. According to a sensory study by Nwosisi et al. (2017) using a semi-trained sensory panel, the least favored cultivars (Japanese Purple) had watery [due to a somewhat lesser dry matter/ moisture content of the white fleshed sweetpotato as reported by Leighton et al. (2010)], sweet, fibrous, vanilla, and dense textural traits. Among other things, the lower acceptance of hardness or dense textural traits can be confirmed from our TPA results. Japanese Purple and Hernandez with the least liked textural traits from their study also showed the greatest hardness and resilience. However, they did not differ significantly from each other and from some of the other cultivars (Put those cultivars in this bracket). Walter et al. (2002) discovered that sensory hardness and density were highly correlated with the value of instrumental measurements while cohesiveness, oiliness and moistness were negatively correlated with the value of instrumental measurements. In an experiment conducted on sweetpotato French fries, consumers preferred the caramel flavor and disliked starch flavor, and first-bite moistness and cohesiveness of mass in texture. On the other hand, there have been other reports that the most essential sensory descriptors affecting consumer acceptability were starch and stickiness as they were more favored by consumers compared to the least preferred types which were neither starchy nor sticky (Tomlins et al., 2004; Nwosisi et al., 2017). Following the use of instruments, a fully-trained sensory panel should thus be set up to confirm the results of our present study as the process of determining the acceptance of a food product is measured from different dimensions (Costell et al., 2010).
Effect of cooking methods on textural characteristics of sweetpotatoes
Springiness, cohesiveness, gumminess and chewiness were highest in the baked treatments (Table 3). While hardness and resilience were observed to be highest in the open cooked treatments, cohesiveness was found to be greatest in the pressure-cooked treatments although it was not significantly different from the baked treatment. Hardness, gumminess, chewiness and resilience were significantly reduced in pressure-cooked sweetpotatoes when compared to the rest of the cooking methods. Springiness was least among the open-cooked treated sweetpotato cultivars.
Table 3 Effect of cooking methods on textural properties of sweetpotato cultivars
|Baking Rank||Open cooking Rank||Pressure cooking Rank|
|Hardness (N)||188.11 b (2)||270.84 a (1)||101.02 c (3)|
|Springiness (%)||0.70 a (1)||0.60 c (3)||0.65 b (2)|
|Cohesiveness (%)||0.10 a (2)||0.07 b (3)||0.11 a (1)|
|Gumminess||23.86 a (1)||19.27 b (2)||9.70 c (3)|
|Chewiness||19.80 a (1)||12.02 b (2)||6.40 c (3)|
|Resilience (%)||0.05 b (3)||0.54 a (1)||0.20 b (2)|
Mean values in a row with different letters indicate significantly different at P<0.05. Values in parenthesis in a row indicate the ranking among the cooking methods with respect to that parameter.
Different methods of cooking are impacted by a blend of various factors, like temperature and time, thus when comparing various cooking techniques, care should be taken as outcomes will fluctuate due to the type of cooking treatment applied and the food product being prepared (Bernad, 2013). The deciding factor for the texture of plant substances are the cell wall’s properties, magnitude and spread of vesicles within the cell’s cytoplasm and the air-spaces located in-between cells (Bach, 2012). In addition, other components such as size and magnitude of food particles, level of heterogeneity, and the association of starch with lipids, protein and fiber would modify the characteristics that arise due to the thermal treatment (Trancoso-Reyes et al., 2016). As water flows down during osmosis into the cell vacuole to fill the cell wall compartment, turgor pressure helps to keep the cells rigid (Bach, 2012). Flaccidity sets in when turgor pressure is lost (Bach, 2012). Cells with high turgor pressure are usually stiff and hard, whereas flaccid cells are rubber-like (Bach, 2012). The sweetpotato flesh is composed mainly of starch, which only swells up by water absorption and then breaks down due to the hydrolysis of the weak bonds (Sugri et al., 2012). Starch granules in the raw state on the other hand are hard, tightly packed, tiny aggregation of starch molecules, which give a chalky feels when chewed out of the cells (Leighton et al., 2010). During cooking of the sweetpotao, the starch granules begin to soften at about 66 oC (this temperature varies in plants), and moisture is absorbed, which impairs their compact structure and the granules swell up to many times their original size and weight (McGee, 2004).
Comparison of the effect of thermal treatments on sweetpotato cultivars
Of all the treatments tested, baked Old Yellow cultivars were the most gummy and chewy (Table 4). Baked Old Yellow sweetpotato was also the springiest, however it di did not vary significantly from baked Japanese Purple sweetpotato cultivar. The maximum viscosity attained during the heating cycle, peak viscosity, shows the swelling ability of the starch granules before they are physically broken down (Ikegwu and Okechukwu, 2010). Truong and Walter (1994) observed in their study that although in baked roots, the microstructure of the cell wall was destroyed completely, many gelatinized starch granules still retained integrity and shape. This finding contradicts with what was reported on Egyptian sweetpotato cultivar (Damir, 1989). There was a complete shape deformation of starch granules baked at 175 ˚C for 60 min. The extent of deformation of starch granules and other contents associated with the structure of baked sweetpotato likely varies among cultivars and may contribute to textural variability (Truong and Walter, 1994). The proportion of amylopectin and amylose in starch may thus account for the texture attributes in food products, including, stickiness, and resistance against shear stress, swelling of starch granules due to heat, solubility, tackiness, stability of gel, cold swelling, and retrogradation. Japanese Purple cultivar prepared using the open cooking method was the hardest, however, they were not different from open-cooked Hernandez sweetpotato cultivar (Table 4). TPA hardness and fracturability showed comparative patterns and were highly correlated with peak force (Truong et al., 1998). It is noteworthy that the strength of the cell wall and cell tugor pressure are the reason for hardness in plant tissue. When heat is applied however, the cell membrane structure is disturbed, and there is loss of turgor pressure wherein water filters from the cells (Bach, 2012). First, the cell tissues loose solidness quickly, a turgor pressure diminishes then the cell wall loses its integrity as a result of a loss of pectic compounds. The open-cooked Hernandez cultivars were also the most resilient, however, they did not vary significantly from the Japanese purple and Old Yellow sweetpotato also prepared using open cooking method (Table 4). Baked Old Yellow sweetpotato were also the most cohesive of all treatments and cultivars tested, however, their cohesive property was not significantly different from Japanese Purple and Old Yellow pressure-cooked sweetpotato, Old Yellow open-cooked sweetpotato and Hernandez baked Sweetpotato. Boiling at high temperatures disturbs cell cohesion and adhesion, bringing about a defect in tissue rigidity (Truong et al., 1998). Asides from this, potatoes with greater dry matter content are softer in texture after they are boiled (Thybo and Martens, 2000).
Table 4 Texture parameters of sweetpotato cultivars as affected by different cooking methods
|Hernandez||532.81 ab||2||0.68 bc||7||0.07 cdef||14||39.47 b||3||26.91 b||3||1.26 a||1|
|Japanese Purple||562.47 a||1||0.68 bc||8||0.07 cdef||12||42.35 b||2||28.68 b||2||0.93 a||2|
|Murasaki||252.32 d||6||0.39 h||18||0.06 def||16||16.10 c||6||6.18 c||8||0.20 b||12|
|Orleans||171.19 e||7||0.49 fgh||16||0.05 f||18||8.33 cd||9||4.06 c||11||0.16 b||4|
|Old Yellow||49.64 gh||14||0.63 bcde||11||0.12 ab||4||5.86 cd||12||3.73 c||14||0.88 a||3|
|O’Henry||56.64 gh||13||0.73 b||3||0.06 ef||17||3.52 d||15||2.58 c||15||0.02 b||11|
|Hernandez||58.67 gh||12||0.71 bc||6||0.10 bcd||6||6.36 cd||10||4.47 c||10||0.02 b||14|
|Japanese Purple||40.03 gh||16||0.58 cdef||13||0.15 a||2||6.25 cd||11||3.75 c||13||0.02 b||13|
|Murasaki||33.69 h||17||0.54 defg||14||0.10 bcde||9||3.42 d||17||1.85 c||17||0.01 b||16|
|Orleans||372.75 c||4||0.66 bcd||10||0.09 bcdef||10||33.38 b||4||22.20 b||5||0.03 b||10|
|Old Yellow||25.77 h||18||0.67 bc||9||0.13 ab||3||3.34 d||18||2.23 c||16||0.01 b||15|
|O’Henry||75.22 fgh||10||0.72 b||4||0.07 cdef||13||5.43 cd||13||3.92 c||12||0.01 b||18|
|Hernandez||103.03 fg||9||0.53 efg||15||0.12 abc||5||12.08 cd||8||6.37 c||7||0.05 b||8|
|Japanese Purple||62.80 gh||11||0.94 a||2||0.07 cdef||11||4.89 cd||14||4.58 c||9||0.04 b||9|
|Murasaki||306.67 d||5||0.72 b||5||0.10 bcd||8||31.34 b||5||23.16 b||4||0.09 b||5|
|Orleans||49.20 gh||15||0.45 gh||17||0.07 def||15||3.49 d||16||1.60 c||18||0.01 b||17|
|Old Yellow||470.67 b||3||0.95 a||1||0.16 a||1||76.41 a||1||73.67 a||1||0.08 b||6|
|O’Henry||136.30 ef||8||0.62 bcde||12||0.10 bcd||7||14.96 cd||2||9.41 c||6||0.07 b||7|
* Values followed by different letters differ significantly at p<0.05 by LSD. #Ranking based on the values from high to low.
The softest sweetpotato were the Old Yellow cultivar type prepared using the pressure cooking method, it however did not vary significantly from many of the other cultivars (Orleans, Murasaki, O’Henry, Japanese and Hernandez) across the various treatments tested (Table 4). An investigation by Truong et al. (1998) revealed that sweetpotato samples immersed in boiling water were softer than the raw sweetpotato as shown by a less steep bend with reduced fracture strength. In a different report by Leighton et al. (2010), decrease in both shear strain and stress was seen in every single cultivar prepared with steaming technique in contrast to the qualities observed for raw sweetpotato. The possible reason could be because of the extent in which starch and cell wall constituents break down during cooking, which then impacts various textural properties among sweetpotato cultivars (Leighton et al., 2010). The least springy sweetpotato was the open-cooked Murasaki cultivar, however its low springiness value was not significantly different from that observed for the open-cooked and baked Orleans sweetpotato (Table 4). The least cohesive, gummy, chewy and resilient cultivars were observed to be the open-cooked Orleans, pressure-cooked Old Yellow, baked Orleans and pressure-cooked O’Henry cultivars, respectively. Leighton et al. (2010) also reported that during boiling, take-up or adsorption of water lessens the cohesiveness and weakens the cell walls. Other than this, pectic polymers that play a part in cell adherence are broken down by β-elimination at higher temperatures, and divalent cations, particularly Ca2+ and Mg2+ can decrease softening during heating, as the particles cross-interface the pectic polysaccharides associated with the cell adhesion Leighton et al. (2010). The conduct of the above parameters is related to the sample properties and composition and essentially to the concentration of starch (Trancoso-Reyes et al., 2016). On heating, the crystalline areas are disturbed, water is taken up and the starch forms a gel. The gelatinised starch in the case of potatoes can at times occupy the whole cell, in which case the potato will be viewed as soft.
Correlations among TPA parameters
The correlation coefficients exhibited a positive relationship between the texture variables (springiness, gumminess, chewiness, resilience and hardness) of the sweetpotato roots (Table 5). Gumminess was significantly correlated with hardness and chewiness, suggesting they have a relationship. Chewiness was significantly correlated with hardness. In support of our results, experimental data examination by Walter et al. (2002) to determine the textural measurements and product quality of restructured sweetpotato French fries, indicated that hardness decreases with calcium concentrations and gel strength. Also, gumminess also appeared to be affected similarly as hardness, but the coefficient of variation was large to make this relationship uncertain (Walter et al., 2002).
Table 5 Correlation coefficients of TPA parameters
* Values in asterisks are significant at p<0.05 by LSD
The texture profile analysis (TPA) was employed to predict the consumer acceptability of organic sweetpotato as affected by different processing methods. The mouthfeel characteristics (hardness, springiness, cohesiveness, gumminess and chewiness) can be predicted by using instruments such as texture analyzer. Chewiness was significantly correlated with hardness. Gumminess was significantly correlated with hardness and chewiness suggesting they have a relationship. Hernandez was found to be the hardest cultivar although not significantly different from Japanese Purple. Old Yellow was the most cohesive, gummy and chewy sweetpotato. The least resilient cultivar, O’Henry, was also the least hard, gummy and chewy cultivar, its chewiness and resilience was however not significantly different from Muraski and Orleans. The springiest cultivar was Old Yellow, though it did not differ significantly from the Japanese Purple cultivar. Hernandez was the most resilient cultivar, however it was not significantly different from Murasaki and Orleans. The different processing conditions such as open cooking, pressure cooking and baking affect the textural parameters differently depending upon the conditions. Springiness, gumminess and chewiness were highest under baking conditions. Hardness and resilience were greatest in open-cooked treatments. Cohesiveness was found to be greatest in the pressure-cooked treatments, although it was not significantly different from the baked treatment. In pressure-cooked sweetpotato however, hardness, gumminess, chewiness and resilience were found to be reduced significantly when compared to the rest of the cooking methods. Springiness had the lowest values among the open-cooked treated sweetpotato cultivars. Across the treatments, open-cooked Japanese Purple was found to be the hardest, although not significantly different from Hernandez open-cooked cultivar. Baked Old Yellow sweetpotato was the most gummy and chewy while the softest cultivar was the pressure-cooked Old Yellow; however it did not differ significantly from many of the other cultivars (Orleans, Murasaki, O’Henry, Japanese and Hernandez) tested. Results of this study indicate that the prediction of mouthfeel characteristics using instruments will reduce the time and energy to conduct sensory evaluations and helps to assess sweetpotato sensory quality, thus setting bench marks for marketability. Although, texture stand out amongst the most essential sensory traits of root crops, it has been described as one of the most difficult attributes to gauge instrumentally. Studying the changes in texture properties of different sweetpotato cultivars prepared under different cooking methods and time would help us improve the sensory properties of each meal we consume as we would be able to determine the desirable changes in the major textural characteristics and the optimum time needed for preparation thereby reducing the time spent cooking. Although, qualitative descriptive analysis is a time-consuming process, however, when applied to promising cultivars, it can provide vital information that can predict potential preference by consumers.
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Food colorants can be classified into synthetic colorants such as quinoline yellow (Zhang et al., 2015) and tartrazine (Xu et al., 2015), as well as natural ones, such as lycopene (Xu et al., 2016), and curcumin (Upadhyaya, et al., 2015). At present, natural pigments comprise 31% of the food pigments market (reach up to US$27.5 billion in 2018 (Mapari et al., 2010). However, due to a few safety hazards, natural yellow pigments from animals, plants, or microorganisms have become more attractive in recent years (Vendruscolo et al., 2016). Among these pigments, microbiology pigments have a good quality for harvest, scale-up of production is easier and they are not subject to the vagaries of nature (Gomes et al., 2016). However, prior to food use, toxicological assessments must be conducted because some fungal species producing pigments are also myco-toxigenic producers (Mapari et al., 2009b). Only Monascus is an important microbial resource now in use to produce pigments in an industrial level.
Thousands years ago, ancient Chinese had been using Monascus-fermented red rice as a food colorant to make red rice wine, red soybean cheese, meat and fish products and so on (Blance et al., 1994; Ma et al., 2000; Wild et al., 2002). Currently, more than 50 patents regarding to utilization of Monascus pigments for food have been issued in Japan, the United States, France, and Germany (Lin et al., 1992; Wang et al, 2007). The pigments produced by original Monascus contains three categories of pigments, yellow (monascin and ankaflavin), orange (monascorubrin and rubropunctatin), and red (monascorubramine and rubropunctamine) pigments (Xiong et al., 2015). Among these, the red pigments have been widely used in Asia for centuries as food colorant and now have been successfully produced by fermentation (Feng et al., 2012). Because of their excellent resistance to photodegradation and their pH and thermal stability (Mapari et al., 2009a), Monascus yellow pigments have been receiving much attention. Except for uses as colorants, Monascus yellow pigments have been reported to possess health benefits, such as in reducing diabetes and obesity (Hsu et al., 2014), hypolipidemic (Lee et al., 2010), anti-obesity (Lee et al., 2013), anti-inflammation (Hsu et al., 2012), antitumor (Su et al., 2005; Lee et al., 2013) and antioxidative stress (Shi et al., 2012), and have wider applications than its in the food industry (Klinsupa et al., 2016). Research continues in the development of improved Monascus yellow pigments yields, as well as in identifying new Monascus yellow pigments (Krairak et al., 2000; Chen et al., 2015). Monascus yellow pigments have been widely researched due to, which are related to the molecular structures of yellow pigments (Su et al., 2005). In the last few decades, 35 Monascus yellow pigments and its derivatives have been identified and characterized (Gong and Zhengqiang, 2016). However, the yellow pigments are still not suitable for industrial production, due to their relatively low production and purity, unavailability of microbial species. Not only microbial genus, but also the environmental conditions play a key influence on monascus yellow pigments production in submerged culture. Environmental conditions include chemical conditions like the type and content of carbon, nitrogen, phosphate, and metals, and physical conditions like mechanical stress, temperature, agitation and pH (Shi et al., 2015; Bo et al., 2009; Hu et al., 2012; Tao et al., 2017). Meantime, fungal morphology, influenced by genotypes of strains and environmental conditions (Kaup et al., 2008; Krull et al., 2010), is also considered as a key bioprocess parameter for submerged, which not only has a significant impact on mixing and mass transfer, but also determines the overall process productivities and subsequent economics (Wucherpfennig et al., 2011; Hyun et al., 2002).
In our preliminary experiments (Bo et al., 2009; 2012; 2014), the pH, aeration and temperature influenced the Monascus yellow pigments production by Monascus anka mutant in 5 L fermenter. Therefore, the aim of this work was to systematically investigate the effect of agitation on the production of the yellow pigments in Monascus anka mutant.
MATERIALS AND METHODS
Organism and cultivation
Microorganism used in this study is Monascus anka mutant, which was screened from physical and chemical combination mutagenesis in our laboratory (Bo et al., 2009). Stock cultures of the mutant were maintained on wort agar slants, which contains 15º wort (provided by Guangzhou Zhujiang Beer Co., Ltd, China) and 20 g/L agar (Difco Labatory, Loveton Circle, USA), and subcultivated periodically. Cultures were reactivated by being transferred onto fresh wort agar slants. After cultivation for 2-3 days at 31 ºC, spores were collected with 5 mL sterilized water, and the corrected spore suspension was used as inoculum preparation. Spores suspension (0.3 mL) was inoculated in 250 mL Erlenmeyer flasks containing 30 mL of seed culture medium which was composed of 30 g/L of corn flour, 3 g/L of NaNO3, 4 g/L of KH2PO4, and 0.01 g/L of FeSO4.7H2O. The seed culture medium (initial pH 6.0) was cultivated at 31 ºC and 200 rpm for 1-2 days and then transferred into a 5 L fermenter (BIOFLO 3000 Batch/Continuous Bioreactor, New Brunswick Scientific Edison, NJ, USA). The submerge fermentation medium ingredients included 10 g/L corn steep liquor, 15 g/L NH4Cl, 5 g/L KH2PO4, 20 g/L glucose and 70 g/L starch. Temperature of 31 ºC and aeration of 1.5 m3/h were maintained during fermentation in fermenter. Various agitation rates from 250 r/min to 450 r/min were applied.
Determination of pigments
According to the similar method of Chinese National Standard, GB15961-2005 and some reported articles (Bo et al., 2009), absorbance was applied to represent the pigments concentration. Five mL of culture broth was mixed with 5 mL of 70% (v/v) ethanol for 1 hour, and then centrifuged at 4,000 rpm for 20 min. The obtained supernatant was filtered through filter paper (45 mm, Xinhua Paper Industry Co., Ltd, Hangzhou, China). The filtrate contained two pigments: yellow pigment and red pigment, whose concentrations were determined by measuring the optical density of the supernatant using a 2802SUV/VIS spectrophotometer (Unicosh Scientific Instrument Co., Ltd, Shanghai, China) at 410 and 510 nm, respectively. Results were expressed as OD units per mL of fermented broth. The linearity equation between absorbance and diluting proportions is y = -0.0054x+1.5462(R2= 0.9907), where y is absorbance and x is dilution proportions (in the range from 100 to 300).
Determination of Dried Cell Weight (DCW)
Fungal biomass was determined by gravimetric analysis after filtration of cell samples through preweighed nylon filters (45 mm diameter, 0.8 μm porosity) and dried to constant weight at 60 ◦C under partial vacuum (200 mm Hg).
Determination of Residual Sugar Concentration (RSC)
The residual glucose in the fermentation broth was determined with a spectrophotometer by the standard 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959), and the calibration curve was prepared using glucose.
Determination of NH4+ Concentration
The Berthelot reaction (Weatherburn, 1967) was used for determination of ammonium ion.
Determination of Soluble Starch Concentration
The soluble starch was measured by the modified method of Teng (Teng and Feldheim, 2001). Briefly, sample (0.5 mL) was hydrolyzed by using 0.5 mL of thermostable α-amylase (EC 184.108.40.206, Novo, Denmark) at 100 ◦C for 60 min and then 0.25 mL of amyloglucosidase (EC 220.127.116.11, Novo, Denmark) at 60 ◦C overnight. The amount of glucose was determined by the DNS method. The starch content was calculated as the amount of glucose×0.9 and expressed as g/L (g starch content/L cultures)
Imaging and Morphological Analysis
Sample preparation was carried out following the method described by Haack et al. (Martin et al., 2006). For image analysis, 2 mL of the sample was taken from the culture broth, and one or two drops of lactophenol blue was added to stop growing and increase the contrast of the images. Image capture wassig accomplished on a Zeiss light microscope. The pellets were distinguished from clumps and dispersed mycelia by the differences in the greyness levels, an approach that has been used to provide a definition for a pellet (Thomas 1992). Morphological measurements were carried out using a CMOS camera (IXUS115; Canon, Japan) and the Image-Pro PLUS software (Media Cybernetics Inc., USA). The average clump diameter was calculated on images obtained using a 4×objective. Data, reported as the mean±SD, were obtained from a population size of approximately 100 events per sample.
The specific growth rate, µ (h-1), was calculated following the equation:, where X is the cell concentration (g/L) at time t (h). The specific production rate of yellow pigments, qy (OD.g-1.h-1), was calculated following the equation: , where Y is the yellow pigments value (OD) at time t (h). All the data shown in Tables and Figures were expressed as mean of triplicates. The statistical evaluation of all data was performed by Origin 8.0.
RESULTS AND DISCUSSION
Effect of agitation speed on yellow pigments production and Monascus anka mutant growth
Significant influence of agitation on monascus yellow pigments production and Monascus anka mutant growth has been noticed in this working (Figure 1). When agitation speed was 250，300，350，400, and 450 r/min, the maximum monascus yellow pigments yield was 100.04, 100.12, 110.23, 125.85, and 105.97 OD units at 144, 126, 126, 96, and 96 hour, respectively (Figure 1A). Maximum of qy was 0.1659，0.2264，0.1741, 0.1792, and 0.2177 OD.g-1.h-1 at 66, 54, 54, 42, and 48 hour, respectively (Figure 1B). mmax was 0.026，0.047，0.034, 0.078, and 0.064 h-1 at 18, 24, 18, 6, and 24 hour , respectively (Figure 1C).
Figure 1 Effects of agitation speed on Monascus yellow pigments production (A), qy(B) and m(C)
These results demonstrated that time for yellow pigments production has been in advanced with high maximum specific production rate by high agitation speed, but higher agitation speed led to faster decrease of qy at fermentation anaphase. Meanwhile, high agitation speed can improve specific growth rate for Monascus anka mutant at fermentation prophase, but negative for Monascus anka mutant growth at fermentation anaphase.
Effect of agitation on substrates utilization by Monascus anka mutant
With the increase of agitation speed, the maximum residual sugar content in culture appeared at 18 hour. Thereafter, the residual sugar content showed a downward trend with increased agitation (Figure 2A). The soluble starch content almost reached stable stage at 18 hours as shown in Figure 2B. The possible reason for this phenomenon is that hydrolysis of soluble starch was faster than the consumption of glucose at the initial stage (Teng et al., 2001). Prolonged fermentation time did not digest starch very much. The soluble starch in the culture with higher agitation speed was lower at the end of the fermentation. (Figure 2B). NH4+ was used as nitrogen source in the present work. Sharp decrease of NH4+ was observed in the first 48 h beside of culture at 250 r/min (Figure 2C).
Figure 2 Effects of agitation on residual sugar (A), soluble starch (B) and NH4+ (C) utilization by Monascus anka mutant
The above results indicated that 400 r/min at fermentation metaphase while 300 r/min at fermentation anaphase was beneficial for substrates utilization. In general, higher agitation speed improved the hydrolysis of soluble starch (Teng et al., 2001) and NH4+ consumption (Bo et al., 2014). From Figure 1A, it seemed that residual glucose in fermentation broth with agitation speed of 450 r/min was higher than that with the speed of 350 r/min. However, it should be noted that the residual soluble starch in fermentation broth with agitation speed of 250 r/min was much higher than that of the fermentation with the speed of 450 r/min. Therefore, fermentation with higher agitation speed metabolized more glucose.
Effect of agitation-shift on yellow pigments production by Monascus anka mutant
From the above experimental results concerning different agitation speed on the yellow pigments production by Monascus anka mutant, stage-divided strategy may improve yellow pigments production and enhance Monascus anka mutant growth with efficient utilization of substrate theoretically. The stage-divided strategy was as follows: 400 r/min was carried out to improve the Monascus mutant growth and yellow pigments production before fermentation of 96 hour, and then 300 r/min was applied to increase yellow pigment production with efficient substrate conversion.
Under agitation-shift strategy in batch monascus yellow pigments fermentation, the maximum yellow pigments yield reached 149.43 OD at 102 hour, which was 49.37%，49.25%，35.56%，18.73%, and 41.01% higher than that of 250，300，350，400 , and 450 r/min, respectively (Figure. 3A). Maximum qy and mmax, 0.2177 OD.g-1.h-1 and 0.0528 h-1, respectively, was achieved at fermentation time of 48 hours and 6 hours (Figure 3B). m and qy could be maintained at 0.01 h-1 and 0.1 OD.g-1.h-1 from 6 hours to 54 hours and 24 hours to 66 hours, respectively. It was impossible to achieve using single temperature-shift strategy (Figure 3B). It showed that the yield of yellow pigments was higher than those of reports (Shi et al., 2015; Klinsupa et al., 2016; Krairak et al., 2000; Bo et al., 2009; Hu et al., 2012; Tao et al., 2017).
Figure 3 Effects of two stage agitation control on Monascus yellow pigments production (A) and Monascus anka mutant growth (B)
Under two stage agitation control, soluble starch was 19.79 and 10.37 g/L at 18 and 96hour, respectively. In the first 18 h, the content of soluble starch was decreased significantly. Meanwhile, reducing sugar content showed the highest at 8 h. The reducing sugar content, soluble starch content and NH4+ content was 8.24 g/L, 7.37 g/L and 0.135 mol/L, respectively at the end of fermentation. The yield of amylase produced by Monascus, which can hydrolyze soluble starch into sugar (Teng et al., 2001), could be increased by the higher agitation speed and therefor the reducing sugar utilization was accelerated. Higher agitation speed significantly enhanced the carbon source (reducing sugar and soluble starch) consumption by Monascus anka mutant (Figure.2). However, NH4+ may be only used for Monascus anka mutant growth because agitation has no obviously influence on change of NH4+ contents in post-fermentation but only in early-fermentation in culture (Figure1-2). All the above results indicated two stage agitation control could obviously improve substrate metabolism (Figure 4) , the yield of product relative to soluble starch and NH4+, and accelerate the Monascus growth and yellow pigments production, leading to a short fermentation time eventually (Table 1) compared with one-stage agitation strategy.
Table 1 Important parameters of yellow pigments production under different agitation condition
|agitation (r/min)||Maxium µ(h-1)||Maxium
( OD .g-1.h-1)
|yield of yellow pigments relative to ammonium ion( OD .mol-1)||yield of yellow pigments relative to starch( OD.g-1)||Culture colour change(h)|
|250||0.026(18 hr)||0.0877(48 hr)||652193.8||2202.67||23|
|300||0.047 (24 hr)||0.1659(66 hr)||649244.5||1990.17||22|
|350||0.034 (18 hr)||0.2264(54 hr)||691400.6||2051.62||21|
|400||0.078(6 hr)||0.1741(54 hr)||774223.3||2211.38||21|
|450||0.064(24 hr)||0.1792(42 hr)||610813.3||1760.16||15|
|Two-stage||0.053(6 hr)||0.2177(48 hr)||915568.9||2458.67||20|
Figure 4 Effect of two stage agitation control on substrate utilization by Monascus anka mutant
Effect of agitation on morphological changes of Monascus anka mutant
Compared with single cell microorganism, there is morphological changes in filamentous fungi lifecycle by liquid and solid fermentation, especially more obvious and complex for submerge culture, then the morphological changes lead to influence on the target metabolites production. Three morphologies of filamentous fungi, such as free filaments, clump and pellet, appear in liquid culture (Thomas 1992). There are two reasons of morphological changes to influence metabolite: 1) secretion mechanisms changes, 2) rheological properties changes of culture, including oxygen mass transfer (Wucherpfennig et al., 2010). Besides incubation, pH, metal, substrate, temperature and oxygen, the agitation is the major factor on morphological changes (Kaup et al., 2008; Krull et al., 2010).
The mycelia morphology appeared free filaments and clump when agitation was 250 r/min and 300 r/min, respectively. Mycelia morphology was between free pellet and clump under 350 r/min. Mycelia morphology appeared well-distributed pellet with 0.52 mm in diameter when agitation was 400 r/min. But pellet was not well-distributed with diameter of 0.57 mm and some filaments appear in culture because of high agitation for 450 r/min. Mycelia morphology appeared to be pellet with diameter of 0.41 mm and yield of yellow pigments arrived 149.43 OD, which was 49.37% and 18.74% higher than that from 250 r/min and 400 r/min, respectively (Table 2). In the meantime, we also found seed age for incubation had an influence on mycelia morphology in this study, when old seed (beginning synthesis yellow pigments) was inoculated into fermenter, the mycelia morphology appeared between free filaments and pellet, or clump with low yellow pigments production. However, when inoculated with fresh seed, mycelia morphology appeared to be pellet with high yellow pigments production (data not shown). The mycelial morphology of Monascus, including the pellet size and hyphal diameter, was significantly influenced by the culture conditions such as the initial pH and shaking speed, which further exerted great impact on the production of yellow pigments. The relationship between the agitation and the fungal morphology has also been revealed in this study for enhanced production of natural yellow pigments (Jun et al., 2017). All result demonstrated pellet of mycelia morphology was benefit for monascus yellow pigments production.
Meantimes, the citrinin was unable to be detected by HPLC method (Bo et al., 2009) in this study namely, the Monascus anka mutant may produce no or just a little citrinin (the detection limit is 0.1mg/L).
Table 2 Effect of agitation on the mycelia morphology and yellow pigments production
|Mycelia Morphology||Mycelia diameter
|300||Free filaments and clump||0.98±0.03b||100.12±9.54c|
|350||Clump and pellet||0.69±0.05c||110.23±9.8b|
|450||Pellet and Free filaments||0.57±0.03c||105.97±8.79c|
Different letters in superscript within the same row indicate significant differences among the oil sample test (Tukey¢s test, p<0.05).
Previously, the yield of Monascus yellow pigment could be improved by controlling pH and nitrogen sources (Shi et al., 2015) or a novel approach of two-stage microbial fermentation in nonionic surfactant micelle aqueous solution (Hu et al., 2012). However, due to its pH-dependent property, relatively low production and purity, it is impossible to scale-up yellow pigment production and application for all kinds of foods. The Monascus genus, which can produce yellow pigments owning pH-dependent property with high yield, is the key factor, such as Monascusr mutant strain KB (Yongsmith et al., 1993; 1994) and our specific strain of Monascus anka mutant (Bo et al., 2009; 2012; 2014). In the current study, Monascus growth and yellow pigments production with high speed and longtime could be achieved by two stage agitation control strategy and short fermentation time to improve the yield of yellow pigments, which can make it possible to scale-up yellow pigment production.
To our knowledge, this is the first report focusing on the agitation optimization of Monascus yellow pigments production in submerge culture in fermenter as a source of natural yellow pigments. In the current study, Monascus anka mutant, which could produce yellow pigments with pH-independent property (Bo et al., 2009; 2012; 2014), was cultured in a fermenter using two-stage agitation controlling strategy. Maximum yellow pigments of more than 149.43 OD were found in submerge culture under optimal agitation conditions: the agitation was 400 r/min to improve the Monascus growth and yellow pigments production before culture 96 hour and then 300 r/min was carried out to continue yellow pigment production with efficient substrate utilization. Future development on yellow pigments should focus on strain improvement for higher production of yellow pigments as well as process scaling up. Hopefully, the results in this paper have very important theoretical and realistic significance for realizing the industrial production of monascus yellow pigments by submerged culture. The data of this work could contribute to making the industrial production of Monascus yellow pigments feasible.
Acknowledgments: This research work was financially supported by the National Natural Science Foundation of China (Nos.31301550), scientific research project of Hunan province department of education (Nos. 14C1185), Program for Science & Technology Innovation Talents of Hunan Province (2017TP1021; KC1704007).
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Depending on the shape of bacteria, they are classified as cocci (spherical or oval cells), bacilli (rod shaped cells), vibrios (comma shaped curved rods), spirilla (rigid spiral forms), spirochetes (flexible spiral forms), actinomycetes (branching filamentous forms) and mycoplasmas (cell wall deficient forms). Helical bacteria have been found in nature under very diverse circumstances, and spirochetes (Treponema spp., Borrelia spp. and Leptospira spp.) are free-living inhabitants of mud and water.
All Aquaspirillum species described in Bergey’s Manual of Systematic Bacteriology share the following characteristics: they are all rigid helical cells, except for Aquaspirillum delicatum which is vibrio and Aquaspirillum fasciculus which is a straight rod. The genus Aquaspirillum, with 13 species, was created for all aerobic freshwater spirilla having a low salt tolerance (Pot, 2006). Leptospires are aerobic spirochetes whose cells are flexuous, motile, tightly coiled and have axial flagella; they are gram negative and there is no visual difference between serogroups. Some are pathogenic, though others are harmless freshwater saprophytes; all requiring oxygen (dissolved in water) to survive. The genus Leptospira sp. includes at least 22 species arranged into three large subgroups based on 16S rRNA phylogeny, ten (10) pathogenic species, seven (7) saprophytic species and five (5) intermediate species (Bourhy et al., 2014; Picardeau, 2017). Saprophytic Leptospira spp. is free-living environmental microorganisms; however, pathogens leptospires can survive several days in fresh water when pH and temperature are adequate (Faine et al., 1999; Trueba et al., 2002). Each bacterium grows and divides independently of any other bacteria, although aggregates of bacteria (biofilms) have been frequently observed, even with members of different species (Ristow et al., 2008). Biofilm formation has been observed between saprophytic and pathogenic leptospires, Azospirillum brasiliensis and pathogenic leptospires, and between Sphingomonas sp. whit Leptospira spp. (Barragan et al., 2011; Kumar et al., 2015; Ristow et al., 2008). These cellular aggregates would give them protection from dynamic environments and even survive in poor nutrient conditions. In other studies, the ability to form bacterial aggregations in vivo was observed in pregnant guinea pigs infected with Leptospira Pomona (Brihuega et al., 2012).
The present research shows the behaviour and growth of Leptospira spp. and Aquaspirillum spp. in aqueous and semi-solid fluid (with and without nutrients) incubated at different temperatures, such as the interaction and biofilm-forming of leptospires with environmental bacteria.
MATERIAL AND METHODS
The stream Callvú Leovú is born in the vicinity of the town of Chillar, Buenos Aires province; and after traveling about 60 km crosses the city of Azul to end at Canal 11 (city of Las Flores), the mentioned channel was built in order to drain the waters of this channel and of other streams towards the Samborombón Bay.
Sample collection and culture
Water samples were collected from Callvú Leovú stream during 2016 years and transported in sterile 500 millilitres glass bottles (Figure 1). Temperature and pH were monitored in the field. Water samples were filtered through a sterile membrane. In this study, a pre-filter technique was applied using Whatman filter paper before filtration through membrane filter with 0.22 μm pore size. All samples were collected in early morning. A sample of the filtered water (one millilitre) was inoculated into Ellinghausen–McCullough–Johnson–Harris (EMJH medium: Difco Laboratories, Detroit Michigan USA) liquid medium without the addition 5-fluorouracil as selective antimicrobial agent. Cultures were incubated by duplicate at 13º C and 28-30º C during 90 days, and bacterial (Aquaspirillum spp. and Leptospira spp.) growth was monitored weekly using dark field microscopy. If leptospires or spirilla were not detectable after 90 days of incubation, the sample was considered to be negative.
Production of pure cultures
To obtain pure cultures of leptospires and Aquaspirillum spp., liquid and semisolid media of EMJH and Thyoglicollate (pure and mixed) with and without addition of 5-fluorouracil (300 μm/ml) were used, in all cases the pH was 7.2.
Characterization of Leptospiral isolates
Multiple Locus Variable number tandems repeat Analysis (MLVA) genotyping
Faine DNA templates were obtained using Chelex Resin-100 (Bio Rad). MLVA was performed using two sets of oligonucleotides specific for pathogenic leptospires (L. interrogans, L. kirschneri and L. borgpetersenii). Oligonucleotides that hybridized to the flanking regions of the VNTR4, VNTR7, VNTR9, VNTR10, VNTR19, VNTR23 and VNTR31 loci were used to discriminate strains of L. interrogans and oligonucleotides that hybridized to the flanking regions of the VNTR4, VNTR7, VNTR10, Lb4 and Lb5 loci were used for L. kirschneri, L. borgpetersenii and L. interrogans strains (Majed et al. 2005; Pavan et al. 2011). The final volume (50 μl) of each reaction mixture contained polymerase chain reaction (PCR) buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCL), 200 μM deoxynucleoside triphosphates, 2 μM each of the corresponding forward and reverse primers, 2 mM MgCl2, 1.25 U of Taq DNA polymerase (Invitrogen) and 5 μl of DNA template. PCR amplifications were carried out in a Thermo Scientific PxE 0.2 Thermal Cycler, using the following cycling parameters: 94º C for 5 min, followed by 35 cycles of denaturalization at 94º C for 30 s, annealing at 55º C for 30 s and extension at 72º C for 90 s, with a final cycle at 72º C for 10 min. The amplified samples were examined by electrophoresis in ethidium bromide-containing 2% agarose gels in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) at 100 V for 50 min. Amplified DNA bands were visualized through ultraviolet light exposure (Uvi Tec transiluminator BTS-20.M, Manufacturer UviTec, St. John’s Innovation Centre, Cowley Road, Cambridge, England). Amplicon sizes were estimated using CienMarker (Biodynamics) and the GelAnalyzer 2010a program. To calculate repeat copy numbers, the following formula was used: number of repeats (bp) = [fragment size (bp) – flanking regions (bp)]/ repeat size (bp). Repeat copy numbers were rounded down to the closest whole number. If the copy number was less than one, it was rounded to zero.
Sequencing and phylogenetic analysis of Leptospiral strains
PCR targeting the 16S rRNA gene was carried out for bacterial identification. The following primers were used: 5′-GGCGGCGCGTCTTAAACATG-3′ and 5′-GTCCGCCTACGCACCCTTTACG-3′; these primers have the ability to amplify all pathogenic and non-pathogenic species of Leptospira sp. (Djadid et al., 2009). After verification of the amplicon by electrophoresis (in an ethidium bromide-containing 2% agarose gel) and visualization upon UV light exposure, PCR products were purified using a commercial kit (Embiotech). The sample was sequenced at the Institute of Biotechnology, National Institute of Agricultural Technology (Argentina) using a 3130xl Genetic Analyzer (Applied Biosystems). For alignment and construction of the phylogeny, the program MEGA version 6.06 was used (Tamura et al., 2013). The dendogram basing in partial sequences of the 16S rRNA gene was constructed using Neighbour-joining with a bootstrap of 100.
Characterization of Aquaspirillum sp. isolates
Real-time PCR amplification of the 16S rRNA gene was performed in a Rotor Gene Q thermocycler (Qiagen, Hilden, Germany) in a final volume of 20 µl using EvaGreen as intercalating fluorescent dye (KAPA FAST, Biosystems, Woburn, USA). Generic primers p201 (5′-GAGGAAGGIGIGGAIGACGT-3′) and p1370 (5′-AGICCCGIGAACGTATTCAC-3′) were used (Tseng et al., 2003). Primers were synthesized at Operon (Huntsville, Alabama, USA). Different PCR amplification conditions were tested, by changing variables such as temperature of annealing, primer concentration, number of cycles, DNA concentration and final reaction volume, in order to produce suitable fluorescence levels for HRM analysis. During the validation process the PCR products were run on agarose gels to check the size of the amplicons. PCR amplifications were carried out in a Rotor Gene Q thermocycler (Qiagen, Hilden, Germany) in a final volume of 20 µl using EvaGreen as intercalating fluorescent dye (KAPA FAST, Biosystems, Woburn, USA). The selected cycling program consisted of an initial denaturation of 2 minutes at 95° C, and 45 cycles of 94° C 10″, 60° C 15″, and 72° C 15″. PCR products were purified and sequenced.
Bacterial strains and growth conditions
The leptospiral and spirilla strains used in this study were isolated from water samples of Callvú Leovú stream during 2016 years. Bacterial cells in logarithmic phase (1-2 x 108 cells / ml) were cultured by quadruplicate in EMJH medium (liquid and semi-solid) and sterile stream water; all tubes were incubated at two temperatures ranges (4-10º C and 28-30º C) during 20 weekend and the growth was monitored periodically using dark-field microscopy. To perform this experiment, three groups were formed: “a” (Leptospires alone: Leptospira spp. strain lepto106), “b” (Spirilla alone: Aquaspirillum spp. strain aquas106), and “c” (Leptospira spp. strain lepto106 whit Aquaspirillum spp. strain aquas106 at equal concentrations).
The concentration considered optimal was standardized by direct microscopy dark field using Neubauer chamber.
RESULTS AND DISCUSSION
Bacterial strains isolates
During the period April-December 2016 six leptospiral strains and six spirilla strains were obtained. All leptospiral isolates (strain lepto104, strain lepto106, strain lepto109, strain lepto110, strain lepto113 and strain lepto114) were negative by Multiple-Locus Variable-number tandem repeats Analysis (MLVA), however, molecular identification by 16S rRNA gene sequences verified that strain lepto106 (used in this experiment) were identified as non-pathogenic leptospires whit sequence similarities of 99% using Blast (results not shown), and closely related to the species L. yanagawae and L. meyeri. All spirilla bacteria isolates (strain aquas106, strain aquas108, strain aquas109, strain aquas110, strain aquas113 and strain aquas114) were identified as Aquaspirillum spp. by molecular identification by 16S rRNA gene sequences.
In moment of waters samples recollection in Callvú Leovú stream, we register temperature and pH; the results obtained can be seen in Table 1.
Leptospires such as spirilla developed both in tubes incubated at 13° C and at 28-30° C. In a sample of water obtained in month of January only the growth of Aquaspirillum spp. was observed in media incubated at 13º C. Generally, in all tubes first the presence of Aquaspirillum spp. (cells of 5-10 μm in length and less than 0.22 μm of diameter characterized by the presence of 3-5 turns and a characteristic movement) was observed, and a few days later the leptospires appeared, characterized by their flexuous motility and morphology typical of the genus Leptospira spp. (cells of 10–20 μm in length and less than 0.22 μm of) under dark field microscopy (Table 2). All strains isolation was maintained in liquid and semisolid EMJH media.
Figure 1 Callvú Leovú stream, Azul, Buenos Aires province, Argentina. Area of collection of water samples.
Table 1. Bacterial strains isolate from waters samples in Callvú Leovú stream, Azul, Buenos Aires province, Argentina.
|Stream water||Growth in EMJH medium (Days)|
|Months||Tº||pH||Leptospira spp.||Aquaspirillum spp.||Leptospira spp.||Aquaspirillum spp.|
Legend: Tº- temperature
Obtaining pure cultures of leptospiral and spirilla bacteria
The EMJH media with the addition of 300 μg / ml of 5-fluorouracil were used to obtain the growth of leptospires in absence of spirilla bacteria. To obtain growth of spirilla bacteria in absence of leptospires, Thioglycollate media with addition of EMJH (10%) and reverse were used. In Thioglycollate media (with 10% EMJH medium) growth of Aquaspirillum spp. (3 x 107 cells / ml) without the presence of leptospires, was observed on the second day of incubation at 28-30° C; in this media biofilm-forming of Aquaspirillum spp. and changes in cell structure (length and number of spires increased) were found. Under these conditions they remained viable for more than 30 weeks.
In EMJH medium (with 10% Thioglycollate media) growth of Aquaspirillum spp. was observed during the second day of incubation at 28-30° C; biofilm-forming and changes in cell structure were not observed.
Table 2 Differential characteristics of leptospires and spirilla bacteria
|Phenotypic characteristic||Leptospira spp. strain lepto106||Aquaspirillum spp. strain aquas106|
|Growth at 13°C||X||X|
|Growth at 28-30°C||X||X|
|Growth at 4-8°C||X||X|
|Growth at pH <7.2||–||–|
|Growth in EMJH medium||X||X|
|Growth in Thioglycollate medium||–||X|
|Helicoidally form||Flexuous helical cells||Rigid helical cells|
|Cells dimensions||10–20 μm in length and less than 0.22 μm of diameter||10-60 μm in length and less than 0.22 μm of diameter|
|Motility||By axial filaments||By polar flagellum|
Incubation and growth at 28-30º C
Leptospires alone (Leptospira strain lepto106): In liquid EMJH medium leptospires reached their maximum development (3 x 107 cells / ml) in the third week of incubation, and from the fourth to the seventh a plateau phase was observed with approximately 2.5 x 106 cells / ml; then declined and viable cells were undetectable after 12 weeks of incubation (Figure 2A). In contrast, in semi-solid EMJH medium the maximum development (2.5 x 106 cells / ml) was observed from the second week of incubation, and remained stable for up to 12 weeks (Figure 2B). In watercourse leptospires behaved similarly to those observed in liquid EMJH media (Figure 2C).
Spirilla alone (Aquaspirillum strain aquas106): In liquid EMJH media spirilla bacteria showed a progressive growth, reaching 3 x 107 cells / ml in the second week of incubation. In the first three weeks of incubation cells were observed to be larger with increase in the number of spires. After the third week spirilla bacteria remained viable for up to 12 weeks (Figure 2A). In semi-solid EMJH medium, Aquaspirillum spp. increased 3 x 107 cells / ml rapidly in the first week of incubation. From the seventh week Aquaspirillum spp. enter a plateau phase up to 12 weeks with approximately 2.5 x 106 cells / ml (Figure 2B); then in death phase cell mobility was not observed. In this fluid, during the first three weeks of incubation, changes in cellular structure (length and number of spires increased) were observed. In stream water Aquaspirillum spp. showed behaviour like that liquid EMJH media (Figure 2C).
Co-culture (Leptospira strain lepto106 and Aquaspirillum spp. strain aquas106): in liquid EMJH medium both bacteria behaved in a similar way, increasing from 2.5 x 106 cells / ml to nearly 5 x 10 6 cells / ml during the first three weeks. Approximately 2.5 x 106 cells / ml, was constant for several weeks. In these medium small cells aggregates until week 14 were visible (Figure 4A); spirilla bacteria maintained mobility but changes in cellular structure (increase in length and number of turns) were observed. In semi-solid EMJH medium, leptospires increased to approximately 5 x 106 cells / ml) in the first week of incubation, however from the third week spirilla bacteria maintained similar cells concentrations (Figure 3B). In this media cellular aggregates on the surface of media were observed until the sixth week (Figure 4). In stream water Aquaspirillum spp. strain aquas106 growth more efficiently than EMJH media, increasing to 3 x 107 cells / ml in the first week up to week five of incubation. In this fluid leptospires growth similarly to that EMJH media (Figure 3C).
Figure 2 Development of Leptospira spp. strain lepto106 and Aquaspirillum spp. strain aquas106 alone in liquid EMJH medium (A), semi-solid EMJH medium (B) and stream water (C) incubated at 28-30° C.
Figure 3 Co-culture of leptospires (Leptospira spp. strain lepto106) and spirilla (Aquaspirillum spp. strain aquas106) in liquid EMJH medium (A), semi-solid EMJH medium (B) and stream water (C) incubated at 28-30° C.
Figure 4 Cell aggregation and formation of biofilm between leptospires and Aquaspirillum spp. in semi-solid fluid incubated at 4° C. (A) surface-attached biofilm in air-liquid interface. (B, C and D) aggregates between Leptospira spp. strain lepto106 and Aquaspirillum spp. strain aquas106 (white arrows) at 40x and 100x magnification. (E and F) dark field microscopy of spirilla bacteria and free leptospires in sectors with no cell aggregates at 100x and 40x magnification, respectively. (G and H) helical shape of Aquaspirillum spp. with variable number of spiras at 200x and 100x magnification, respectively. (I) leptospires (white arrows) and Aquaspirillum spp. (black arrows) at 100x magnification.
Incubation and growth at 4-8ºC
Leptospires alone (Leptospira strain lepto106): leptospires developed at similarly in all media used. Although no significant development (2.5 x 106 cells / ml) was observed; bacteria were viable and mobility after 12 weeks of incubation (Figure 5A, B and C). In liquid media leptospires increased their size, and even their hooks were more notorious under observation by dark field microscopy.
Spirilla alone (Aquaspirillum strain aquas106): cells were undetectable in the first three weeks of incubation in all media used, however, when tubes were incubated at 28-30° C for one week, spirilla bacteria increased to approximately 3 x 107 cells / ml (Figure 6A and B) and 5 x 106 cells / ml (Figure 6C). In semi-solid EMJH media Aquaspirillum spp. strain aquas106 showed a more extensive stationary phase than liquid media, in addition cells with atypical mobility and shape were observed. No viable cells were seen after the eighth week in stream water media; however, in viscous media 2.5 x 106 cells / ml were observed.
Co-culture (Leptospira strain lepto106 and Aquaspirillum spp. strain aquas106): in liquid media (EMJH and stream water) no development was observed in the first 3 weeks, however, when tubes were incubated at 28-30° C for one week, spirilla (3 x 107 cells / ml) and leptospires (2.5 x 106 cells / ml) were observed (Figure 7A and C), remaining viable after 10 weeks in liquid EMJH medium. However, in stream water (after incubation at 28-30º C) 2.5 x 106 leptospires / ml and 5 x 106 spirilla / ml were found, from the fifth week the number of cells / ml was reversed. In semi-solid EMJH media leptospires increased to 3 x 107 cells / ml and spirilla to 2.5 x 106 cells / ml in the first week of incubation at 4-8° C (Figure 7B), this concentration was constant after 16 weeks of incubation. The formation of cellular aggregate between leptospires and spirilla bacteria was observed from the first week of incubation (Figure 4).
Figure 5 Development of Leptospira spp. strain lepto106 in liquid EMJH medium (A), semi-solid EMJH medium (B) and stream water (C) incubated at 4-8° C. In the grey column the period of incubation at 28-30° C is observed.
Figure 6 Development of Aquaspirillum spp. strain aquas106 in liquid EMJH medium (A), semi-solid EMJH medium (B) and stream water (C) incubated at 4-8° C. In the grey column the period of incubation at 28-30° C is observed.
Figure 7 Co-culture of leptospires (Leptospira spp. strain lepto106) and spirilla (Aquaspirillum spp. strain aquas106) in liquid EMJH medium (A), semi-solid EMJH medium (B) and stream water (C) incubated at 4-8° C. In the grey column the period of incubation at 28-30° C is observed.
In water samples from Callvú Leovú stream, spirilla and saprophytes leptospires were isolated; the growth in liquid EMJH media was on average of 4.6 days at 13° C and 6.9 days at 28-30° C. The pH recorded in water samples was always above 7.2, being optimum for the growth of helical bacteria; in winter period the temperature of stream water was not lower than 11.5° C, and did not inhibit the bacterial development in specific media. Ours results suggest the importance of 5-fluorouracil as specific antimicrobial when leptospires are to be isolated from surface water samples. Spirilla (Aquaspirillum spp.) they are rigid helical bacteria not retained by membranes filter whit a pore diameter size of 0.22 μm. These bacteria could be confused with leptospires at low magnification under dark field microscopy. The differential characteristics between Leptospira spp. and Aquaspirillum spp. are detailed in the table 2. The viability of leptospires and spirilla bacteria in all media used according to incubation temperature is detailed in the table 3.
Table 3 Viability of the bacteria in the media used according to incubation temperature, using dark field microscopy.
|Media||Leptospira spp.||Aquaspirillum spp.||Leptospira spp. + Aquaspirillum spp.|
|4-8 °C||28-30 °C||4-8 °C||28-30 °C||4-8 °C||28-30 °C|
|Liquid EMJH medium||168||84||77||84||140||98|
|Semi-solid EMJH medium||168||168||84||98||217||168|
In specific media for the isolation of leptospires as in stream water, spiral bacteria always reached a higher number of cells in the first week of incubation at 28-30° C compared to leptospires. Spirilla bacteria was undetectable in the first three weeks of incubation at 4-8° C in all media used, however, in a face of thermal stimulus (incubation at 28-30° C for one week) cells increased to approximately 3 x 107 cells / ml, and in semi-solid EMJH medium to remain viable after 12 weeks. Spirilla bacteria and leptospires were able to remain viable for three weeks at low temperatures until the environment conditions are optimal.
Leptospires developed in all media used and remained motile for 112 to 168 days (at 4-8° C of incubation) in stream water and liquid EMJH medium respectively; however semi-solid EMJH media was more efficient at 28-30° C. These results are like those observed by Trueba et al., (2004) who described the survival of leptospires for 110 days (aqueous media) and 347 days (semi-solid medium) in distilled water (Trueba et al., 2004). In liquid EMJH medium viable cells of leptospires were undetectable after 84 days of incubation at 28-30° C, possibly by depleting nutrient medium.
Leptospires and spirilla bacteria (co-culture) growth in liquid EMJH media incubated at 28-30° C, in this medium, Aquaspirillum spp. strain aquas106 maintained mobility for several weeks, although changes in cell structure (increase in length and number of turns) were observed. The co-culture in liquid media (EMJH and stream water) incubated at 4° C, Aquaspirillum spp. strain aquas106 did not develop during three weeks. In semi-solid EMJH media, Leptospira spp. strain lepto106 showed more cell numbers / ml in the first week of incubation respect to Aquaspirillum spp. strain aquas106, and this concentration was constant after 16 weeks. In stream water Aquaspirillum spp. strain aquas106 showed more efficient growth than EMJH medium.
In semi-solid EMJH medium, cell aggregates between leptospires and spirilla bacteria were observed on the surface, which persisted up to the sixth week. Ristow et al., (2008) observed that L. interrogans serovar Lai strain Lai 56601 formed a halo attached to the wall of glass tubes at the air–liquid (Ristow et al., 2008).
Cell aggregation and formation of biofilm between Leptospira spp. strain lepto106 and Aquaspirillum spp. strain aquas106 was observed in semi-solid fluid incubated at 4° C as 28-30° C. In our study, the formation of cellular aggregate between leptospires and spirilla bacteria was independent at incubation temperature. Other studies showed the ability of L. biflexa to form biofilm at three different temperatures (Ristow et al., 2008). Viscosity may favour the aggregation of leptospires by providing a matrix that holds the cells together, facilitating motility and therefore chemotaxis (Trueba et al., 2004). Cell aggregation may be a mechanism that facilitates the adaptation of leptospires to different environmental conditions. Two types of biofilm architecture were observed by Ristow et al., (2008), one consisting of large, distinct mound-shaped microcolonies (L. interrogans) and the other showing smaller microcolonies with a flatter structure that were linked together by a complex network of L. biflexa (Ristow et al., 2008). This mechanism has been observed in saprophytic leptospires as well as in pathogens leptospires (Barragan et al., 2011; Brihuega at al., 2012; Ristow et al., 2008; Trueba et al., 2004).
In natural water sources there is a great diversity of environmental bacteria that could interact with the leptospires. This interaction has been with Sphingomonas consortium and Azospirillum brasilense (Barragan et al., 2011; Kumar et al., 2015). Leptospires and spirilla bacteria share similar habitats in nature, the presence of Aquaspirillum spp. in water would help to increase the average life of leptospiras in the environment. All isolates were negative by Multiple-Locus Variable-number tandem repeats Analysis (MLVA), however, molecular identification by 16S rRNA gene sequences verified that all isolates were identified as non-pathogenic leptospires. Further studies will aim to meet serogroups circulating leptospires in surface waters.
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Poultry industry alleviates the poverty by offering enormous opportunities to millions of people in the country. Availability of poultry meat is 3.90 Kg per capita in Pakistan, 55 Kg per capita in Kuwait, 50 Kg per capita in USA and 12 Kg per capita in the world per annum. This condition is similar regarding the consumption of eggs. There is a big gap in the consumption of the poultry meat and eggs. This is due to many problems which are being faced by the poultry industry of Pakistan. One of the major problems is economic losses caused by infectious diseases. The main threats are the diseases caused by Mycoplasma species (Marois et al., 2001). The main pathogenic species of Mycoplasma are Mycoplasma gallisepticum (M. gallisepticum) and Mycoplasma synoviae (M. synoviae) (Umar et al., 2017). Mycoplasma causes immense losses in the poultry industry by decreasing eggs production, reducing growth and increasing condemnation at slaughter house (Kleven, 2008; Ferguson-Noel and Williams, 2015). About 10-20% losses in eggs production has been reported in the flocks affected from Mycoplasmosis (Bradbury and Morrow, 2008). Mycoplasmas are free living and self-replicating bacteria which are known to have the smallest genome and have low G+C content about 23-40%. (Nicholas and Ayling, 2003). Cell wall is absent in Mycoplasmas. The cell membrane of these organisms is incorporated with sterols which differentiates them from other organisms (Kleven, 2008). Based on 16S rRNA analysis Mycoplasma belongs to phylum Firmicutes, class Mollicutes and family Mycoplasmataceae (Ley, 2003; Ley, 2008). In birds out of 22 known species of Mycoplasma, the four common pathogenic species include M. gallisepticum, M. synoviae, M. meleagridis and M. iowae (Bradbury, 2001). Of all avian Mycoplasma pathogens, M. gallisepticum and M. synoviae are important species due to high prevalence in different types of poultry and M. gallisepticum is being considered the most pathogenic (Umar et al., 2017). Other than chicken turkeys, quails, partridges, pheasants and pigeons are also the natural hosts of M. gallisepticum (Ley, 2003). M. gallisepticum causes chronic infections in both chickens and turkeys and is the most virulent of all the Mycoplasma species (Liu et al., 2001). M. gallisepticum and M. synoviae cause respiratory disease in both chickens and turkeys while M. iowae and M. meleagridis cause diseases only in poultry (Fan et al., 2011).
M. gallisepticum causes chronic respiratory disease (CRD) in the chickens and its incubation period is 16-21 days (Siddique et al., 2012). Gasping, respiratory rales, coughing, nasal discharge and rhinitis are the major signs of CRD. Sometimes M. gallisepticum causes arthritis, salpingitis, conjunctivitis and fatal encephalopathy (Much et al., 2002). In the egg type birds, it causes marked decrease in eggs production and embryo mortality (Mukhtar et al., 2012). M. synoviae is very important poultry pathogen worldwide with respect to the economic losses caused by it such as decreased eggs production, growth retardation and condemnation of poultry meat at slaughterhouse. It usually causes infectious synovitis (respiratory infection) in chickens and may result in sub clinical infection. At present, M. synoviae causes infectious synovitis less frequently and air sacculitis more frequently in chickens and turkeys (Benčina et al. 2001; Jacob et al., 2014). It causes air sacculitis which can also be the result of co-infection with M. gallisepticum and E. coli. When infection becomes systemic, it causes inflammation of synovial membranes of joints and tendon sheaths causing synovitis, tenovaginitis and bursitis (Kleven, 2008).
.CHRONIC RESPIRATORY DISEASE (CRD)
M. gallisepticum and M. synoviae both cause CRD in all types of chickens (Bradbury and Morrow, 2008). The primary causative agent of CRD is M. gallisepticum and it causes disease under stress and poor management conditions or when a bird is suffering from some other respiratory problem (Papazisi et al., 2002). In the expanding poultry industry, M. gallisepticum is the most virulent avian pathogen and causes worldwide outbreaks leading to immense economic losses (Evans et al., 2005). It primarily damages respiratory tract by colonizing it and then secondary bacteria like E.coli and viruses invade there and cause severe infections (Liu et al., 2001; Peebles et al., 2015). Extensive antibiotic treatment is used to keep Mycoplasma under check and attenuated vaccines are used to prevent the disease but complete eradication of pathogen is very difficult. M. gallisepticum is the only avian Mycoplasma specie which is invasive in vitro as well. This is the reason it not only resists host defense and antibiotics but also enters the blood and causes systemic infection (Winner et al., 2000; Umar et al., 2017 ).
M. gallisepticum causes disease in birds of all ages but immunocompromised birds are more susceptible to this pathogen (Nunoya et al., 1995). In case of infection, the organism first colonizes the respiratory tract and in different strains of M. gallisepticum the tissue tropism, cell injury, attachment and pathogenicity varies (Sun et al., 2017). It is vertically transmitted through eggs and disseminated in hatchery. Decreased hatchability and low eggs production count for the major economic losses caused by M.gallisepticum. Infected birds produce low quality day old chicks and slower growth rate. This also leads to increased medication and control procedure costs in the farming (Ley, 2003). The pathogenesis of Mycoplasmas in poultry is summarized in Figure 1 and 2.
Sialoglycoprotein receptors in the respiratory epithelium are required for the attachment of the Mycoplasma to the epithelial cells and initiation of the disease. The process is mediated through cyto-adherence. To escape the innate host defense, attachment is very important process. Since many metabolic pathways are absent in the Mycoplasma, so for their survival it needs very close interaction with the host cell (Simecka et al., 1992). Mycoplasma species have the ability to cause direct cell injury, although exact mechanism of cell injury is not being understood. Mycoplasmosis causes cell injury by depriving nutrients, producing toxic substances and alteration in the host cell metabolites. Mycoplasma species produce enzymes like phospholipases, proteases and nucleases. These enzymes cause membrane damage to host cell and increase the chances of genetic alteration in host cell which may lead to auto immune disease (Bhandari and Asnani, 1989; Umar et al., 2017). Mycoplasma species produce hydrogen peroxide which play very important role in cell injury as well as damage to cell membrane and facilitates the entry of Mycoplasma during adherence process. As shown in figure 2 that hydrogen peroxide released by Mycoplasma causes oxidative stress to host cell and may also cause hemolysis. Nascent oxygen (O–2) is produced from hydrogen peroxide by catalase enzyme. This nascent oxygen (O–2) causes oxidative damage inside the host cell and responsible for major cell injury. To counter this oxidative damage antioxidant enzymes like glutathione (GSH) and superoxide dismutase (SOD) are produced by the host cell. In this way host cell directs its energy for producing these enzymes to encounter oxidative damage caused by Mycoplasma (Razin et al., 1998; Razin, 2006; Xu et al., 2015).
Figure 1 Pathogenesis of Mycoplasma gallisepticum
Figure 2 Sequence of events for oxidative damage in host cell caused by Mycoplasma (Razin, 2006).
Mycoplasma is transmitted vertically through eggs and horizontally through close contact, air borne droplets and contaminated dust particles (Papazisi et al., 2002; Umar et al., 2017). Increased population of poultry in an area due to rapid expansion increases the risk of transmission of Mycoplasma. It is one of the reasons why it is difficult to maintain Mycoplasma free flocks (Lysnyansky et al,. 2005).
The clinical signs in the birds infected with M. gallisepticum include open mouth breathing, rales and respiratory sounds. Nasal discharge, coughing and sometimes conjunctivitis are also seen in the infected birds (Saif et al., 2003). Lacrimation and depression is also observed in infected birds (Forrester et al., 2011). Sometimes fatal encephalopathy, arthritis and salpangitis are seen in M. gallisepticum infected birds (Much et al., 2002).In the case of infected broiler breeder and commercial layer, sharp decrease in eggs production takes place. There is marked increase in embryo mortality in eggs of infected birds (Ley, 2003). The clinical signs in M. synoviae infection are somewhat similar to M. gallisepticum. M. synoviae causes subclinical upper respiratory tract infection and synovitis in chickens and turkeys is one of very important clinical findings. M. synoviae causes air sacculitis more frequently than infectious synovitis (Benčina et al., 2001: Khalifa et al., 2013). M. synoviae disseminates very quickly after it is introduced at farm because of its lateral spread both by direct contact and between cages is very quick (Kleven, 2008).
The major pathological finding in M. gallisepticum infection is the air sacculitis while in some birds upper respiratory tract infection may also be present (Hong et al., 2005). Pathogenic mechanisms of Mycoplasmas are controlled by number of pathogenic factors which include ability of pathogen to attach to host cell, type of cell injury and ability to resist host immune response.
IMMUNITY IN MYCOPLASMOSIS
Immune system of host fails to deal effectively with Mycoplasma specie because of chronic nature of the infection. Mycoplasmas evade the host immune response by antigenic variation of surface proteins. M. gallisepticum and M. synoviae are transmitted either by vertical method or by direct contact between sick and susceptible birds (Marois et al., 2001). Ability of Mycoplasma to survive within the host cell allows the pathogen to resist immune response of host and anti-microbial therapy (Winner et al., 2000). Age of bird, size of flock and locality of poultry farm are the factors which affect the severity of the disease. Great economic losses occur due to Mycoplasmosis in broiler, breeder and layer birds in terms of condemnation of carcass, reduced eggs production, feed efficiency, hatchability losses and increased cost for the treatment of the infection (Hassan et al., 2012). For treatment and control of Mycoplasmosis early and timely diagnosis is necessary. Isolation of M. gallisepticum and M. synoviae is not reliable due to least tolerance in adverse environment and the fastidious nature of the organism. In vitro cultivation of Mycoplasma is very difficult, expensive and time consuming. It requires three to four weeks to grow and even after that there can be mixed growth or no growth. In the cultures Mycoplasmas are over grown by the fast growing or apathogenic species of Mycoplasma. Serological tests and molecular techniques are reliable methods for diagnosis of the disease. Serological tests like serum plate agglutination (SPA) test, ELISA and direct haemagglutination inhibition (HI) tests are used. SPA test is a quick tool for flock screening although it may give false positive results because of cross reactivity of M. gallisepticum and M. synoviae (Kleven, 2008). While conducting serology of M. gallisepticum and M. synoviae cross reactivity of antigens is common problem (Ehtisham-Ul-Haque et al., 2011). Polymerase chain reaction (PCR) essays are commonly used for rapid detection of the M. gallisepticum and M. synoviae (Ahmed et al., 2009). Most effective control measure to control Mycoplasmosis is to cull seropositive birds from flock, but this is expensive practice, hence impossible.
Since Mycoplasma lacks cell wall, so cell wall synthesis inhibiting antibodies like penicillin etc are ineffective against the pathogen. Antibiotics that inhibit metabolic processes of microorganisms like macrolides, tetracyclines, fluoroquinoles and others are effective against Mycoplasma (Ley, 2003).Tylosine and gentamycin are effective in higher doses. Tylosine may be toxic to embryos and reduce the hatchability (Nascimento et al., 2005). Tilmicosin has lowest minimum inhibitory concentration (MIC) followed by tylosine for the Mycoplasma species (Hassan et al., 2012).
DIAGNOSIS OF MYCOPLASMOSIS
For the diagnosis of Mycoplasmosis a number of methods including isolation and identification, serological methods and molecular techniques have been used. Cultivation on laboratory media is most reliable method for the confirmatory diagnosis of Mycoplasma (Ley, 2003; Umar et al., 2017).). Due to the limitations of diagnostic tests and the similarities in the disease caused by Mycoplasmas, specific diagnosis is very difficult. It is very important to characterize and identify the Mycoplasma species and strain variability. Brief review of various methods used for diagnosis of Mycoplasma is given below.
Isolation and identification
Direct isolation and identification of Mycoplasma is not part of routine procedure used for diagnosis of Mycoplasma (Zain and Bradbury, 1996). The main reason for this is the fastidious and slow growing nature of the Mycoplasma species. Mycoplasmas require one to three weeks or even more time for their growth and identification (Umar et al., 2017). Another major problem in isolation of Mycoplasma is the growth of fast growing non-pathogenic Mycoplasma species and growth of other bacteria and fungi (García et al., 2005). Selective pressures on populations of Mycoplasmas that differ substantially in vivo and in vitro are also an important factor. Pathogenic properties of the strain may be lost during passages in the culture media. Mycoplasma has very small genome and size of genome is 996 kilo base pairs (Papazisi et al., 2003). Mycoplasma has little capacity of biosynthesis and is dependent on host cell for its requirements. Mycoplasma is dependent on host for cholesterol, amino acids, fatty acids, vitamins, nucleotides and other nutrients, that is why in vitro growth is very difficult. Mycoplasmas do not have regulatory genes involved in gene expression and cannot respond to the changing environmental conditions in vitro, it makes extremely fastidious to work with this organism (Razin et al., 1998). Mycoplasma once isolated from their host tends to die rapidly if not placed in suitable medium and environment (Zain and Bradbury, 1996). Handling of samples between collection and inoculation is a critical step for isolation of Mycoplasmas. Swabs dipped in Mycoplasma broth and placed at 4◦C are more viable than dry swabs. Due to these reasons isolation of Mycoplasma is laborious, time consuming, expensive and difficult task. Small size and lack of cell wall make morphological characterization of Mycoplasma very difficult and may not give true picture of in vivo presentation.
In order to overcome deficiencies of Mycoplasma, very complex media are used for in vitro cultivation. Generally growth media for Mycoplasma is composed of protein digest and meat infusion base. The media is also supplemented with horse or swine serum, yeast extract and glucose. To inhibit the growth of bacteria and fungi, bacterial inhibitors and antibiotics are also added (Hong et al., 2005). Thallium acetate and ampicillin are added in media as inhibitors of bacterial growth. Mycoplasmas are resistant to thallium acetate while thallium acetate prevents the growth of gram positive and gram negative bacteria (OIE, 2008). Ampicillin inhibits the growth of bacteria by inhibiting the call wall synthesis and cross linking of peptidoglycans. Ampicillin has no effect on Mycoplasma due to the absence of cell wall. Biochemicals, physiological and morphological characteristics of Mycoplasmas are affected by composition of media and cultural conditions. Lipid content, nutritional quality and osmotic strength of the medium are very important factors which affect the morphology of Mycoplasma. Mycoplasmas are rapidly mutating organisms and changes may occur in short periods during growth. This ability of diversification plays important role in the pathogenesis of disease caused by Mycoplasma. During the cultivation, pathogenic characteristics of Mycoplasma are lost due to rapidly occurring mutations (Wise et al., 1992). All these attributes of Mycoplasmas should be kept in mind while attempting isolation and cultivation of Mycoplasma. Typical egg fried, small and clear colonies of Mycoplasmas are observed on solid medium as shown in figure 3. Colonies are clear with central whitish raised parts (Kleven, 2008).
Figure 3 Egg fried colonies of Mycoplasma gallisepticum (Metwally et al., 2014).
Many serological tests are routinely used for sero monitoring of flocks against Mycoplasma. Serological tests are easy, give fast detection and require less expertise. These tests include SPA, ELISA, and HI (Kleven, 2008). Although serological tests are quick and fast yet they have their own disadvantages and limitations.
Serological tests are based on detecting antibodies in the serum produced in response to antigens and subsequent detection of these antibodies. To prevent the spread of infection, rapid diagnosis of Mycoplasma is necessary through serological screening. Serological methods do not detect the sub clinical or early infections. As antibodies are produced after minimum one week of infection and it requires three weeks post infection to conduct HI test (Kempf et al., 1993). Another major problem of serological tests is their sensitivity and specificity. Sensitivity and specificity of SPA test are almost same as HI test and ELISA. ELISA is not feasible for sero monitoring because it is more time consuming and costly (Higgins and Whithear, 1986). False positive results can give a very high prevalence by SPA test which are because of cross reactivity, use of inactivated vaccine, contaminated sera and age of the flock (Luciano et al., 2011). Major constraint in the use of SPA test for diagnosis is its low specificity (Pourbakhsh et al., 2010). SPA can be used for screening flocks but not for screening individual birds. For proper diagnosis and control, programs based on sero conversion may be inadequate and sero monitoring should be combined with culture and molecular techniques (Luciano et al., 2011).M. gallisepticum is shown to be cross reactive with closely related M. imitans that would also lead to aberrance in prevalence of specific Mycoplasma species (Bradbury et al., 1993). This is because both M. gallisepticum and M. imitans have many similarities including same antigenic and phenotypic properties and same terminal attachment structure (Abdul-Wahab et al., 1996). Flocks showing no clinical signs may be serologically positive if the flock recovered from the infection at younger age (Ley, 2003).
Due to high sensitivity and increasing specificity of the polymerase chain reaction (PCR), it has become valuable tool in the diagnosis of Mycoplasma species. Since PCR is dependent on the target, its specificity is highly flexible. PCR can be species specific or strain specific by targeting unique gene in a particular specie or conserved region in a specific strain. For four main avian pathogenic Mycoplasma species PCR essays are developed in 1990s (Raviv and Kleven, 2009). Earlier PCR methods targeted 16S ribosomal DNA (16SrDNA) region but the recent PCR essays target the species specific regions and the surface proteins (Liu et al., 2001; García et al., 2005; Raviv et al., 2007). PCR essays that target 16S rDNA region are less specific and may cross react with the other avian Mycoplasmas because 16S rDNA region is highly conserved among phylogenetically related groups (García et al., 2005). Those PCR essays are less sensitive which target surface proteins because of high levels of intraspecific genetic polymorphism (Raviv et al., 2007). For detection of M. gallisepticum many PCR methods are applied including commercial kits produced by IDEXX Laboratories, Genekam Biotechnology AG etc. PCR essays are developed to target various genes including 16S rRNA gene, pvpA, gap A, lipoprotein, mgc2 and 16S-23S intergenic spacer region (Domanska-Blicharzet al., 2008). PCR developed by targeting 16S rRNA gene has its own limitations and shortcomings. Although this region is highly conserved but 16S rRNA gene of M. gallisepticum and M. imitans is very much similar and both organisms are amplified (García et al., 2005). Keeping in mind the above mentioned limitations of PCR essays based on 16S rDNA region and 16S rRNA gene, we can say that PCR cannot be solely used to identify M.gallisepticum without possibility of false positive results.
Surface proteins on which PCR essays are based help the Mycoplasma cell to bind to the host cell membrane-receptors. These proteins which mediate the attachment are called cytoadhesins. After the firm attachment of Mycoplasma to host cell, pathogenesis and host cell alterations occur (Goh et al., 1998). One of the important cytoadhesins is encoded by mgc2 gene (Boguslavsky et al., 2000). For the detection of M. gallisepticum, mgc2-PCR is highly specific and sensitive (García et al,. 2005).
In M. gallisepticum, mgc2 gene is fairly conserved and is used for molecular detection of isolates. Essay based on mgc2 gene is able to differentiate between field strain and the vaccine strain (Lysnyansky et al., 2005). Other cytoadhesins are encoded by gapA gene (Goh et al., 1998), PvpA gene (Boguslavsky et al., 2000) and MGA 0319 gene (García et al., 2005). In a study 42.4% tracheal samples were found positive when Mycoplasma specific primers were used. The reason for the high prevalence by PCR is the detection of DNA from both viable and non viable Mycoplasma (Marois et al., 2001). As compared to culture isolation, PCR is fast, less expensive, effective and more reliable.
CONTROL OF MYCOPLASMOSIS
The three main components of control program for Mycoplasma include biosecurity, treatment and vaccination. Rapid expansion of poultry industry and high concentration of multi aged birds in the close proximity are the other main reasons for the high incidence of Mycoplasma. Due to these factors and poor biosecurity measures, it is difficult to maintain Mycoplasma free flock (Lysnyansky et al., 2005). Vertical transmission is one of the major reason for ineffective control of Mycoplasma (Papazisi et al., 2002). First step towards the control of Mycoplasma is the acquisition of fertile eggs and poultry birds which are free from Mycoplasma. This can be achieved by antibiotic treatment of fertile eggs and heating the eggs at 46°C for 12-14 hours (Umar et al., 2017).
In the areas where complete eradication is difficult, live vaccines are used as alternative control strategy. There are five commercially available live vaccines for the control of M. gallisepticum which include the F strain, K-strain, MS-H strain , Ts-11 and 6/85 strain (Liu et al. 2001; Ferguson-Noel and Williams, 2015). The MG-F strain was described as typical pathogenic and naturally occurring strain. A single dose of MG-F strain vaccine is needed to protect the birds against M. gallisepticum (Ley, 2003). The F strain is highly virulent and immunogenic, but is responsible for post vaccination clinical outbreaks. The Ts-11 and 6/85 strain originated from Australia and U.S.A respectively (Ferraz and Danelli, 2003). Ts-11 and 6/85 are live vaccines and contain poorly transmitted strains which makes them safer vaccine than MG-F strain vaccine. Ts-11 and 6/85 strain show post vaccination mild respiratory stress but induce lower immunity as compared to MG F strain vaccine (Peebles et al., 2015; Umar et al., 2017).
Inactivated oil-emulsion bacterins and recombinant vaccines are also used to protect the poultry birds from Mycoplasma. These vaccines are quite successful and have shown minimal resistance against local infections (Jacob et al., 2015). A recombinant fowl pox (rFP-MG) vaccine that possesses and express protective MG antigen is shown to be less effective than live vaccine, but there is no risk of introducing live pathogen (Jacob et al., 2014).
Control of Mycoplasmosis is generally based on the elimination of these organisms from poultry flocks. It is only possible in those flocks where prevalence is low like in grandparent flocks. In layers such approach is not feasible. Medication and vaccination are the parts of control strategy of mycoplasmosis. Poultry industry is expanding fast worldwide. Very close location of the poultry farms, rearing of mixed avian species in close milieus, mixed commercial poultry farming and presence of wild birds in close proximity have made the control of this disease very difficult and almost impossible to maintain Mycoplasma free flocks. Establishment of Mycoplasma free breeding flocks is required for the control of Mycoplasmosis as it is transmitted by vertical method. Before adding to the flock poultry birds should be tested. Breeding stock should be purchased from certified infection free sources. Poultry birds should be hatched and reared in a way to reduce the horizontal transmission by preventing the contact with infected flocks. The poultry equipments and premises should be disinfected and cleaned on regular basis. To eliminate the infection from flock, repeated testing and culling of carrier birds can be helpful. Compounds containing phenols and quaternary ammonium based compounds should be used for effective disinfection. Keeping in mind the economic importance and high incidence of the mycoplasmosis, there is dire need to design the prevalence study to define and quantify the load of avian mycoplasmosis in the region.
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Honey is a product that contains a blend of complex carbohydrates, mostly monosaccharides glucose and fructose. Others are present in lower amounts, according to the botanical origin. Moreover, other compounds such as organic acids, lactones, amino acids, mineral salts, vitamins, enzymes, pollen, wax and pigments are present (Fallico et al., 2004).
The enzymes are secreted by bees (invertase, glucose oxidase and amylase) or by plants (amylase, catalase and phosphatase) (Vorlova and Čelechovská, 2002). Honey contains small amounts of different enzymes, the most important of which are diastase (-amylase), invertase (-glucosydase), glucose oxidase, catalase and acid phosphatase (White, 1975). They are sensitive to heat and therefore are able to indicate overheating of the product and the degree of conservation (Ahmed et al., 2013). The activity of enzyme in honey also depends on age of the bees, stage of the colony, nectar ﬂow, environmental conditions and the beekeeping practices (Karabournioti and Zervalaki, 2001).
According to several authors the activity of invertase in honey is used mainly in Europe for the evaluation of monofloral honeys, as well as for the determination of the characteristics related to the geographical origin of the different types of honey (Oddo et al., 1999; 2004; Bartakova et al., 2007; Serrano et al., 2007).
Honey freshness is generally evaluated by determining the value of parameters that increase or decrease with overheating and/or ageing. The most commonly used are hydroxymethylfurfurale, diastase and invertase (Oddo et al., 1999). However, excessive heat treatment leads to the formation of 5-hydroxymethylfurfuraldehyde (Nozal et al., 2001). Hydroxymethylfurfural (HMF) is a cyclic aldehyde produced as a result of sugar degradation (Cervantes et al., 2000). HMF value is virtually absent or very low in fresh honey and is high in honey that has been heated, stored in non-adequate conditions and old honey (Nozal et al., 2001; Khalil et al., 2010). At room temperature, the action of normal honey acidity on reducing sugars can possibly produce HMF. It has a toxic effect and also induces reactive oxygen species (De Smet et al., 2015).
The Alimentarius Codex (2001) and International Honey Commission (Bogdanov et al., 1997), set the maximum concentration of HMF to 40 mg.kg-1for honey from non-tropical regions and high values of HMF (80 mg.kg-1) from countries or regions with tropical ambient temperatures. Extremely high values of HMF (>500 mg.kg-1) demonstrate adulteration with invert syrup (Coco et al., 1996).
Usually the heating process is used to reduce viscosity, and to prevent crystallization or fermentation (Singh et al., 1988). According to Bakier (2006), the effective liquification of honey requires heating for at least 10 min at 52–55 °C. Honey heating is carried out in two different ways: in air-ventilated chambers, at 45–50 °C for 4 – 7 days or by immersion of honey drums in hot water. Although, the second heating method is more efficient, the first is the most common (Belitz and Grosch, 1999). Algeria is a broad territory extends over an area of 2,381,741 km2 and is the second largest country in Africa (Haouam et al., 2016), in this country immersion of honey bottle in hot water for liquefaction is more used.
In this context, the aim of this study is to test the quality of honey and the efficiency of the traditional method of heating by analyzing the HMF content, the diastase activity and invertase activity in twenty honey samples from north of Algeria, treated by the traditional heating method (immersion the bottle of honey in hot water) and compare their levels by while five treatment periods.
MATERIAL AND METHODS
Twenty multifloral honey samples of Apis mellifera intermissa were produced in various regions from north of Algeria (Table 1) and (Figure 1) and were collected from beekeepers. All samples were collected in airtight plastic containers while the same year and then they have been stored in a refrigerator at 4 – 5 °C until analysis.
Table 1 Botanical and geographical origin of honey samples
|Samples||Honey type||Geographical origin||Region|
|H6||Oum- El- Bouaghi||Meskianna|
|H10||Limit Tebessa- Tunisie|
|H14||Oued El Aineb|
|H19||West Ben Mhidi|
|H20||Bounamousa- Ben Mhidi|
Figure 1 Distribution of honey samples from north of Algeria
Each honey sample was divided into six sub-samples of about 5g in glass bottles. One portion was immediately analyzed and five portions in closed bottles were undergo a thermal processing (conventional heating in a water bath- isothermal heating) without stirring, during five periods of treatment (2, 4, 6, 8 and 16 min). The conventional heating procedure was as follows: 2, 4, 6 and 16 min at 100 °C and cooled down in a room temperature. The temperature of the analysis was selected on the basis of the traditional heating (dilution the bottle of honey in the saucepan with water boiling). Heat-treated samples were subjected to Hydroxymethylfurfural (HMF), diastase activity and invertase activity analyses.
HMF (5-(hydroxymetyl-) furan-2-carbaldehyde) was determined by reverse phase HPLC Agilent 1200 (Ramsey, Minnesota, USA) equipped with UV detector, according to the harmonized by the European Honey Commission (Bogdanov et al., 1997). Five gram of honey sample was weighed into a 50 mL beaker and dissolved in 25 mL HPLC grade water. The solution was transferred into 50 mL volumetric ﬂask and ﬁlled to the mark with HPLC grade water. Then the solution was centrifuged and poured into sample vials for chromatographic separation. The HPLC condition was the following: mobile phase, 90% water and 10% acetonitrile; ﬂow rate 1ml/min; injection volume 20 µl. HMF content of the sample was calculated by comparing the corresponding peak areas of the sample and those of the standard solutions, taking into account the dilution factor. Results were expressed in mg.kg-1.
Diastase activity analysis
Diastase activity was measured by Phadebas, according to the harmonized Methods of the European Commission of Honey (Bodganov et al., 1997; Tosi et al., 2008), using spectrophotometric method. Diastase activity is the one unit corresponds to the enzyme activity of 1 g honey that can hydrolyse 0,01g of starch in 1 h at 400 C (Oddo et al., 1999). According to Oddo and Pulcini (1999) the number of diastases (ND) is calculated with the following equation: ND = – 4.37 x (Δ A620)2 + 3.38 x (Δ A620) + 0.03 and the results were expressed in Schade units.
Invertase activity analysis
Invertase was determined using the Siegenthler method, according to the harmonized by the European Honey Commission (Bogdanov et al., 1997). The enzyme activity is evaluated photometrically, by measuring the decomposition of the substrate p-nitrophenyl α-D glucopyranoside into the product p-nitrophenol (which has a maximum absorbance at 400 nm). The results were expressed as invertase number (IN). The IN indicates the amount of sucrose per gram hydrolysed in 1h by the enzymes contained in 100g of honey under test conditions (Oddo et al., 1999).
A one-way analysis of variance (ANOVA) was performed to examine the effects of heating at five period of treatment on HMF, diastase activity and Invertase activity with their initial values. F-test was used to estimate the statistically significant differences (P-value <0.05) among honey samples. The differences among the means were determined for significance at the 5% level using Tukey’s test. All the analyses were carried out at least in duplicate, and the results are expressed as mean values ± standard deviations (SDs). All data were analyzed using the Statistica 8.0 software for windows from Statsoft.
RESULTS AND DISCUSSION
The variation of HMF contents, diastase activity and invertase activity according to initial value and different period of treatment of honey are reported in Table 2. As well as the number of samples exceeding limit presented in Table 3.
Table 2 Variation of HMF, diastase activity and invertase activity during the period of treatment (n = 20).
|Time||HMF ( mg.kg-1)||Diastase activity (Schade units )||Invertase activity (IN)|
|p –value||rang||mean±sd||p –value||range||mean ± sd||p –value||range||mean ± sd|
|0 min||–||0.69 – 09.50||3.72 ± 2.45a||–||9.62 – 29.47||19.37 ± 5.35a||–||38.01 – 163.91||101.93 ± 37.23a|
|2 min||ns||0.15 – 10.08||4.69 ± 3.22b||ns||10.12 – 28.72||18.95 ± 5.01a||**||11.79 – 162.39||71.41 ± 39.04b|
|4 min||ns||0.58 – 11.23||4.18 ± 3.31b||ns||7.60 – 28.26||15.96 ± 4.74 a||***||0 – 115.15||23.54 ± 36.12c|
|6 min||ns||0.52 – 12.13||4.82 ± 3.46b||***||0.47 – 28.26||9.12 ± 8.17b||***||0 – 53.91||1.10 ± 18.63c,d|
|8 min||ns||0.18 – 12.59||4.60 ± 3.69b||***||0.00 – 25.47||5.62 ± 7.50b,c||***||0.0||0 ± 0d|
|16 min||**||0.55 – 23.85||8.20 ± 4.97b||***||0.11 – 15.45||2.16 ± 3.81c||***||0.0||0 ± 0d|
HMF – hydroxymethylfurfural, n – number of samples, ns –not signiﬁcant, sd – standard deviation, **signiﬁcant at p < 0.01, ***signiﬁcant at p < 0.001, With different letters are signiﬁcantly different
Table 3 Variation of HMF, diastase activity and invertase activity during the period of treatment (n = 20).
|Time||HMF (mg.kg-1)||Diastase activity (Schade units )||Invertase activity (IN)|
|International standard limit||Samples exceeding limit||Samples conforming limits (%)||International standard limit||Samples below limit||P Samples conforming limits (%)||International standard limit||Samples below limit||Samples conforming limits (%)|
HMF content in untreated honey
Hydroxymethylfurfural (HMF) is absent or present in trace amounts in fresh honeys, since it is a parameter of honey freshness (Sodre et al., 2011). HMF level in fresh honey samples (at time 0 min) varied between 0.69 and 9.50 mg.kg-1, these results were indicated HMF contents were below 10 mg.kg-1, similar results were observed by Getu and Birhan (2014) for Ethiopian honey (HMF ranged between 0.5 and 3.2 mg.kg-1). According to Al-Farsi et al. (2018) high quality honey should present low HMF contents. All fresh honey samples studied contained HMF within the recommended food authority limit (40 mg.kg-1). According to White (1994) proposed the HMF level as the only reliable heating/storage index in honey.
Effect of heating on HMF contents
HMF, this product of fructose decomposition, increases with storage and prolonged heating of honey (Al-Farsi et al., 2018). During heating from initial value (0 min) to 8 min all honey samples showed not formation of HMF their values maximum varied from 9.50 to 12.59 mg.kg-1 (Figure 2). These results are in agreement with those of Fallico et al. (2004), at high temperature (100 °C) no difference, related to HMF formation, can be measured among honeys of different origin. At time 16 min all samples had a slight increase to the maximum 23.85 mg.kg-1. In this time there is a remarkable HMF formation, moreover a signiﬁcant difference (p<0.001) was also observed between honeys, but still below the international standard limit (40 mg.kg-1). The same authors Fallico et al. (2004) showed that the HMF levels in honey samples, heated at 100 °C, were signiﬁcant correlated only with time of heating. Singh and Bath (1997) reported that with increasing heating time of 0–30 min, an increased in intensity of HMF formation for three monofloral honeys from India at 65 °C.
Figure 2 Variation of HMF during heating
Diastase activity in untreated honey
The diastase activity is a very interesting enzyme to know the freshness of honey (Oddo et al., 1990). The diastase content in our samples ranged between 9.62 and 29.47 Schade units. Similar values for diastase reported in Argentina honey which averaged 19.7 Schade units. (Cantarelli et al., 2008). According to the European Codex Honey Standards, a well-processed and ready to be consumed honey must contain diastase number ID ≥ 8 Schade units. We noted that 100% of fresh honey samples studied contained ID ≥ 8 Schade units. The enzyme activity in honey from the same ﬂoral origin can possibly vary, due to the plant’s nectar secretion quality and quantity, which was influenced by the contribution of the environment and the presence of different geographical races of bees, which is mainly governed by biotic and abiotic factors (Adgaba et al., 2017; Belay et al., 2017).
Effect of heating on diastase activity
During heating from initial value (0 min) to time 4 min (Figure 3), the diastase activity of all honey samples showed some differences were seen. The mean values decrease from 19.37 ± 5.35 Schade units to 15.96 ± 4.74 Schade units, but is not signiﬁcant because only one sample reports a value below the limit. At time 6 min all samples had a slight decrease to 9.12 ± 8.17 Schade units, in addition a very highly significant difference (p<0.001) was observed, therefore 11 samples of multiﬂora were unconﬁrmed to the European Codex Honey Standards limit (diastase number “ID” ≥ 8 Schade units). According to Bogdanov et al. (1999) and White (1994) the honeys are also used as a quality parameter even though some have a lower level of enzymes intrinsically. Honeys with lower level of enzyme, needs to consist essentially a maximum of 15 mg.kg-1of HMF Zappala et al. (2005). At time 8 and 16 min the mean values of the diastase activity was also decrease from 5.62 ± 7.50 Schade units to 2.16 ± 3.81 Schade units respectively therefore lower of 8 Schade units. In addition a very highly significant difference (p<0.001) was also been noticed in these times (8 and 16 min), therefore 13 samples and 18 samples of multiﬂoral respectively were below to limit, with the exception of two samples that have values above the limits at 16 min.
Figure 3 Variation of diastase activity during heating
Invertase activity in untreated honey
In particular, invertase is the enzyme responsible for converting sucrose, maltose, mélézitose, raffinose, cellobiose and tréhalose to fructose and glucose which are the main sugars in honey (White, 1975; Parvanov, 2012). The mean value of invertase activity in fresh honey simples is 101.93 ± 37.23 IN with a minimum value observed is 38.01 IN. The invertase activity is variable in the different types of honey, the minimum values of its activity have been proposed by the International Honey Commission (IHC): ≥50 IN for normal honeys, ≥20 IN for honeys with a low enzymatic activity and ≥10 IN for monofloral honeys (from Arbutus sp., Robinia sp. and Erica sp. (Bogdanov et al., 1997). Therefore we noted that 100% of fresh honey samples studied contained the value of invertase activity IN ≥ 20 IN.
Effect of heating on invertase activity
During heating the invertase activity of all honey samples showed some differences were seen (Figure 4), the mean values decrease from 101.93 ± 37.23 IN to 71.41 ± 39.04 IN at time 2 min and to 23.54 ± 36.12 IN at time 4 min, according to Karabournioti and Zervalaki (2001), the decrease of invertase is very fast and starts from the temperature of 35oC, is the temperature that in many countries can be obtained during the summer. In addition the results of one-way analysis of variance (ANOVA) showed a highly significant difference (p<0.01) at 2 min and a very highly significant difference (p<0.001) at 4 min, but still above the International Honey Commission limit (invertase activity ≥ 10 IN). At time 6 to 16 min a very highly significant difference (p<0.001) was also observed and the mean values of the invertase activity are respectively 1.10 ± 18.63 IN, 0 ± 0 IN and 0 ± 0 IN however lower of 10 IN. Therefore the number of samples below limit was respectively decreased from 14 samples to 20 samples. These results showed that the values of the invertase activity are inversely proportional to the heating period. Invertase is considered the best indicator of freshness that diastase because it is more sensitive to heat (Oddo et al., 1999). According to the European Honey Commission the invertase activity could serve as a criterion for determining whether honey is stored long-term or heated at high temperatures (Bogdanov et al., 1997).
Figure 4 Variation of invertase activity during heating
We concluded that hydroxymethylfurfural (HMF), diastase activity and invertase activity concentrations in fresh multifloral honey samples from north of Algeria were within the internationally recommended range. The traditional heating (Conventional heating in the water bath at 100 °C) effect on the three parameters studied during five periods of treatment (0, 2, 4, 6, 8 and 16 min) were found to be signiﬁcantly different in HMF at 16 min, diastase activity at 6 min and invertase activity at 4 min, but still within the international standard limit. The results show that invertase is more heat-sensitive and heating time than diastase and HMF. It is obvious that heating is not the only factor influencing HMF formation in honey and the destruction of enzymes, but the most important is the heating time.
Acknowledgments: This work was funded by grants from the “ Direction Générale de la Recherche Scientiﬁ que et du Développement Technologique” (DGRSDT). Thanks are given to all the beekeepers that have generously provided the honey samples to carry out this study.
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Medicinal plants have been used extensively as alternative agents for the treatment of various infections and diseases for thousands of years (Jamshidi-Kia et al., 2018). Traditional herbs have received much attention as a source of novel drugs since they are considered safe for human use (Yixuan et al., 2007). Plant-based medicines are widely used for primary health care in many developing countries (Abu-Irmaileh et al., 2003). As a result, it is found that about 60–80% of the world population relies on traditional treatment. The contribution of medicinal plants and natural products as drugs or as sources of using drugs in medicine is unquestionable (WHO, 2002). Numerous secondary metabolites such as alkaloids, tannins, and flavonoids extracted from different medicinal plants have shown antioxidant (Rauf et al., 2013), anticancer (Yizhong et al., 2004; Costa-Lotufo et al., 2005), anti-inflammatory (Latifi et al., 2015), antibacterial potential (Mahboubi et al., 2012).
The Slovak Republic is situated in Central Europe. It is placed in the climatically welcoming mild zone of Northern Hemisphere. Since time traditional medicinal plants have proved their important part in the content of therapeutic and various preparations used in popular human health medicine in Slovak. In past centuries about 600 to 800 species were used for curative reasons (Salamon, 2014). Nowadays about 200 medicinal plants are used in the official therapy and in popular doctoring, respectively (Salamon, 2015). It is approved by the years of positive experience of people carrying explicit contact with nature. Over the years this experience has been validated in practice, improved and categorized, folk herbalist knowledge got formed and passed through generations. This study focused on 7 plants form 5 plant family as useful in traditionally managing various human diseases. We have summarized the data obtained from published literature and the uses of chosen studied plants in folk medicine in Table 1.
Table 1 Short summary of traditional medicinal uses of studied plants
|Common name||Botanical name||Family||Use in traditional medicine||References|
|Yellow toadflax||Linaria vulgaris L.||Plantaginaceae||To treat coughs and asthma. Possesses uterine stimulatory activity, expectorant, antiseptic, antiperiodic and anthelminthic properties||(Hua et al., 2002, Bruhn, 1982)|
|Yellow bedstraw||Galium verum L.||Rubiaceae||Used as preventive and/or a concomitant therapeutic approach in head and neck cancer||(Schmidt et al., 2013)|
|Perforate St John’s-wort||Hypericum perforatum L.||Hypericaceae||The popular treatment for anxiety, depression, cuts, and burns.||(Gaster et al., 2000)|
|Black locust||Robinia pseudoacacia L.||Fabaceae||Demonstrates laxative, antispasmodic, diuretic effect||(Subramoniam, 2016)
|Hungarian thyme||Thymus pannonicus L.||Lamiaceae
|Popular against coughs and other respiratory complaints, as well as some cases of gastrointestinal disorders||(Maksimović et al., 2008)|
|Lemon balm||Melissa officinalis L.||Lamiaceae||Menstrual problems, hypertension, migraines, vertigo and fever, depression and melancholy, bronchitis and asthma, eczema and gout, epilepsy, paralysis, Bell’s palsy, and arthritis. Advanced researches showed its neuroprotective, anxiolytic, antispasmodic, antihyperlipidemic and hepatoprotective effects.||(Lopez et al., 2009, Kennedy et al., 2004, Cases et al., 2011)|
|Sage||Salvia officinalis L.||Lamiacea||Used in the treatment of digestive and circulation disturbances, bronchitis, cough, asthma, angina, mouth and throat inflammations, depression, excessive sweating, skin diseases, and many other diseases||(Rami et al., 2011, Walch et al., 2011, Khan et al., 2011)|
Infectious diseases are a major cause of death and disability in humans as they prevail constant and rapid change. With microorganisms emerging new preventions and treatments evolve as well. It has been reported that almost 25% of the population experienced 2-3 episodes of infection every year (Shuman et al., 2018). Many foods primarily deteriorate because of microbes that give rise to the loss of quality and safety, on which people worldwide are concerning more because foodborne diseases are subject to outbreak due to pathogenic and spoilage microorganisms in foods (Rawat, 2015). Moreover, for a long time considered to be non-pathogenic to humans, some bacteria have been sporadically identified as responsible for various infectious diseases in humans. Numerous diseases, even those that were once easily healed, are becoming a huge problem. Noteworthy, the frequency of sporadically infections can also be underestimated. Hence, search for novel antimicrobial compounds or alternative therapy for these resistant infectious agents is inevitable.
Antibacterial compounds provided widely by herbal species may have crucial applications in the nearest future as native antimicrobial components not only in the food industry but also for the medicinal purpose. The antimicrobial activity mechanisms of important herbal species demonstrate the process of bacteria growth inhibition that may involve cytoplasmic membrane destabilization and further excessive permeabilization, extracellular microbial enzymes inhibition, etc. Antimicrobial mechanism of action of herbs may also be connected with antiadherence of bacteria to epithelial cells, being essential means for colonization and infection of many pathogens (Davidson et al., 2015).
In the present study, we used 10 strains of Gram-negative and Gram-positive bacteria, with known and/or recently found pathogenesis against humans and animals. Data summarized in Table 2.
Table 2 Studied bacteria strains and their reported pathogenic effects
|Bacillus thuringiensis||+||Bacillaceae||Nongastrointestinal infections in mammals||(Celandroni et al., 2014)|
|Micrococcus luteus||+||Micrococcaceae||Septic arthritis, meningitis, and prosthetic valve endocarditis||(Miltiadous et al., 2011)|
|Staphyloccocus epidermidis||+||Staphylococcaceae||Devastating effects on certain organs such as kidney, liver, intestine, stomach, and spleen which, depending on their severity, could be fatal||(Akinkunmi et al., 2014)|
|Staphyloccocus aureus||+||Staphylococcaceae||Bacteremia and infective endocarditis as well as osteoarticular, skin and soft tissue, pleuropulmonary, and device-related infections.||(Tong et al., 2015)|
|Citrobacter koseri||–||Enterobacteriaceae.||Neonatal meningitis and brain abscess with high mortality rates||(Lin et al., 2011)|
|Escherichia coli||–||Enterobacteriaceae||Urinary tract infections, meningitis, pneumonia, septicemia, and other types of infections||(Russo and Johnson, 2003, Smith et al., 2007, Fratamico, 2016)|
|Pseudomonas proteolytica||–||Pseudomonadaceae||Not found in the literature||(Chauhan et al., 2015)|
|Hafnia alvei||–||Enterobacteriaceae||Gastroenteritis, meningitis, bacteremia, pneumonia, nosocomial wound infections, endophthalmitis, and a buttock abscess||(Günthard and Pennekamp, 1996)|
|Salmonella enterica||–||Enterobacteriaceae||Gastroenteritis, bacteremia, enteric fever, and an asymptomatic carrier state||(Ryan and Ray, 2004)|
|Yersinia enterocolitica||–||Enterobacteriaceae||Diarrhea in the inoculated animals followed by lethality in guinea pigs and mice, but negative for autoagglutination test, calcium dependency, conjunctivitis, and positive for heat-stable enterotoxin production||(Igumbor et al., 1993)|
The aims of the present study were (1) to evaluate total polyphenol, phenolic acid and flavonoid content in ethanolic extract of selected medicinal herbs (2) to assess the antioxidant activity of these samples; (3) to detect the antibacterial activities of samples against gram-positive and gram-negative bacteria.
MATERIAL AND METHODS
Chemicals and Reagents
The HPLC phenolic standards were apigenin, daidzein, kaempferol, resveratrol, rutin, quercetin, vitexin, neochlorogenic acid, chlorogenic acid, trans-p-caffeic acid, trans-p-coumaric acid, trans-p-sinapic acid, trans-p-ferulic acid, rosmarinic acid. All other chemicals and reagents were of analytical grade and were purchased from Reachem (Slovakia) and Sigma Aldrich (Germany and/or Switzerland). Cynaroside as a standard was kindly provided by Prof. Peter Rapta, DSc (Institute of Physical Chemistry and Chemical Physics, Bratislava, The Slovak Republic).
Mature plant samples were harvested in May 2016 from the spontaneous flora in Slovak Republic (locality Zobor, DMS N 48° 19′ 46.676”E 18° 6′ 2.003”) and identified. Aerial parts of the collected plants were separated from undesirable materials and dried in open air under shade for 2 weeks. The dried plants were powdered with a mechanical grinder to obtain a coarse powder, stored in an airtight container and kept in a cool, dark, and dry place until analysis commenced.
Preparation of extracts
Content of selected polyphenolic compounds (apigenin, cynaroside, daidzein, kaempferol, resveratrol, rutin, quercetin, vitexin, neochlorogenic acid, chlorogenic acid, trans-p-caffeic acid, trans-p-coumaric acid, trans-p-sinapic acid, trans-p-ferulic acid, rosmarinic acid) was performed by HPLC method (according to Novakova et al. 2010), as described below.
Preparation of calibration solutions: the standard stock solution was prepared by dissolving 0.5 mg each of them with methanol in 10 ml volumetric glass flasks. All standard solutions were kept at 6°C in the dark for a maximum of 3 h. prior to injection, the solutions were filtered through syringe filter Q-Max (0.22 µm; Frisenette ApS, Knebel, Denmark).
Sample preparation: Dried plants (2.5 g) after homogenization in a mortar were extracted with 25 ml of 80% ethanol (v/v) at laboratory temperature for 8 h (Unimax 2010, Heidolph Instrument, Schwabach, Germany). The extract was filtered through Munktell No 390 paper (Munktell & Filtrac, Barenstein, Germany) and stored in closed 20 ml vial tubes. Prior to injection, the extracts were filtered through syringe filter Q-Max (0.22µm; Frisenette ApS, Knebel, Denmark).
HPLC analyses: The phenolic compounds were analyzed using an Agilent 1260 Infinity high-performance liquid chromatography (Agilent Technologies, Waldbronn, Germany). Separation was achieved on a Purosphere reverse-phase C18 column (4 mm×250mm×5µm) Merck KGaA, Darmstadt, Germany). The solvents were: (A) acetic acid in methanol (50/1000ml), (B) acetic acid in HPLC grade water (50/1000ml). the following gradient program was employed: 0-5 min isocratic elution (20% A and 80 % B), 5-11 min linear gradient elution (60% A and 40% B), and 80% A and 20 % B 11-20 min. the initial flow rate was 1 ml/min. column oven temperature was set up to 30°C and the samples were kept at 4°C in the sample manner. The PDA detection was conducted at 330nm for quantitative purposes with data acquisition rate of 5 Hz. Detection was carried out in a diode array detector (DAD), within 210-400 nm as the preferred wavelengths.
Radical scavenging activity
Radical scavenging activity of extracts was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sanchéz-Moreno et al., 1998). The sample (0.4 ml) was mixed with 3.6 ml of DPPH solution (0.025 g DPPH in 100 ml methanol). An absorbance of the reaction mixture was determined using the spectrophotometer Jenway (6405 UV/Vis, England) at 515 nm. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid) (10-100 mg/L; R2=0.989) was used as the standard and the results were expressed in mg/g Trolox equivalents.
Reducing power of extracts was determined by the phosphomolybdenum method of Prieto et al. (1999) with slight modifications. The mixture of sample (1 ml), monopotassium phosphate (2.8 ml, 0.1 M), sulfuric acid (6 ml, 1 M), ammonium heptamolybdate (0.4 ml, 0.1 M) and distilled water (0.8 ml) was incubated at 90°C for 120 min, then rapidly cooled and detected by monitoring absorbance at 700 nm using the spectrophotometer Jenway (6405 UV/Vis, England). Trolox (10-1000 mg/L; R2=0.998) was used as the standard and the results were expressed in mg/g Trolox equivalents.
Total polyphenol content
Total polyphenol content of extracts was measured by the method of Singleton and Rossi (1965) using Folin-Ciocalteu reagent. 0.1 ml of each sample was mixed with 0.1 ml of the Folin-Ciocalteu reagent, 1 ml of 20% (w/v) sodium carbonate, and 8.8 ml of distilled water. After 30 min. in darkness the absorbance at 700 nm was measured using the spectrophotometer Jenway (6405 UV/Vis, England). Gallic acid (25-300 mg/L; R2=0.998) was used as the standard and the results were expressed in mg/g gallic acid equivalents (mg GAE/g).
Total flavonoid content
Total flavonoids were determined using the modified method of Willett (2002). 0.5 ml of sample was mixed with 0.1 ml of 10% (w/v) ethanolic solution of aluminium chloride, 0.1 ml of 1 M potassium acetate and 4.3 ml of distilled water. After 30 min. in darkness the absorbance at 415 nm was measured using the spectrophotometer Jenway (6405 UV/Vis, England). Quercetin (0.520 mg/L; R2=0.989) was used as the standard and the results were expressed in μg/g quercetin equivalents (mg QE/g).
Total phenolic acid content
Total phenolic acids content was determined using the method of Farmakopea Polska (1999). A 0.5 mL of sample extract was mixed with 0.5 mL of 0.5 M hydrochloric acid, 0.5 mL Arnova reagent (10% NaNO2 + 10% Na2MoO4), 0.5 mL of 1 M sodium hydroxide (w/v) and 0.5 mL of water. Absorbance at 490 nm was measured using the spectrophotometer Jenway (6405 UV/Vis, England). Caffeic acid (1-200 mg.L-1, R2=0.9996) was used as a standard and the results were expressed in mg/g caffeic acid equivalents (mg CAE/g).
Ten strains of microorganisms were tested in this study, Gram-negative bacteria: Escherichia coli CCM 3988, Salmonella enterica subsp. enterica CCM 3807, Yersinia enterocolitica CCM 5671, Citrobacter koseri CCM 2535, Pseudomonas proteolytica CCM 7690, Hafnia alvei CCM 2636 and Gram-positive bacteria: Bacillus thuringiensis CCM 19T, Stapylococcus aureus subsp. aureus CCM 2461, Micrococcus luteus CCM 732, Staphylococcus epidermidis CCM 4684. All tested strains were collected from the Czech Collection of microorganisms. The bacterial suspensions were cultured in the nutrient broth (Imuna, Slovakia) at 37°C.
Disc diffusion method
Antimicrobial activity of each plant extract was determined by a disc diffusion method. Briefly, 100 μl of the test bacteria were grown in 10 ml of fresh media until they reached a count of approximately 10E5 colony-forming units (cfu)/ml. Then 100 μl of the microbial suspension was spread onto Mueller Hinton agar plates. The extracts were tested using 6 mm sterilized filter paper discs. The diameters of the inhibition zones were measured in millimetres. All measurements were to the closest whole millimetre. Each antimicrobial assay was performed in at least triplicate. Filter discs impregnated with 10 μl of distilled water were used as a negative control.
Microbroth dilution method
MICs were determined by the microbroth dilution method according to the Clinical and Laboratory Standards Institute recommendation (CLSI, 2015) in Mueller Hinton broth (Biolife, Italy). Briefly, the DMSO plant extracts solutions were prepared as serial two-fold dilutions obtaining a final concentration ranging between 0.5-512 μg/ml. After that, each well was inoculated with the microbial suspension at the final density of 0.5 McFarland. After 24 h of incubation at 37°C, the inhibition of microbial growth was evaluated by measuring the well absorbance at 450 nm in an absorbance microplate reader Biotek EL808 with a shaker (Biotek Instruments, USA). The 96 microwell plates were measured before and after an experiment. Differences between both measurements were evaluated as growth. Measurement error was established for 0.05 values of absorbance. Wells without plant extracts were used as negative controls of growth. Pure DMSO was used as negative control. This experiment was done in eight-replicates for a higher accuracy of the MICs of used medical plant extracts.
All experimental measurements were carried out in triplicate and are expressed as an average of three analyses ± standard deviation. The correlations coefficient (R2) between the parameters established by regression analysis.
RESULTS AND DISCUSSION
Total polyphenolic, flavonoids and phenolic acids content
Total polyphenolic content, total flavonoid content and total phenolic acids content of the seven extracts considered in this study are presented in Table 3. Total polyphenolic content under the applied conditions varied from 60,94±6.68 (L.vulgaris) to 10.05±0.17 (R.pseudoacacia ) mg GAE per g of sample. According to the Table 3, the polyphenolic content of 3 species from Lamiaceae family (M.officinalis, S.officinalis, and T.pannonicus) were quite similar (20.90 ± 1.06, 20.01 ± 0.71 and 23.98 ± 1.37 mg GAE per g of sample respectively).
The flavonoid content of studied extracts varied from 83.72 ± 1.29 (L.vulgaris) to 11.56 ± 0.15 (R.pseudoacacia) mg QE per g of sample. The flavonoid contents of species from Lamiaceae family varied lightly but all were less than 20 mg QE per g of sample – M.officinalis (11.56±0.15), S.officinalis (14,35±0.49) and T.pannonicus. (19,35±1.22) mg QE per g of sample.
The total phenolic acids content of the studied ethanolic extracts varied within the samples, from 2.50 ± 0.74 (R.pseudoacacia) to 52.25 ± 2.61 (L.vulgaris) mg CAE per gram of sample. Lamiaceae family species namely lemon balm, sage and thyme had the values (24.24±2.80, 18.52±7,73 and 16.74±7.56 mg CAE per gram of sample) respectively.
In general, L.vulgaris and H.perforatum had the highest content of polyphenols, flavonoids, and phenolic acids while R.pseudoacacia had the lowest.
Table 3 Total polyphenolic, flavonoids and phenolic acids content of studied samples
|Ethanolic extracts||Total polyphenolic content (mg GAE/g)||Total flavonoid content
|Total phenolic acids content
|Linaria vulgaris L.||60.94±6.68||83.72±1.29||52.25±2.61|
|Galium verum L.||37.53±1.90||48.83±0.81||29.41±4.84|
|Thymus pannonicus L.||23.98±1.37||19.35±1.22||16.74±7.56|
|Hypericum perforatum L.||50.79±2.02||72.40±2.45||46.06±7.26|
|Robinia pseudoacacia L.||10.05±0.17||15.27±0.97||2.50±0.74|
|Melissa officinalis L.||20.90±1.06||11.56±0.15||24.24±2.80|
|Salvia officinalis L.||20.01±0.71||14.35±0.49||18.52±7.73|
GAE – gallic acid equivalent; QE – quercetin equivalent; CAE –caffeic acid equivalent; mean ± standard deviation
Antiradical and antioxidant activity
DPPH is a widely used free radical for simple and fast estimation of the antiradical capacity due to its stability, reliability and the simplicity of the assay (Ali, 2013). DPPH radical scavenging capacity of ethanolic extracts is presented in Table 4.
The total antioxidant assay based on the reduction of phosphate-molybdenum (VI) to phosphate-molybdenum (V) gives the information on the presence of antioxidant components in the sample predicting the antioxidant activity of crude extracts on the total basis. The total antioxidant activity of ethanolic extracts was evaluated, and the results were expressed as mg Trolox equivalents/g (Table 4).
Table 4 DPPH scavenging effect vs total antioxidant capacity of studied ethanolic extracts. Values are expressed as mean ± standard deviation (n=3), the correlation between total antioxidant capacity and total antiradical activity R2 = 0.23.
|Ethanolic extracts||Reducing power
|Radical scavenging activity (mg TEAC/g)|
|Linaria vulgaris L.||8.38±0.08||134.49±3.63|
|Galium verum L.||8.00±0.46||63.61±7.62|
|Thymus pannonicus L.||8.23±0.19||52.71±4.08|
|Hypericum perforatum L.||8.56±0.07||135.47±42.21|
|Robinia pseudoacacia L.||3.96±0.40||17.92±3.36|
|Melissa officinalis L.||8.99±0.04||51.43±4.46|
|Salvia officinalis L.||8.96±0.04||48.08±2.07|
TEAC – Trolox equivalent antioxidant capacity; mean ± standard deviation
The correlation between the antioxidant, antiradical activities and total polyphenolic, phenolic acids and flavonoid contents of the studied medicinal plants ethanolic extracts were calculated (Table 5).
Table 5 the correlation between antioxidant and antiradical capacity and phenolic, phenolic acids and flavonoid contents of studied ethanolic extracts
|Ethanolic extracts||Reducing power
|Radical scavenging activity (mg TEAC/g)|
|total phenolic content (mg GAE/g)||R2 = 0.92||R2 = 0.34|
|total flavonoid content (mg QE/g)||R2 = 0.86||R2 = 0.05|
|total phenolic acids content (mg CAE/g)||R2 = 0.93||R2 = 0,19|
GAE – gallic acid equivalent; QE – quercetin equivalent; CAE –caffeic acid equivalent; TEAC – Trolox equivalent antioxidant capacity
The total flavonoid, polyphenol and phenolic acids content correlated with the phosphomolybdenum assay (R2 = 0.86, 0.92 and 0.93, respectively). The results suggested that the phenolic compounds contributed significantly to the antioxidant capacity of the medicinal herbs. However, a direct correlation between radical scavenging activity and total flavonoid, polyphenolic, and phenolic acids contents failed to demonstrate linear regression.
Quantification of polyphenolic compounds by HPLC
Targeted metabolic profiling of studied ethanolic extracts using HPLC has resulted in the characterization of seven phenolic acids namely neochlorogenic acid, chlorogenic acid, trans-caffeic acid, trans-p-coumaric acid, trans-p-sinapic acid, trans-p-ferulic acid and rosmarinic acid and eight flavonoids namely apigenin, cynaroside, daidzein, kaempferol, rutin, quercetin and vitexin in various quantities (Table 6).
Of them, cynaroside, neochlorogenic and trans-p-coumaric acid were found in all the samples. Resveratrol was not observed in any extracts.
The persistent presence of cynaroside was observed in all the strains and the maximum content was 1434.14±2.40 in extracts of G.verum followed by 1385.55±1.57 in T.pannonicus and 1360.19±1.51 in R.pseudoacacia. \ L.vulgaris sample contained the least amount of cynaroside (55.36±0.49) in comparison to its content in other samples.
The content of neochlorogenic acid varied from minimum (113.14±8.07) in M.officinalis. to maximum (612.98±0.71) in R.pseudoacacia.
The minimum content of trans-p-coumaric acid was as low as 0.08±0.02 in T.pannonicus and 0.14±0.06 in L.vulgaris. More significant contribution of the phenolic acid was in Salvia officinalis Mill. (285.62±4.58).
Rutin was found in all the extracts except M.officinalis. The content was as high as 491.93±1.65 (in G.verum) and 220.06±17.13 (in L.vulgaris), an as low as 27.06±3.83 in T.pannonicus and 23.63±0.57 in R.pseudoacacia.
S.officinalis and M.officinalis contained the highest content of rosmarinic acid – 2906.73±150 and 6914.1±779 correspondingly.
The maximum content of chlorogenic acid (2886.92±11.82) was found in G.verum, trans-p-caffeic acid (472.0.5±62.5) in S.officinalis, trans-p-sinapic acid (110.91±0.09) in L.vulgaris, trans-p-ferulic acid (86.34±0.05) in H.perforatum.
Among other polyphenols, the presence of quercetin was detected in various quantities in 5 strains, apigenin, daidzein and vitexin in 3 strains, and kaempferol in 2 strains.
Table 6 The content of standard samples in mg per kg of the total dry extracts or the dry plant material. Values are expressed as mean ± standard deviation (n=3)
|Extract||Linaria vulgaris L.||Galium verum L.||Thymus pannonicus L.||Hypericum perforatum L.||Robinia pseudoacacia L.||Salvia
The serial microdilution results were analyzed using the Analysis of Variance (ANOVA). Single factor statistical tool indicated that there is a significant difference in the sensitivity of the tested microorganisms to the various extracts. The MIC50 ranged from 1.33 to 204.80, MIC90 from 2.92 µg/mL to 304.16 µg/mL (Table 7). The inhibition zone was as high as 12 mm (by G.verum against Yersinia enterocolitica CCM 5671). In general, there was not observed remarkable correlation between MICs and the inhibition zone parameters. Gram-positive bacteria seemed to be more resistant to all the extracts that gram-negative strains – MIC50 was not lower than 25.58 µg/mL and MIC90 was not lower than 27.20 µg/mL.
Among them Yersinia enterocolitica CCM 5671 was the most succeptible, it was inhibited by G.verum with MIC50 (1.33 µg/mL) and MIC90 (2.92 µg/mL) and the highest inhibitory zone (12.80mm). S.officinalis had a bit higher MICs (5.44 µg/mL and 8.59 µg/mL correspondingly), followed by T.pannonicus and H.perforatum (MIC50 = 12.80 µg/mL, MIC90 = 13.64 µg/mL).
Ps.proteolytica was also effectively inhibited by G.verum, T.pannonicus and H.perforatum (MIC50 = 0.80 µg/mL, 12.80 µg/mL,12.80 µg/mL and MIC90 = 0.86 µg/mL, 13.64 µg/mL, 13.64 µg/mL) recpectively. Among Gram –negative bacteria H.alvei was the most succeptible to S.officinalis (MIC50 = 10.09 µg/mL, MIC90 = 28.00 µg/mL).
Gram-positive species were less affected by studied extracts. Stapylococcus aureus subsp. aureus CCM 2461 showed succeptibility to H.perforatum (MIC50 = 12.80 µg/mL, MIC90 = 13.64 µg/mL). In general, the microorganisms of the species Ps.proteolityca and Yersinia enterocolitica CCM 5671 were the most susceptible to most plant extracts tested in this work (with MICs <25 µg/mL).
Table 7 The antimicrobial activity of medical plant extracts expressed in MICs (µg/mL) and inhibition zones (mm)
|Microorganism||Extracts effect||Linaria vulgaris L.||Galium verum L.||Thymus pannonicus L.||Hypericum perforatum L.||Robinia pseudoacacia L.||Salvia
|Micrococcus luteus CCM 732||Zone (mm)||8,00
|Staphylococcus epidermidis CCM 4684||Zone||3,00
|Stapylococcus aureus subsp. aureus
|Citrobacter koseri CCM 2535||Zone||2,00
|Escherichia coli CCM 3988||Zone||1,00
|Pseudomonas proteolytica CCM 7690||Zone||1,67
|Hafnia alvei CCM 2636||Zone||2,33
|Salmonella enterica subsp. enterica
mean ±standard deviation
It has been reported that the wild plants are the most abundant and cheapest source of food for the human community and they are also used for the medicinal purpose as a source of antioxidant compounds. Antioxidants like phenolic acids, polyphenols, and flavonoids scavenge free radicals inhibiting the oxidative stress that leads to a number of human diseases (asthma, inflammatory arthropathies, diabetes, Parkinson’s and Alzheimer’s diseases, cancers as well as atherosclerosis). Due to Armatu et al. (2010) low content of polyphenols proves a weak or no antioxidant activity and vice versa. Moreover there is no universal rule for expressing the antiradical and antioxidant capacity so they are reported either as IC50 (the antioxidant concentration required to reduce the DPPH absorbance by half), % loss or original absorbance or Trolox/ascorbic acid equivalent (e.g. mg TEAC/100 g FW, mg/g TE, etc). The latter is just a number for comparison, the former at least considers some concentration dependence. On one hand, this incompatibility makes it difficult to compare the results on the other hand however indirect comparison enables to determine the health benefits and public health relevance of polyphenols promising future for the powerful antioxidants and those who consume them.
DPPH and power reducing assays were used in our work to evaluate the antiradical and antioxidant activity of ethanolic plant extracts. In agreement with other authors the results varied depending on the test applied.
The antioxidant and antiradical activities of Linaria vulgaris were studied were poorly. Thus, Vrchovska et al. (2008) have investigated the ability of L. vulgaris lyophilized infusion to act as a scavenger of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, reactive oxygen species (superoxide radical, hydroxyl radical, hypochlorous acid) and nitric oxide and proposed the potent antioxidant activity present in the infusion to be ascribed by ﬂavonoid derivatives. Our results show that among other extracts L.vulgaris demonstrated comparatively high antioxidant and antiradical activity.
G.verum samples collected at the flowering period at various parts of Europe, and the extracts prepared in ethanol at different temperatures and different time of extraction exhibit very potent antioxidant activities. Our findings of the antiradical ability of G.verum’s ethanolic extract are in agreement with those reported by Lakič et al. (2010), Vlase et al. (2014), Al-Snafi (2018). In addition Lakič et al. (2010) observed quite high scavenging ability of G.verum ethanolic extract against hydroxyl radical and hydrogen peroxide, as well as potency to inhibit lipid peroxidation.
Different antioxidant assays were utilized to evaluate free radical scavenging activity and antioxidant activity of Hypericum perforatum L. The plant was reported to be an effective scavenger in quenching DPPH and superoxide radical;, metal-chelating capacity was proposed to attribute to its antioxidant mechanisms (Zou et al., 2004). Mašković et al. (2011) report that the ethanolic extract of H. perforatum (from central Serbia) possesses higher antioxidant activity in comparison to the H.perforatum acetonic extract, as well as to such standards as ascorbic acid and butylated hydroxytoluene (BHT). In our work, St John’s-wort ethanolic extract showed very high antiradical activity while the total antioxidant assay based on the reduction of Phosphate-Molybdenum (VI) to Phosphate-Molybdenum (V) showed a low presence of antioxidant components in the extract. In addition, Radulovic et al. (2007) showed that the antioxidant capacity of H. perforatum methanolic extract was highest in the case of the flowers (in comparison to leaves and stems) and also was the highest with the comparison to eight other local (South Serbia) Hypericum species.
According to literature Black Locust possesses a very good melliferous potential (class IV-VI): it represents an excellent source of nectar for honey production (Persano-Oddo et al., 2004). Furthermore, this plant’s wood is widely used in the wine industry as it was found to increase antioxidant activity of media and is a very effective means of enriching wines and other beverages in functional phytochemicals. The researches show that at higher concentrations (0.016 mg/mL), the values of antioxidant activity for ethyl acetate fractions of heartwood and bark of R. pseudoacacia from Iran were very close to those observed for very important physiological antioxidant – vitamin C while leaves had the lowest (Hosseinihashemi et al., 2016). On the other hand, Marinas et al. (2014) compared the 70% ethanol extracts obtained from Robinia pseudoacacia’s leaves, seeds and sheaths. The highest content of polyphenols was found in the leaf extract followed by seed extract. Sheath extract showed the lowest content of polyphenols. In accordance with polyphenolic content leaf extracts also showed the strongest antioxidant capacity. On the contrary, our work shows that ethanolic extract of flowers of R. pseudoacacia contains a very little amount of polyphenols, phenolic acids and flavonoids (10,05±0,17 mg QE/g) and shows the lowest antioxidant and antiradical capacity compared to other studied plants.
All Lamiaceae family plants possess high amounts of polyphenols, high antioxidant, and antiradical activities. The three studied species, namely S. officinalis, M.cofficinalis, and T.pannonicus, confirmed the good antioxidant and antiradical potential of the Lamiaceae species exhibiting almost similar records for radical scavenging (48.08±2.07, 51.43±4.46 and 52.71±4.08 mg/g Trolox equivalent, correspondingly) and antioxidant (8.96±0.04, 8.99±0.04 and 8.23±0.19 /g Trolox equivalent, correspondingly) capacities probably due to a similar quantity of polyphenols, flavonoids and phenolic acids,
Also, HPLC fingerprints of the extracts were analysed in order to determine phenolic compounds from plant material.
Cheriet et al. (2015) reviewed the data on different Linaria species including the data published as early as 1907 (Klobb, 1907), and the phytochemical content of Linaria species was revised very carefully.. In addition to a long list of flavonoids that have already been detected, we have found some other compounds not mentioned before, that are apigenin, cynaroside (luteolin-7-O-glucoside), daidzein and rutin. Quercetin was also found in agree with Pethes et al. (1974). Vitexin seemed to be not typical to Linaria species as it was not mentioned to be observed as Linaria species constituent and was also not detected in our experiments. Some other polyphenols reported in the bibliography were identiﬁed by reversed-phase HPLC analysis as protocatechuic acid, gallic acid, p-hydroxybenzoic acid, vanillic acid and salicylic acid, caffeic acid, p-coumaric acid, ferulic acid, homoprotocatechuic acid, O-hydroxyphenylacetic acid, gluco-syringic acid and p-methoxybenzoic acid (Cheriet et al., 2015). We were the first to detect also rosmarinic, sinapic and chlorogenic and neochlorogenic acids in L. vulgaris ethanolic extract. On the other hand, ferulic and caffeic acids were not detected in contrast to works by Sokolowska-Wozniak et al. (2003).
In recent years, the consumption of products derived from Hypericum perforatum L., the plant being one of the most popular of medicinal plants worldwide, has increased dramatically,. H.perforatum-derived products are available as phytopharmaceuticals, nutraceuticals, teas, tinctures, juices, and oily macerates (Gaedcke, 2003). The constituents of St John’s wort (Hypericum perforatum L.), compiled from several sources (Barnes et al., 2001, Ganzera et al., 2002, Rusalep et al., 2016), may let us consider hyperforin (a prenylated phloroglucinol) and hypericin (a naphthodianthrone) to be the major active constituents of the plant. Besides them, H.perforatum contains additional biologically active compounds such as rutin, quercetin, and chlorogenic acid (Hans, 1998). The chromatographic analysis of H.perforatum extracts also confirmed the presence of kaempferol, luteolin, quercitrin glycosides (hyperoside, quercitrin, and rutoside) (Stuart, 2014). In addition to the above-mentioned constutuents, we also observed cynaroside (luteolin-7-O-glycoside). Some amount of phenolic acids such as neochlorogenic and trans-p-ferulic acids were detected, rosmarinic, trans-p-sinapic, trans-p-coumaric and trans-p-caffeic acids were detected in minority.
HPLC analysis of 70% ethanolic extracts of R. pseudoacacia revealed the presence of catechin, rutin, resveratrol and quercetin in the leaf extract, and catechin, epicatechin and rutin in the seed extract; None of these compounds were identified in the sheath extract. (Marinas et al., 2014). In flowers extract we detected rutin and quercetin, as well as a quite high amount of cynaroside and traces of vitexin which were not mentioned to be present in Black locust before. Resveratrol, however, was not detected. For the first time, the presence of chlorogenic and neochlorogenic acids was detected, as well as a little amount of trans-p-ferulic, trans-p-caffeic, trans-p-sinapic and trans-p-coumaric acids.
Lamiaceae species are ones of the oldest and still the most popular medicinal plants. Although previous studies also reported chemical constituents of the extracts investigated herein, the results presented now may show novel aspects of the plants’ composition. Thus S.officinalis and M.officinalis were not reported to contain sinapic acid which is in correspondence with other researches (Hernandez-Saavedra et al., 2015). Furthermore, Roby et al. (2013) showed the chemical profile of S.officinalis from Shambolia farm located at Fayoum area in Egypt to contain p-coumaric, caffeic, ferulic and rosmarinic acids. M.officinalis was also reported to contain caffeic, ferulic, chlorogenic, rosmarinic acids, the latter being the major constituent (Arceusz and Wesolowski, 2013). On the contrary, chlorogenic acid s was not observed in our samples. The analysis of published literature proves that the presence of neochlorogenic acids observed in S.officinalis and M.officinalis seems to be reported for the first time by us (Zheng and Wang, 2001). On the other hand we showed that another species of Lamiaceae family – Thymus pannonicus – had the highest amount of sinapic acid after Linaria vulgaris. Rosmarinic acid naturally occurring in plants of the Lamiaceae family had the highest content in sage and lemon balm while not being observed in thyme. Contrarily, Boros et al. (2010) showed that T. pannonicus grown and sampled at Soroksár, Hungary showed the highest content of rosmarinic acid than other phenolic acids.
There is renewed interest in antimicrobial activities of herbal plants as potential source of polyphenols reported to be effective antimicrobial substances against a wide variety of microorganisms. Seven species from five popular medicinal herbs were tested against Gram-positive and Gram-negative bacteria in terms of the size of inhibition zone (mm), MIC50 (µg/mL) and MIC90 (µg/mL).
In our work L. vulgaris ethanolic extract did not show any significant effect against neither Gram-positive nor Gram-negative bacteria. Publications screening did not reveal data on L.vulgaris antimicrobial studies. However the biological study on antibacterial activity was reported to be observed for Linaria corifolia, endemic to Irano-Turanian region (Gonuz et al., 2005). Assuming inhibitory zone assay they showed that the ethanolic extracts of aerial parts of the plant were more effective against Gram-positive bacteria (especially St. epedermidis ATCC 12228 and St.aureus ATCC 6538P) in comparison to Gram-negative bacteria. On the other hand, Gul et al. (2017) reported that ethanolic crude extract of L.corifolia aerial parts was only effective against B.cereus as well as yeast Candida albicans while ethanolic extracts of undersoil parts had an antimicrobial effect against St.aureus.
Antibacterial studies of Galium verum was not reported to possess very strong antibacterial activity against both Gram-positive and Gram-negative bacteria. However, it is likely that the nature of solvent might play important role in antimicrobial properties of G.verum. Thus Vlase et al. (2014) compared antimicrobial properties of four ethanolic (70%) extracts of Galium. Their results show that the antimicrobial activity of G.verum was lower than the effect of G.odoratum or G.mollugo. However, G.verum demonstrated some activity against Gram-negative bacteria (S.typhimurium, E.coli) and a moderate antibacterial activity against Gram-positive L.monocytogenes and St.aureus. Extract of G. verum in our work was based on 80% ethanol and possessed significant activity against Y.enterocolitica with broad inhibition zone and low MICs. High inhibition zone was also observed for M.luteus and C.koseri, and there were very low MICs for Ps.proteolitica. In addition to other works, Galium verum extract (in 96% ethanol) exhibited high activity in relation to Proteus vulgaris, Pseudomonas aeruginosa, Bacillus subtilis (Shynkovenko et al., 2017). Furthermore in Ilyina et al. (2016) work lipophilic (chloroform) extract of G. verum showed a significant level of antimicrobial activity. It has given the basis for a further search of antimicrobial substances among chloroform complexes obtained from different species. Summarizing our results and bibliography G.verum displays more efficiency in reference to Gram-negative microorganisms and slightly less efficiency in reference to Gram-positive strains.
A number of studies are available in the literature regarding in vitro antibacterial activity of Hypericum perforatum and the extracts are reported to be more active than decoctions (Kolesnikova, 1986). The aerial parts of H. perforatum are reported to exhibit more pronounced activity against Gram-positive bacteria than Gram-negative bacteria (Reichling et al., 2001, Avato et al., 2004). The antibacterial activity of ethanolic extract of H.perforatum tested in our work showed variations in activity against tested strains. Ps.proteolytica and Y. enterocolytica were more susceptible with terms of MICs among Gram-positive bacteria, B.thuringiensis had the highest inhibition zone, and St.aureus had a moderate susceptibility. According to the review by Saddiqe et al. (2010), St.John’s wort’s antibacterial effect varied significantly depending on solvents, proposing that organic solvents were more suitable for extracting antibacterial plant components. Thus the water extract was active only against S.oxford while petroleum ether, chloroform, and methanolic extracts had high MICs and were active against on P.aeruginosa, S.aureus, S.oxford, S.mutans, S.sanguis, E.coli, P.vulgaris. On the other hand, even 30% ethanol solution (Lasik et al., 2007) evaluated antagonistic properties against four bacteria – Enterococcus faecium, Bifidobacterium animalis, Lactobacillus plantarum and E.coli isolated from the human large intestine.
Literature data reports the antibacterial effect of aqueous extracts (in contrary to methanol) of black locust flowers against P.putida, B. subtilis, E.coli, S.cerevisiae and P.myxofaciens (Cioch et al., 2017). In additionMarinas et al. (2014) showed that the alcoholic extract of the R. pseudoacacia leaves potentiated the antimicrobial activity against the nine tested bacterial strains of E.coli, K. pneumoniae, B.subtilis, S.aureus, and P.aeruginosa while seeds and sheaths extract possessed very poor effect. Oppositely, the studied flower alcoholic extract did not evaluate any significant antimicrobial effect against test strains. Acetone extract from S.officinalis was active against St.aureus, E.coli, P.aeruginosa, B.subtilis, E.cloacae, K.pneumoniae, Pr.mirabilis. St.aureus strains were found to be the most sensitive bacteria to aqueous ethanolic and aqueous methanolic sage extracts (Kozłowska et al., 2015). The distinct antibacterial activity of aqueous ethanolic and aqueous methanolic extracts of S.officinalis was also observed against S.epidermidis and B.bronchiseptica with a MIC value of 0.5 mg/mL, and B.subtilis and G.stearothermophilis with MIC value of 0.25 mg/mL. Recently, the antimicrobial activity of S.officinalis was shown against vancomycin-resistant enterococci (Horiuchi et al., 2007). On the contrary, our work did not show any significant antibacterial effect of sage.
Ehsani et al. (2017) revealed a significant antimicrobial effect of M.officinalis against S.typhimorium, E.coli, L.monocytogenes and S.aureus. According to our results, the M.officinalis ethanolic extract possesses moderate antibacterial activity against gram-negative Ps.proteolyticca, H.alvei, and Y.enterocolitica. Our results, however, are not supported by some other works like Rabbani et al. (2015) showing that lemon balm extract had significant antibacterial activity against Gram-positive bacteria such as S. aureus and St. epidermidis.
The previous researches have shown that most aspects of thyme medicinal applications are related to the various levels of thymol and/or carvacrol, phenolic derivatives with strong and wide-spectrum of antimicrobial activity (Maksimović et al., 2008, Nabavi et al., 2014). Though the publications on a crude extract of T.pannonicus are scarce, the works on its volatile oils‘ antimicrobial activity revealed noteworthy antimicrobial potential against bacteria and yeasts. Our work, however, showed that among all the studied plants T.pannonicus had the highest antibacterial effect against Ps.proteolitica and Y.enterocolitica with regard to inhibition zone that correlated well with MICs.
We have examined the antioxidant, antiradical and antimicrobial activities for seven plant ethanolic extracts and the results complete the lack of literature data with new information concerning the polyphenolic compounds and their bioactivity. Our data demonstrate the difference in antioxidant activities of the reference antioxidants and selected phenolic and flavonoid compounds in different assays. This may be due to the fact that the different antioxidant capacity determining methods have different specificities for different solvents, reagents, pH conditions, or hydrophilic and hydrophobic substances. There seems to be no rule as to the variation of the antioxidant capacity, with the activity also being dependent on the identity of the species and also the site and date of collection. The observed antimicrobial activity confirms evidence of the effectiveness of the traditional use of these herbs drug against various pathogens. In general, the differences in the antiradical, antioxidant and antimicrobial activities may be due to different geographical environment, an age of the plant, the different method followed for isolation, cultivar type, seasonality, etc. Furthermore, the extracts are very complex mixtures of many variable compounds with distinct activities. So we believe that carefully designed studies to standardize methods of extraction and in vitro testing would be advantageous so that the search could be more systematic and interpretation of results would be facilitated.
Acknowledgments: This research was supported Erasmus Mundus project, CASIA III. The authors thank Prof. Peter Rapta (Slovak University of Technology in Bratislava) for kindly provided cynaroside standard.
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Monascus pigment from angkak has been used as a coloring agent in foodstuffs, texture industries, pharmacology, medicine and cosmetics as well as used as a folk medicine to improve food digestion, blood circulation and lowering blood cholesterol levels. Angkak is also known as red koji, Hung-Chu, monascal rice, Hong Qu, ang-kak, ankak rice, red mold rice, and Beni-Koji. Monascus pigment is not only as a natural food coloring but also the different antioxidant potentials, i.e. its abilities donating a hydrogen atom and/or an electron, chelating redoxactive metals and inhibiting lipoxygenases decades (Ramarathnam et al., 1995; Hadjipavlou-Litina et al., 2010). Commonly in Monascus fermentation, solid state fermentation (SSF) is a popular fermentation method used to produce the pigmentation and/or the antioxidant activities of angkak as well as a distinguished antioxidant, i.e. monacolin K (Yang et al., 2004; Yang et al., 2006; Kongbangkerd et al., 2014). The most important bioactive compound isolated from Monascus is monacolin K, which is identical to the potent cholesterol-lowering, antiatherosclerotic drug lovastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. The enzyme produces mevalonyl-CoA, which is the important rate determining step to synthesise cholesterol, resulted in the reduction in blood pressure (Kongbangkerd et al., 2014).
However, a limitation of SSF affecting Monascus fermentation is residual reducing sugars around 2,000 mg.kg-1 substrate in monascal products, especially glucose, after the end of the conventional Monascus fermentation (Babitha et al., 2007). Moreover, Kongbangkerd et al. (2014) reported that the reducing sugar contents were still remained at 8.00 mg.g-1 dry weight obtained from after the conventional fermentation and were rapidly hydrolyzed until absent after 2-step fermentation.
Germinated brown rice (GBR) is called as sprouted brown rice. The process of germination increases the bio-availability of important substances by neutralizing phytic acid. The neutralizing phytic acid is able to release the proteins, vitamins, and enzymes, allowing these important nutrients to be absorbed during digestion (Patil and Khan, 2011). Choi et al. (2006) reported that, beyond 24 h germination of brown rice, the enhanced contents of fructose, reducing sugars and γ -aminobutyric acid (GABA) appeared in GBR were higher 3.4 times, 2.75 times, and 7.97 times, respectively, than those appeared in the non-GBR. A study suggests that orally administered GABA increases the amount of human growth hormone (HGH) (Powers et al., 2008). GABA directly injected to the brain has been reported to have both stimulatory and inhibitory effects on the production of growth hormone, depending on the physiology of the individual. Certain pro-drugs of GABA (ex. picamilon) have been developed to permeate the blood–brain barrier, then separate into GABA and the carrier molecule once inside the brain. This allows for a direct increase of GABA levels throughout all areas of the brain, in a manner following the distribution pattern of the pro-drug prior to metabolism (Powers et al., 2008).
Therefore, a combination of 2-step fermentation and GBR, as a substrate for Monascus purpureus, used to produce angkak will be expected to enhance antioxidant activities and GABA contents in an angkak product.
MATERIAL AND METHODS
Lyophilised Monascus purpureus TISTR 3090 was purchased from the Thailand Institute of Scientific and Technological Research (TISTR). The strain was cultivated on Potato Dextrose Agar (PDA; Merck, Darmstadt, Germany) at 25°C for 7 days or until 106 spores mL-1 . After a pure culture was obtained, the mycelium was reinoculated into PDA slant and incubated at 25°C for 7 days or until 106 spores mL-1 before being used for angkak production.
Conventional fermentation method and 2-step fermentation of angkak
Conventional fermentation, brown rice (Oryza sativa) seeds were germinated at different times, i.e. 0, 12, 24, 36 and 48 hours. Then, a 100 g of germinated brown rice (GBR) was put into a flask 500 mL and was sterilized in an autoclave at 121°C for 15 min and then left until cool down. About 5 mL of 106 spores mL.spore suspension-1 of M. purpureus obtained from actively growing slants in sterile water was inoculated into sterilized GBR and incubated at 25°C for 12 days. In conventional fermentation, it indicated that Monascus was appeared in the dead phase of growth curve; thus, Monascus was reinoculated again in step 2 fermentation in order to ferment the substrate continuously. Fermentation of step-2, angkak produced from GBR with a period time for 48 hours (obtained from the conventional fermentation) was then reinoculated with the same volume and spore suspension contents and continuously fermented with the same condition as the conventional method for another 12 days (Kraboun et al., 2013). Then, the product was dried in an oven at 40°C for 24 h. A fine powder (20 mesh) was obtained using a mill (Retsch ultracentrifugal mill and sieving machine, Haan, Germany) (Kongbangkerd et al., 2014). The sample was determined Trolox equivalent antioxidant capacity (TEAC), DPPH free radical scavenging ability, reducing sugars, monacolin K, GABA and citrinin contents.
Sample extraction for antioxidant activity assay
The extraction method described by Yang et al. (2006) was used with some modifications. A 10 g sample was extracted in a shaker with 100 mL of methanol at 170 rpm for 24 h, and the solution was filtered through Whatman no. 4 filter paper. The residue was then extracted with two additional 100-mL portions of methanol as described above. The combined methanolic extracts were then evaporated at 40°C under vacuum condition to dryness. The dried product was used for analysis of antioxidant activities.
Reducing sugars analysis
The analysis of reducing sugars contents was evaluated spectrophotometrically by a slightly modified method of Re et al. (1999). The reducing sugars released in hydrolysis were analyzed using the DNS assay (Doner and Irwin, 1992). It contained a 1:1:1:1 volumetric mixture of 3,5-dinitrosalicylic acid 1%, Rochelle salt 40%, phenol 0.2%, potassium disulphide 0.5%, all in sodium hydroxide 1.5%. Typically, to 100 µL sample mixture 100 µL DNS reagent were added. The mixture was incubated in a boiling water bath for 5 min. After cooling to room temperature, the absorbance of the supernatant at 540 nm was measured.
Trolox equivalent antioxidant capacity (TEAC)
For ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay, antioxidant activity of angkak extracts against ABTS+ radical was evaluated spectrophotometrically by a slightly modified method of Re et al. (1999). The TEAC assay is based on the scavenging of ABTS+ radical converting into a colourless product. The degree of decolourisation induced by a compound is related to that induced by Trolox, giving the ‘TEAC value’. The ABTS+ radical was produced by the reaction between 2 mL of 7 mM ABTS solution and 40 µL of 2.45 mM potassium persulphate solution and stored in the dark at room temperature for 16 h. Before usage, the ABTS+ solution was diluted to get an absorbance of 0.700+0.025 at 734 nm with ethanol. For the assay, the resulting solution was mixed with 300 µL of sample of each monascal waxy corn extract (1–20 mg/mL). The absorbance was read at 30 °C after exactly 6 min. The obtained absorbance of samples was compared with a standard curve from the corresponding readings of trolox (0.4-0.04 mM). The total antioxidant capacities (TAC) were estimated as Trolox equivalent antioxidant capacity (TEAC) by interpolation to 50% inhibition (TEAC50).
DPPH radical scavenging activity
The scavenging activity (H/e-transferring ability) against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) was measured spectrophotometrically by following Velazquez et al. (2003). The extract (40 µL) with varying concentrations (1-20 mg/mL) was mixed with 200 µL of 0.02 mM DPPH solution and methanol 4 mL. Samples were kept for 15 min at 25 °C and the absorbance was measured at 517 nm. The absorbance of a blank sample containing the same amount of solvent was also measured. The extent of decolourisation is calculated as a percentage reduction of absorbance, and this is determined as a function of concentration and calculated relative to the 0.1-0.01 mM of equivalent Trolox concentration. The radical scavenging activity is expressed in mmol of equivalent trolox per gram of sample (mmol Trolox equivalent /mL) with interpolation to 50% inhibition (IC50).
Monacolin K analysis
An 0.5 g sample was extracted with 25 mL of 70% ethanol by using a shaker at 50°C for 2 h, followed by filtration through a 0.2 µm membrane and the extract was analysed by HPLC. The HPLC system consisted of Shimadzu LC-10AT VP Liquid Chromatograph, a FCV-10AL VP pump, an LDC Analytical SpectroMonitor 3100 detector set at 238 nm and an LDC Analytical CI-4100 integrator. A chromatography column Ascentis C18, 5µm, 250×4.6 mm was connected to a 20 µL loop injector. An isocratic mobile phase of acetonitrile:water in the ratio of 65:35 (by vol.) was used. The flow rate and temperature were 1.0 mL/min and 28°C, respectively (Chayawat et al., 2009). Monacolin K dissolved in 70% ethanol was used as a standard.
One gram of dried angkak powder was extracted with 5 ml water at 60°C for 2 h with vigorously shaking. After 12,000 x g cencentrifuging for 20 min at 4°C, 400 μl aliquot of supernatant (or standard solution of GABA) was vacuum-dried. The residue was dissolved in 50 μl ethanol-water-triethylamine (2:2:1) solution, and the mixture was then evaporated to dryness under vacuum until dry and redissolved again in 40 μl ethanol-water-triethylamine-phenylisothiocyanate solution (6:1:1:1). The final mixture was allowed to react for 20 min at room temperature to form phenylisothiocyanate-GABA (PTC-GABA).
Procedure of HPLC analysis described by Wang et al. (2004) was slightly modified. Briefly, the dry residue containing PTC-GABA was dissolved by adding 400 μl mobile phase that consisted of 80% solution A (aqueous solution of 8.205 g sodium acetate, 0.5 ml triethylamine, 0.7 ml acetic acid, and 5.0 ml acetonitrile in 1000 ml distilled water, pH 5.8) and 20% solution B (acetonitrile-water, 60:40, pH 5.8). Chromatographic separation was conducted on a Shim-pack VP-ODS C18 column (4.6 × 150 mm i.d., 5 μm). The eluent was pumped at a flow rate at 0.6 ml/min. Temperature of column oven was 46°C and UV detection wavelength was set at 254 nm.
Citrinin analysis was described by Lim et al. (2010). A 1 g sample was extracted with a solution (acetone : ethyl acetate = 1:1, v/v) at 65oC for 90 min under vigorous shaking. The supernatant was obtained by centrifugation at 1,600 x g for 10 min followed by filtration through a 0.45 µm PTFE (Polytetrafluoroethylene) filter unit (National Scientific, Rockwood, TN). The citrinin was determined by HPLC using a chromatography column Ascentis C18 column (4.6 x 250 mm). The mobile phase consisted of methanol/acetonitrile/ 0.1% phosphoric acid (3:3:4, v:v) and the analysis was performed with a fluorescence detector set at excitation and emission wavelengths of 330 and 500 nm, respectively. The flow rate was 0.6 mL/min and the sample was spiked to confirm the presence of citrinin.
All determinations were performed in triplicate and results were expressed as the mean+standard deviation calculated using spreadsheet software Microsoft Excel. This was carried out in a completely randomized experimental design (CRD) and the data were analysed by an analysis of variance (p<0.05) and means were compared using Duncan’s new multiple range test. The results were processed by SPSS 16.0 (SPSS Inc., Chicago, IL, USA) for Windows.
RESULTS AND DISCUSSION
Reducing sugars and pigment intensity of angkak produced from GBR with different germination times and different fermentation methods
The contents of reducing sugars and pigment intensity of angkak using germinated brown rice (GBR) with different germination times as a substrate are shown in Table 1. The contents of reducing sugars of angkak obtained from the conventional fermentation decreased with increasing pigment intensity when using GBR with increased germination times. Moreover, using GBR with a germination period for 48 hours, Monascus purpureus could produce the highest pigment intensity and use the highest contents of reducing sugars indicating reducing sugar decreased 28.08 %. This was a cause of M. purpureus applying the variously significant substances such as vitamins, minerals and dietary fibers as well as the antioxidants (GABA and phenolic compounds) occurred during the seed germination process. Furthermore, reducing sugars also used as a substrate so that the significant substances were continuously produced affecting an increase of pigment intensity (Chung et al., 2009).
Therefore, the angkak produced from GBR with a germination period for 48 hours via the conventional method was continuously fermented by M. purpureus. In 2-step fermentation, 99.43 % of the contents of reducing sugars were applied by M. purpureus and they were still residual at 0.01 mg/g substrate; whereas, the pigment intensity was increased to 3,500.89 unit /g substrate. This result indicated that 2-step fermentation led to increasingly used contents of reducing sugars to be a substrate for M. purpureus affecting higher pigment intensity (Kongbangkerd et al., 2014). This was in agreement with Kongbangkerd et al. (2014) who reported that the pigment intensity of monascal waxy corn from 2-step fermentation was 3,500 unit /g substrate and the contents of reducing sugars were exhausted compared with those of monascal waxy corn from the conventional method (500 unit /g of substrate of pigment intensity and 8 mg/g of reducing sugars). In addition, the 2 kinds of hydrolyzing enzymes such as α-amylase and glucoamylase were continuously appeared during the conventional and 2-step fermentations so that this may be another cause to enhance hydrolysis of the substrates for pigment production (Babitha et al., 2007).
Table 1 Contents of reducing sugars and pigment intensity of angkak produced from germinated brown rice (GBR) with different germination times and different fermentation methods
|Fermentation methods||Germination times of brown rice (hour)||Reducing sugars (mg/g substrate)**,***||Decreased percentages of reducing sugar (%)*||Pigment intensity (unit /g substrate)**,***|
|1.78 ± 0.00e
1.68 ± 0.02d
1.53 ± 0.00c
1.33 ± 0.05b
1.28 ± 0.33b
0.01 ± 0.00a
|200.89 ± 2.50a
250.78 ± 9.00b
369.85 ± 3.50c
459.79 ± 5.70d
500.98 ± 4.90e
3,500.89 ± 15.34f
*Decreased percentages of reducing sugars are the used contents compared with the contents of reducing sugars angkak using GBR with a germination period for 0 hour.
**Different letters within the same column indicate statistical differences (one-way ANOVA and Duncan test, p < 0.05).
***Values are mean ± S.D of triplicate determinations.
IC50 of DPPH and ABTS of angkak produced from GBR with different germination times and different fermentation methods
The IC50 values of DPPH and ABTS of angkak produced from GBR with different germination times and different fermentation methods are shown in Figure 1. It was found that, in the conventional method, the IC50 values of DPPH and ABTS of angkak were significantly lower when using GBR with increased germination times (p < 0.05). The pigment of angkak using GBR with 48 hours of germination time was lower IC50 values of DPPH and ABTS than that from GBR with 0 hour of germination time (without germination), which had the lowest IC50 values of DPPH and ABTS showing 0.15 and 0.17 mmol Trolox equivalent /mL, respectively. In 2-step fermentation, the IC50 values of ABTS and DPPH of pigment from angkak were 0.07 and 0.06 mmol Trolox equivalent /mL, respectively, which were 2 times lower than those from angkak using GBR with a germination period for 48 hours through the conventional method. It seemed that the pigment extracted from angkak produced from a combination of GBR and 2-step fermentation might have the presence of the glycone part, which masked hydrogen donation property of the pigment, indicating an important feature for free radical scavenging (Bhanja et al., 2008). Moreover, monacolin K and/or total phenols occurred in the pigment from angkak affected the inhibition of the formation of ABTS• by one-electron oxidants, showing an effectiveness as an electron donor (Hagerman et al., 1998; Yang et al., 2006).
Figure 1 IC50 values of DPPH and ABTS of angkak produced from germinated brown rice (GBR) with different germination times and different fermentation methods.This was carried out in a completely randomized experimental design (CRD) and the data were analysed by an analysis of variance (p<0.05) and means were were compared using Duncan’s new multiple range test.
Monacolin K, GABA and citrinin of angkak produced from GBR with different germination times and different fermentation methods
The contents of monacolin K, GABA and citrinin of angkak produced from GBR with different germination times and different fermentation methods are shown in Figure 2. As compared to the angkak products using GBR with different germination times and the conventional method, the contents of monacolin K, GABA and citrinin were significantly higher when using GBR with increased germination times (p < 0.05). In angkak obtained from GBR with a germination period for 48 hours, the highest contents of monacolin K, GABA and citrinin were 77.16 and 120.59 mg/kg dry weight and 10.17 µg/kg dry weight, respectively. The contents of monacolin K (99.75 mg/kg dry weight), GABA (154.19 mg/kg dry weight) and citrinin (12.37 µg/kg dry weight) of angkak via 2-step fermentation were higher than those of GBR with a germination period for 48 hours through the conventional method. The monacolin K, GABA and citrinin contents of angkak via 2-step fermentation were in agreement with those reported for monascal waxy corn (Kongbangkerd et al., 2014). In this experiment, the fermentation temperature was 25oC which was appropriate for monacolin K and GABA (Tsukahara et al., 2009). Su et al. (2003) confirmed that solid state fermentation (SSF) was a type of fermentation that could lead to not only the yields of the products but also a low energy requirement, which reduced the production costs. In addition, this experiment using SSF which was conducted by incubation at 25oC indicating the highest yield of monacolin K. Furthermore, Pengnoi et al. (2017) comfirmed that a temperature of 25°C was appropriate for the angkak production due to increased to 93.07% of monacolin K content. However, angkak is frequently contaminated with citrinin. Contamination with citrinin is a problem influencing acceptability because it is a mycotoxin which damages the liver and kidneys of mammals (Kongbangkerd et al., 2014). Although the contents of citrinin of angkak from 2-step fermentation were 12.37 µg/kg dry weight, the citrinin contents were not exceeded following the maximum allowance level in red fermented rice. According to the legislation of many countries, Japan has issued an advisory limit of 200 µg kg-1 of citrinin in commercially agricultural products. The limit set by the Chinese Food and Drug Administration is 20 µg kg-1 of citrinin, while the European Union has recommended a citrinin limit of 100 µg kg-1 (Shi and Pan, 2011). This study result was a success using a selected condition, which produced the low contents of citrinin and the high contents of monacolin K and GABA to be potential for providing safe functional food.
Figure 2 Monacolin K, GABA and citrinin of angkak produced from germinated brown rice (GBR) with different germination times and different fermentation methods. This was carried out in a completely randomized experimental design (CRD) and the data were analysed by an analysis of variance (p<0.05) and means were compared using Duncan’s new multiple range test.
The increasing time of germination of GBR impacted on reducing sugars usage by Monascus purpureus. Hence, the used contents of reducing sugars of angkak obtained from a combination of GBR with a germination period for 48 hours and 2-step fermentation was 99.43 %; therefore, the highest contents of monacolin K and GABA were produced as well as the lowest IC50 values of DPPH and ABTS. Moreover, the citrinin contents were not exceeded following the maximum allowance level in the red fermented rice product as well.
Acknowledgement: This work was funded by Rajamangala University of Technology Krungthep. Moreover, The author would like to thank all staffs of the faculty for maintenance and operation of the scientific laboratory and equipment.
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Manufacture of most of cheese varieties involves a combining of four ingredients: milk, rennet, microorganisms and salt, which are processed. The common steps of cheese-making include gel formation, curd whey expulsion, acid production and salt addition, followed by a period of ripening. A variation in ingredient blends and processing has led to the evolution of cheese varieties. While variations in processing parameters such as processing temperature and curd handling techniques play a major role in production of each cheese type, but the cheese microflora play a critical and pivotal role in the development of the unique characteristics of each cheese variety (Beresford et al., 2001). Traditional raw-milk cheeses are highly valued for their flavors, while large-scale products are often perceived by the consumer as ‘‘boring’’ (Law, 2001). This difference is a consequence of the elimination of raw milk microflora by pasteurization that has a key role in flavor development. To compensate the sensory characteristics of product the food industry looks for alternative LAB (Lactic Acid Bacteria) cultures capable of improving products flavor (Leroy and De Vuyest, 2004). However, the LAB are only a part of the complete microflora of raw milk (Kongo et al., 2007). Complex approach then the addition of LAB is associated to other technological methods such as pressing allows the production of diverse of traditional cheeses (Parguel, 2011). The raw-milk microbiota also represents the contamination from the environment (air, utensils, the animal skin), and the load and its diversity will vary with location, season and livestock species and milking procedures.
Food spoilage is an enormous economic worldwide problem. Approximately one-fourth of the world’s food supply is lost through microbial activity alone (Huis in’t Veld, 1998). Milk is highly nutritious food that serves as an excellent growth medium for a wide range of microorganisms (Ruegg, 2003; Rajagopal et al., 2005). The microbiological quality of milk and dairy products is influenced by the initial microbiota of raw milk, the processing conditions, and post-heat treatment contamination (Richter et al., 1992). Undesirable microbiota that can cause a spoilage of dairy products includes Gram-negative psychrotrophs, coliforms, lactic acid bacteria, yeasts, and molds. In addition, the various pathogens of public health concern such as Salmonella spp., Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolitica, pathogenic strains of Escherichia coli and enterotoxigenic strains of Staphylococcus aureus may also be found in milk and dairy products (Tatini and Kauppi, 2003; Al-Sahlany, 2016; Verma and Niamah, 2017). This is one of the reasons why the increased emphasis should be focused on the microbiological examination of milk and dairy foods. Microbiological analyses of milk and milk products are critical for assessment of quality and safety, conformation with standards and specifications, and regulatory compliance (Vasavada, 1993).
The aim of this study was to evaluate microbiological quality of the traditional Slovak non-smoked and smoked cheese “Parenica” made from cow milk and to identify bacterial strains with MALDI-TOF MS Biotyper.
MATERIAL AND METHODS
There were 50 samples of the Slovak national cheese “Parenica“ examined in this study. The cheese samples included non-smoked cheese (n=25) and smoked cheese (n=25). Additionally, a total of 50 cow milk cheese samples from the Slovak producers located in the western and the middle part of Slovakia were collected (Bánovce nad Bebravou, Liptovský Mikuláš, Červený Kameň, Važec). All samples were placed in sterile sample containers and transported to laboratory on ice for microbiological investigations. Samples were kept in a refrigerator (4±1°C) until the testing began.
The primary dilution of the milk products was made for preparing the samples for testing. For that a 5 ml of sample material was added to 45 ml of 0.87 % sterile saline, then the serial dilutions (10−1 to 10−4) were done and a 100 µl of each dilution was plated out.
Isolation of total count of bacteria
Plate count agar (PCA, Sigma-Aldrich®, St. Louis, USA) for total count bacteria enumeration was used. Inoculated plates were incubated at 30 °C for 24-48 h and then examined for the presence of bacterial colonies.
Isolation of coliform bacteria
Violet red bile lactose agar (VRBGA, Sigma-Aldrich®, St. Louis, USA) for enumeration of coliforms bacteria was used. Inoculated plates were incubated at 37 °C for 24-48 h and then examined for the presence of typical colonies.
Isolation of enterococci
Enterococcus selective agar (ESA, Sigma-Aldrich®, St. Louis, USA) for enumeration of enterococci was used. Inoculated plates were incubated at 37 °C for 24-48 h and then examined for the presence of typical colonies.
Isolation of Lactic Acid Bacteria (LAB)
MRS (Main Rogose agar, Oxoid, UK), MSE (Mayeux, Sandine and Elliker in 1962, Oxoid, UK), and APT (All Purpose TWEEN® agar, Oxoid, UK ) agars were used for enumeration of LAB including lactobacilli, leuconostocs and lactic acid streptococci as well as other microorganisms with high requirements for thiamine (Sigma-Aldrich®, St. Louis, USA). Inoculated agars were incubated at 30 °C for 72 h anaerobically and then the bacterial growth was evaluated.
Sample preparation and MALDI-TOF MS measurement
Prior to identification, the bacterial colonies were subcultured on TSA agar (Tryptone Soya Agar, Oxoid, UK) at 37°C for 18-24 h. One colony of each bacterial isolate was selected. Subsequently, the identification was performed using the Maldi TOF MS Biotyper described by Kluga et al. (2017). Totally, a number of 512 isolates were identified with score higher than 2.
RESULTS AND DISCUSSION
Number of isolated bacterial group
Cheeses are fermented dairy products whose manufacturing involves different types of bacteria (Montel et al., 2014; Irlinger et al., 2015). Cheese producing is a process when a nutrient-rich substrate as milk is colonized by adventitious and deliberately inoculated microorganisms. Two different habitats of bacteria in cheese may be considered: the interior of the cheese and the cheese rind. The rind microbiota can be considered as an interesting model system for the field of ecosystems biology (Wolfe et al., 2014).
Total count of bacteria in non-smoked cheese ranged from 5.25 to 5.58 log cfu.g-1. Enterococci were not identified in the studied samples. Coliform bacteria counts ranged from 1.25 to 1.80 log cfu.g-1, but lactic acid bacteria counts ranged from 4.12 to 4.51 log cfu.g-1. Total count of bacteria in smoked cheese ranged from 5.45 to 5.85 log cfu.g-1. Enterococci and coliform bacteria number of bacteria were not identified in the samples studied. Lactic acid bacteria counts ranged from 4.12 to 4.48 log cfu.g-1.
Kačániová et al., (2018) found similar results in cheese samples, and the total count of bacteria in non-smoked cheese ranged from 3.15 to 3.58 log cfu.g-1. Enterococci were not identified in the studied samples. Coliform bacteria counts ranged from 1.12 to 1.52 log cfu.g-1, but lactic acid bacteria counts ranged from 2.12 to 2.51 log cfu.g-1. Total count of bacteria in smoked cheese ranged from 2.14 to 2.58 log cfu.g-1. Enterococci and coliforms bacteria were not identified in the studied samples. Lactic acid bacteria counts ranged from 1.12 to 2.18 log cfu.g-1.
Total counts of bacteria are the most useful indicator for the overall microbiological quality of the cheese. High viable count often indicates a contamination of the raw material, unsatisfactory sanitation, or unsuitable time and temperature during storage and/or production. The attention has been focused on coliform bacteria because of their public health importance. Coliforms are widely distributed in nature. They gain entry to milk and milk products through the water supply, equipment, unhygienic conditions of production and handling (El-Leboudy et al., 2014).
Isolated bacteria with MALDI-TOF MS Biotyper
Table 1 Isolated species of bacteria from smoked and non-smoked cheese “Parenica”
A total of 53 species of 30 bacterial genera (18 gram-negative G– and 12 Gram positive G+) were identified in smoked and non-smoked cheese by MALDI-TOF Mass Spectrometry. The percentage representation of each bacterial group (G– and G+) were 42.58% for G– (218 isolates) (and 57.42% for G+ (294 isolates). (). Isolated species of bacteria from smoked and non-smoked cheese “Parenica” are shown in Table 1.
Percentages of the number of isolates of each species for G– and G+ are shown in Table 2. The most abundant G– bacterium was Escherichia coli, Klebsiella oxytoca and Klebsiella pneumoniae. Lactobacillus was the most abundant within 12 different species of G+ bacteria with Lactobacillus casei, L. delbrueckii and L. sakei were the most distributed.
Nevertheless, many LAB species were found in both kinds of French cheeses, e.g. L. plantarum, L. paracasei, L. curvatus, L. rhamnosus, L. fructivorans, L. parabuchneri, L. brevis (Nacef et al., 2017). As previously were reported, some Lactobacilli are present in the natural microflora of dairy products and arise from animals, farms and dairies: L. casei ssp. casei/L. paracasei ssp. paracasei, L. rhamnosus, L. plantarum, L. fermentum, L. brevis, L. buchneri, L. curvatus, L. acidophilus and L. pentosus (Corbo et al., 2001, Gobbetti et al., 2002, Medina et al., 2001). Lactobacilli, especially L. curvatus, represents a type of milk microbiota that is resistant to pasteurization. Moreover the presence of LAB could be also attributed to contamination occurring after pasteurization (Martley and Crow, 1993).
Kačániová et al. (2018) found in microbiological analysis of 50 cheese samples three main groups of microorganisms: gram-negative and gram-positive bacteria and fungi. Althogether, 47 species of 18 bacterial genera (17 Gram negative G– and 12 Gram positive G+) and 10 species of yeasts of 5 genera were identified with MALDI-TOF Mass Spectrometry. The percentage representation of each microbial group (G–, G+ and yeasts) from a total of 669 isolates, reached the following values: 166 isolates of G– (24.81%), 297 isolates of G+ (44.39%), and 206 isolates of yeasts (30.79%).
Table 2 Number of isolates identified with MALDI-TOF MS Biotyper in cheese
|Microorganisms||Non-smoked cheese||Smoked cheese||Total|
|Microorganisms||Non- smoked cheese||Smoked cheese||Total|
|Streptococcus salivarius ssp. thermophilus||8||2||10|
Figure 1 Percentage of isolated bacterial species in cheese samples
Mounier et al. (2006) found out that the microorganisms that developed on the cheese surface were an adventitious microflora from the cheese environment (brine, ripening shelves, and personnel) which rapidly outnumbered the commercial cultures. Several hypotheses have been advanced to explain these findings. These ripening cultures may be unfit for the cheese habitat, or negative interactions may occur between them and the adventitious microflora (Maoz et al., 2003).
Microbiological analysis of 100 cheese samples revealed the two main groups of microorganisms comprising 53 species of 30 bacterial genera (18 Gram-negative G– and 12 Gram-positive G+) identified with MALDI-TOF Mass Spectrometry. The percentage representation of each bacterial group (G– and G+) were 42.58% (218 isolates of G–) (and 57.42% (294 isolates of G+). Fast microbial identification is becoming increasingly necessary in industry to improve microbial control, reduce biocide consumption, to avoid cost-intensive recall of contaminated products and damage to brand reputation. While MALDI-TOF-MS has revolutionized speed and precision of microbial identification for clinical isolates, in contrast few performance studies have been published so far focusing on suitability for particularly industrial applications.
Acknowledgement: Work was supported by the grants APVV-16-0244 “Qualitative factors affecting the production and consumption of milk and cheese”.
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Trehalose, also known as α-D-glucopyranosyl, α-D-glucopyranoside, is a disaccharide molecule made up of two glucose molecules linked with a 1,1 glycosidic linkage. It is mostly found in a variety of organisms i.e., bacteria, fungi, invertebrates etc (Himei 2008). Although trehalose was well known for carbon and energy source of plants and animals for years, the researches in the last two decades showed that trehalose is a multifunctional molecule as well. It is found in cell wall glycolipid as a structural component (Arguelles, 2000; Richards, 2002). Trehalose is very stable under hot and acidic conditions. Mizumoto et al (2004) showed that trehalose can be used as a bulk agent due to its stability towards heat and hydrolysis. Moreover, it does not caramelize and undergoes Maillard reactions and it is safe for human consumption and widely accepted by the European regulation system (Richards et al., 2002; Schiraldi, Di Lernia, & De Rosa, 2002). It can also stabilize enzymes in vegetables (Aga et al.,1988), suppress bitterness and enhance sourness (Oku, 1995), suppresses foul odor (Kubota, 2005), suppresses oxidation (Himei, 2008) reaction as well. Colaco & Roser (1995) reported that it can be used as an additive for food preservation. Zdzieblo & Synowiecki(2006) suggested that trehalose can be used for food processing because of its certain unique properties; mild sweetness, low carcinogenicity, good solubility in water, stability under low pH conditions, low hygroscopicity, depression of freezing point, high glass transition temperature and ability to protect proteins. Although trehalose is abundant in many microorganisms, it’s commercial production for the industries has been a big challenge until several enzyme synthesis systems in microorganisms have been discovered (Lama et al. 1990; Nakada et al.1996; Di Lernia et al. 1998) which opened a new dimension in commercial trehalose production in the industries. Avonce (2006) reported that there are five main enzymatic pathways of trehalose biosynthesis has been identified so far. Two of which are very popular for commercial production of trehalose; MTS-MTH pathway (two novel enzymes maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase convert maltodextrin into trehalose in a two-step reaction) and TreS pathway (Trehalose synthase isomerizes α1- α4 bond of maltose to α1- α1 bond resulting in trehalose).The later one requires less energy, more simple, fast and cost-effective. Acidiplasma sp. MBA-1 is a novel acidophilic, cell wall-less archaeon, excretes a significant amount of trehalose into the culture media. A new gene for trehalose synthase has been identified from Acidiplasma sp MBA-1(GenBank). In this study, we hypothesized that the gene could be expressed into an E. coli expression system and using this enzyme trehalose could be produced commercially. We also aim to purify and characterize the trehalose synthase from Acidiplasma sp MBA-1. To the best of our knowledge, this is the first report on purification and characterization of trehalose synthase (TreS) from this bacterial strain.
MTERIALS AND METHODS
The column resin for recombinant Acidiplasma sp. MBA-1TreS purification, the chelating Sepharose Fast Flow, was obtained from GE (Uppsala, Sweden). Electrophoresis reagents were purchased from Bio-Rad. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) and all chemicals for the assay were from Sigma-Aldrich (St. Louis, MO, USA). Standard trehalose was bought from Sinopharma Ltd., China. The reconstructed plasmid was synthesized by Generay Biotech Co., Ltd. (Shanghai, China).
Gene Cloning and expression of recombinant TreS
According to information from the NCBI, the whole genome of Acidiplasma sp. MBA-1 was sequenced by Bulaev A.G. in 2015 and was released into the Gen Bank (NCBI) with the accession number KJE50039.1. The target DNA gene (gene locus_tag: TZ01_03000) and the gene encoding the hypothetical protein of RDH (protein ID number WP_048101287.1) was synthesized and cloned into the pET-22b(+) vector with NdeI and XhoI sites and an in-frame fusion His6-tag sequence at the C-terminus was provided in the reconstructed plasmid. The plasmid was named pET-TreS and was transformed into E. coli BL21 (DE) for TreS overexpression. The E. coli BL21 (DE) cells harboring the pET-TreS plasmid were cultured in Luria Bertani medium supplemented with the antibiotic (kanamycin) to a final concentration of 100 μg mL−1 and incubated at 37 °C. After the culture reached an optical density of 0.6– 0.8 at 600 nm, IPTG was added to the culture to a final concentration of 1 mM, and TreS was induced at 28 °C for 6 h.
Purification of recombinant TreS
The cells were collected by centrifugation at 8,000 × g for 10 minutes and then washed with 50mM sodium phosphate buffer (PBS) with a pH of 7.5. The washed cells resuspended in the lysis buffer were disrupted by ultrasonication at 4 °C using a Vibra-Cell™ 72405 Sonicator (BioBlock Scientific, Illkirch, France). The disrupted cells were removed by centrifugation (10,000 × g for 30 minutes at 4 °C). The collected supernatant (crude enzyme) was loaded onto a chelating Sepharose Fast Flow resin column (1.0 × 10.0 cm) charged with Ni2+ and equilibrated with the binding buffer (50 mM PBS, 500 mM NaCl, pH 7.5). Unbounded proteins in the column were removed with the washing buffer (50 mM PBS buffer, 500 mM NaCl, 50 mM imidazole, pH 7.5). TreS was subsequently eluted with the elution buffer (50 mM PBS buffer, 500 mM NaCl, 500 mM imidazole, pH 7.5). The collected pure enzyme was dialyzed for 24 h at 4 °C against the dialysis buffer (50 mM sodium phosphate buffer, pH 7.5).
Activity assay of TreS
The activity was determined by measuring the amount of trehalose produced from maltose. The total volume of the standard reaction was 1ml consisting of 900 µl 50 mM sodium phosphate (pH 6.5) as a substrate solution (1% maltose) and 100 µl of purified enzyme. The mixture was incubated for 1h at 40°C. After that, the reaction mixture was heated at 100°C in boiling water for 10 minutes to stop the reaction.
Trehalose was detected by High-Performance Liquid Chromatography (HPLC) system equipped with a refractive index detector and an NH2 column (Waters Spherisorb® 5µm, 46×250 mm). The flow rate of the mobile phase was 1ml/min. The mobile phase consists of 77.5% acetonitrile, 15% methanol and 7.5% ddH2O.
Effect temperature on TreS
The effects of temperature on the activity of TreS was determined at various temperatures (20-60 °C). To check the stability of the TreS enzyme against temperature, 100 µl of purified TreS were preincubated with 50mM sodium phosphate buffer (pH 6.5) for 1h at different temperature ranging from 20-60 °C. Finally, a standard reaction was carried out at 40 °C for 1h adding 1% substrate (maltose) into the preincubated purified enzyme. The residual activity was measured by the HPLC system.
Effect of pH on TreS
The effect of pH on the activity of TreS was determined at various pH (5-8.5) using 50mM sodium phosphate buffer at 40 °C. The standard reaction was carried out for 1h. The residual activity was measured by the HPLC system.
Effect of Metal Ions and EDTA activity
The enzyme solution was incubated with various metal ions Mn2+, Ni2+, Cu2+, Mg2+, Ba2+, Zn2+, Al3+, Fe2+, Li+, Co2+ and a chelating reagent EDTA at a final concentration of 1mM. The residual activity was measured by the HPLC system. The measured activities were compared to the enzyme activity without the addition of metal ions (control) under the same conditions.
Total protein concentration
The total protein concentration was measured according to the Bradford method (Bradford, 1976). Bovine serum albumin was used as a standard.
Different sugars have been used as a substrate to check the substrate specificity of the enzyme TreS. We have used Glucose, Lactose, Sucrose, Fructose, Mannose, β-cyclodextrin, Starch, Cellobiose and Galactose as a substrate. The reaction was carried out at optimum conditions with 1% maltose as a substrate. The relative activity was determined by the HPLC.
To determine the conversion rate of the TreS enzyme, a series of standard reactions were carried out using 1% maltose as a substrate. The standard 1ml reaction mixture containing 900 µlsodium phosphate buffers (50mM) as a substrate solution and 100 µl purified enzyme was used. The reaction was carried out at a different time (0-10h). The residual activity was measured by the HPLC system.
As described by Laemmli, the subunit molecular weight of recombinant TreS was examined by using the denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 5% stacking gel and a 12% separating gel. Gels were stained with Coomassie Brilliant Blue 250 and de-stained with an aqueous mixture of 10% (v/v) methanol/10% (v/v) acetic acid.
Acidiplasma sp MBA-1 amino acid sequence released from the gene sequence was compared with similar enzymes from other organisms using the NCBI web site tool BLAST and the sequence alignment tool ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html).
Gene Cloning and expression
The genome of Acidiplasma sp. MBA-1 released in the gene bank with accession number KJE50039.1 was analyzed and the existence of a trehalose synthase with protein ID WP_048101287.1 that convert maltose to trehalose was potentially identified. According to this analysis, the gene was synthesized and the target gene was sub-cloned into pET-22b (+) and named pET-TreS. This construct was transformed into E. coli BL21 (DE3) cells and Acidiplasma sp. MBA-1 trehalose synthase (TreS) overexpression was induced by IPTG. Using the NCBI amino acid sequence of the Acidiplasma sp. MBA-1 showed sequence identities of 89, 66, 63 and 59% with Picrophilus torridus [accession number is WP_011176870.1], Bacteriam JKG1 [accession number is WP_029315667.1], Kouleothrix aurantiaca [accession number is KPV52019.1], Myxococcus xanthus [accession number is WP_011553702.1], respectively (Fig.1).
Figure 1 Acidiplasma sp MBA-1 amino acid sequence comparison with Trehalose synthase from different organisms. The amino acids marked by asterisks are sequence identical in all sequences. Amico acid marked by colons and dots are strongly and weakly conserved, respectively
TreS gene encodes a polypeptide of 562 residues with a calculated molecular mass of 66.09 kDa. Target recombinant protein purification was carried out using nickel affinity column chromatography. SDS-PAGE analysis gave a strong protein band with a molecular mass of 65.9 kDa. The specific activity of this protein was 3.568 Umg-protein−1 in the purified enzyme.
Figure 2 SDS-PAGE analysis of the recombinant protein. Lane 1 purified recombinant TreS (arrow indicates purified enzyme) Lane 2 Crude extract of the recombinant TreS. Lane M Molecular weight standards (116.0, 66.2, 46.0, 35.0, 25.0,18.4 kDa)
Effect of pH on recombinant TreS
Figure 3. shows the effect of pH on recombinant TreS activity. TreS showed the highest relative activity at pH 6.5 whereas the relative activity was 91.35% and 53.28% at pH 7.0 and 7.5, respectively. At higher pH value the activity of TreS dropped sharply.
Figure 3 Effect of pH on recombinant TreS activity. Values are the means of three replicates ± standard deviation.
Effect of Temperature on recombinant TreS
As shown in figure 4, the enzyme showed maximum activity at a temperature of 40 °C, whereas at 35, 30 and 25 °C the relative activity decreased to 81.2%, 69.7%, and 42.4%, respectively. The enzyme activity increased up to 40 °C and then gradually decreased. The activity was dropped to 9.3% at 60°C.
The thermal stability of the enzyme was examined at pH 6.5 in a standard buffer (50mM Sodium phosphate buffer). As shown in figure 4, the relative activity of the enzyme was almost constant up to 40°C and the relative activity dropped significantly to 90, 71, 22 and 0% after incubation for 1h at 45, 50, 55 and 60 °C, respectively.
Figure 4 Effect of temperature on recombinant TreS activity. (■) effect of temperature on the enzyme activity (●) effect of temperature on the enzyme stability. To examine the thermal stability of TreS, the enzymes were pre-incubated at various temperatures (20–60 °C) for 1h at pH 6.5. The residual activities were measured at 40 °C. Values are the means of three replicates ± standard deviation.
Effect of metal ions on recombinant TreS
The recombinant TreS was assayed in the presence of various metal ions, which were incubated with an enzyme solution at a final concentration of 1 mM. The enzyme assay showed that magnesium, EDTA and Lithium raised the enzyme activity by 10.9%, 9.6%, and 6.7% respectively. Enzyme activity did not significantly change when incubated with manganese. The enzyme activity decreased significantly when incubated with cobalt, zinc, and nickel to 62.5%, 41.8%, and 33.34%, respectively, whereas copper completely inhibited the TreS enzyme activity.
Figure 5 Effect of metal ions on recombinant TreS activity. Values are the means of three replicates ± standard deviation.
Only maltose showed substrate specificity with TreS. The other substrates (Glucose, Lactose, Sucrose, Fructose, Mannose, β-cyclodextrin, Starch, Cellobiose and Galactose) showed no specificity (data not shown) for TreS as they did not produce any trehalose in the reaction.
Table 1 Substrate Specificity for TreS
*presence of Trehalose was checked in substrate specificity reaction
The purified enzyme (100 µl) was incubated in 900 µlsodium phosphate (50mM) buffer (pH 6.5) at 40 °C for 0–10 h, using 1% maltose as a substrate. All the reactions were stopped by boiling them for 10 min before the samples were analyzed by the HPLC system. After 9h of reaction, the conversion rate of trehalose and glucose were 43.62%, and 22.01% respectively.
Figure 6 Conversion rate of trehalose and glucose from maltose with a different time (0-10h)
We have confirmed that the gene (gi= 765468230) from Acidiplasma sp. MBA-1. encoded a functional enzyme, trehalose synthase, and it could catalyze the conversion of maltose to trehalose. The optimum temperature of TreS found 40 °C which similar to those trehalose synthases coming from Actinoplanes SN223/29(Lee et al., 2008). TreS maintained a high relative activity up to 45°C while checking the stability of the enzyme against temperature. The optimum pH was 6.5, similar to several trehalose synthases reported in past studies (Liang et al., 2013; Wu et al., 2009). TreS activity was increased by Mg2+ and Li+. The trehalose synthase from Pseudomonas sp (Gao et al., 2013), Deinococcus sp. (Jiang et al., 2013), Deinococcus radiodurans (Filipkowski et al., 2012) is also reported to increase their activity by Mg2+. EDTA has slightly enhanced the activity of the enzyme. We have not found any obvious reasons for that. It is most likely, EDTA is chelating metal ions that affect that binding site of TreS. TreS activity was strongly inhibited by Cu2+. It is probably because copper ions were interfering with the binding site of TreS and making it inactive in the reaction. Other studies are also suggesting the same (Yan et al., 2013; Zhu et al., 2010). Our experimental data showed that TreS could convert about 43.62% maltose to trehalose, accompanied by about 23.85% glucose as a byproduct after 10h of incubation. Other studies suggest that most TreS enzyme could produce glucose as a by product except Pseudomonas stutzeri CJ38 (Lee et al., 2005). It is reported that glucose normally can inhibit the enzyme activity (Chen et al. 2006) and lowers the conversion rate from maltose to trehalose (Wei et al. 2004). Several other studies suggest that the trehalose synthases that produce less or no glucose as a byproduct have a higher production for trehalose of about 70% to 80% (Lee et al. 2005; Chen et al., 2006; Nishimoto et al., 1995,Nishimoto et al., 1996). As TreS possess a weak hydrolytic activity (Zhu et al., 2010), it could be the reason as to why a high amount of glucose is produced. TreS could produce trehalose from maltose with a single step. Maltose is relatively cheap and this pathway could be an alternative method for industrial trehalose production. A number of Trehalose Synthase enzymes from different bacterial strains (Nishimoto et al., 1995; Nishimoto et al., 1996 ; Chen et al., 2006; Zdzieblo and Synowiecki, 2006; Wei et al., 2004; Gao et al., 2013; Yan et al., 2013; Jiang et al., 2013; Liang et al., 2013; Filipkowski et al., 2013) have been identified and characterized. This study provided the characteristics of trehalose synthase from Acidiplasma sp. MBA-1 for the trehalose catalysis metabolism.
In our experiment, enzyme TreS produced from Acidiplasma spMBA-1 can catalyze a considerable amount of maltose into trehalose in a single step reaction. We know that maltose is a relatively cheap substrate. Hence, TreS could be used as an alternative commercial enzyme to produce trehalose commercially. There is a drawback though. A significant amount of glucose is being produced as a byproduct which hinders the production of commercial trehalose. If it is possible to suppress glucose production by genetic modifications, it could enhance trehalose production. Beside, enzyme immobilization technique can be used to improve trehalose production further.
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Biologically active compounds play an important role in the regulation of seed germination and sprout growth and development, particularly hormones (abscisic acid, ethylene) (Alberdi, Corcuera, 1991; Hermann et al., 2007), polyphenols (hydroxi-cinnamic acids, flavonoids, tannins, flavolignans) (Blazhey, 1977; Pourcel et al., 2006), coumarins (Adkins, Bellairs, Loch, 2002) and saponins (Vasilieva, 2000). Pericarps of sugar beet seed ball formed from walls of fruit during the formation of the embryo vary with the formation of various tissue structures: exocarp, mesocarp, and endocarp (Comparative anatomy of seeds, 1991), which functions are due to the peculiarities of anatomical structure, chemical nature and distribution of metabolites. It is shown that highly active compounds that can significantly affect germination of sugar beet seeds (Beta vulgaris L.) can be found predominantly in pericarp (Chiji, Tanaka, Izawa, 1980; Taylor et al., 2003), especially in mesocarp tissue (Juntilla, 1976).
The known secondary metabolites of sugar beet are mainly represented by isoflavones, flavonones, dihydroflavonols and their glycosides (betavulgarin, betagarin, irisone B) having the properties of phytoalexins (antifungal and antibacterial activity); betalamine alkaloids (betalamic acid as a predecessor of betalaines and xanthines), which are pigments and antioxidants; simple indoles and bisindole alkaloids, isoindoles (inhibitors of germination for Beta sp.); diterpenoids and triterpenoids (saponins), for example, glycosides of oleanolic acid, caryophyllene, hederagenin and bisdesmosites (with antibacterial, ihtiotoxic antiulcerogenic action) (Dictionary of Natural Products, ver.22., 2014).
The purpose of this research was to separate the water-soluble compounds from the residues of sugar beet pericarps into fractions and to investigate their biological activity.
MATERIAL AND METHODS
Extraction of polar compounds
The polar compounds from the pericarp residues of Ivanivskyi MS 33 hybrid were extracted in double-distilled water at 40 °C for 24 h (1:10). Gel filtration was carried out to separate substances in a column of 9 mm in diameter and 150 mm in height filled with Sephadex G-25.
Secondary metabolite profiling was performed by DAD-RP-HPLC on Agilent 1100 system using 2-eluent scheme (eluent A = 0,05 M aqueous solution of H3PO4; B = acetonitrile), column – Agilent Poroshell® 120, 2.7 μm, 2.1×150 mm, temperature control 20°C, sample volume 5 μl, flow rate 0.2 ml/min, analysis time up to 80 min, elution profile – wide range linear gradient from 0% B in A to 100% B in 30 min, then isocratic B with flow accelerated to 0.6 ml/min and column temperature increased to 40 ° C. Wavelength detection at 206, 254, 300, 350 and 450 nm was used to determine the most organic compounds (including terpenoids), most of substances with aromatic structure in molecule, phenylpropanoids (mainly cinnamic acids), flavonoids (flavones and flavonols), carotenoids and chlorophylls, respectively. The spectra were recorded at peak maximum in the range 200-800 nm in order to elucidate the nature of secondary metabolites and attribute to certain groups of substances. This is not an exact chemical identification but the assumption based on the chromatographic behavior and spectra of separated components. Thus, flavones and flavonols are characterized by two distinct maximums at 260 and 350 nm. Lots of phenylpropanoids (hydroxycinnamic acids) and also isoflavones, flavanones, dihydroflavonols – by large maximum (often with shoulder) at 300-320 nm. Cinnamic itself, hydroxybenzoic acids and lignans have an absorption maximum in the range 280-300 nm [Dictionary of Natural Products, ver. 22.2 Copyright © 2014 Taylor & Francis Group. – URL: http://dnp.chemnetbase.com (2014)]. Reproducibility of HPLC analysis was monitored using the mixture of nine alkylphenones (Sigma-Aldrich) from acetophenone to myristophenon. The error of sample injection does not exceed 2%, while the retention time deviation mostly ranges within 5%. Processing and visualization of chromatograms and absorption spectra were carried out using Agilent ChemStation® and CorelDraw® software.
Determination of phenolic compounds
The total content of phenolic compounds in the extracted fractions was determined with the aid of spectrophotometric method (scanning spectrophotometer, Optizen Pop, South Korea) using the Folin & Ciocalteu’s reagent (Sigurbjoorsson, 1995). The calibration graph was drawn in terms of gallic acid (Merk, Germany).
Determination of flavonoids
The content of flavonoids was determined spectrophotometrically (at λ = 419 nm) through successive addition of 200 μl of the separated fractions of the extract to 200 μl of 0.1 M solution of AlCl3 and 300 μl of 1M CH3COONa. The calibration graph was drawn in terms of quercetin (Merk, Germany) (Laboratory manual on pharmacognosy, 2003).
Determination of phytotxic activity of metabolites
The phytotoxic activity of metabolites from the pericarp residues was studied on pure test cultures of Chlorella vulgaris 106 on solid nutrient media. Aqueous extract of the pericarp residues (30 μl) was introduced into cells of 6 mm in diameter. Test culture was incubated in a thermostat at +25 °C. The biological activity of the extracted substances was evaluated in terms of growth inhibition as following; on the 5th, 7th and 12th day (Grodzinskii, 1983).
Determination of bacteriostatic effect of the extracts
To determine the bacteriostatic effect of the extracts, 0.1 g, 0.5 g and 1.0 g of pericarp residues of Ivanivskyi MS 33 hybrid were added to 10 g of wet soil, thoroughly mixed and incubated in a thermostat at + 25°C for 24 h in sterilized test tubes. Soil sampling for the experiment was carried out in agricultural plots from a depth of 3˗5 cm. The experiment was carried out in five replication. The bacteria from the soil samples were cultured under sterile conditions on the Zvyagintsev nutritional medium (Zvyagintsev, 1987).
The results of biotests were analysed using the specialized program Image Pro-Premier 9.1. Statistical data was processed using Statistica 6.0.
RESULTS AND DISCUSSION
Extraction and study of biologically active substances from pericarp
According to published data, polar chemical compounds of sugar beet pericarp are known to have the ability to inhibit seed germination in many plant species. Medium polar inhibitors include flavonoids, bisalkaloids and isoindole compounds (Morris, Grierson, Whittington, 1984). In previous research, we found that in most studied genotypes of sugar beet, pericarp of seed ball contains complexes of compounds with clear biologically active action. Most of those compounds are salts of carboxylic acids and phenolic compounds: condensed tannins (proanthocyanidins, isoflavones, dihydroflavonols, flavones, flavonols and their glycosides, as well as simple indoles, isoindoles (inhibitors of germination), bisindole alkaloids, diterpenoids and triterpenoids (glycosides of caryophyllene, hederagenin and bisdesmosites) (figure 1).
Figure 1 HPLC of sugar beet pericarps extract: there are component designations: 0 – components with no retention (hydrophilic substances restrained pool (free organic acids, amino acids, etc.); B – betalamic acid; P – condensed tannins (proanthocyanidins); F1 – isoflavones, flavanones, dihydroflavonols and their glycosides (viz. irisone B, betavulgarin, betagarin); F2 – flavones, flavonols and their glycosides; I1 – simple indole and bisindole alkaloids; I2 – isoindoles (Beta germination inhibitor); T –di- and triterpenoids (saponins), viz. oleanolic acid and hederagenin glycosides and bisdesmosides; S – sterols and their esters. Abscissa – retention time, min, ordinate – signal detector, mAU /milli-absorbance unit. Detecting wavelengths designated five channels nm.
Having accumulated in the tissues of the mesocarp and endocarp of seed ball the biologically active substances form endogenous (tissue) and exogenous (phytogenic spheres) biochemical barriers, the ecological significance of which is active regulation of the processes of seed waking up and suppressing phytopathogenic microorganisms at the stage of the formation of sugar beet sprouts (Klyachenko et al., 2015). However, quantitative indices do not give any unambiguous answer to questions about the influence of the multicomponent system of endometabolites of sugar beet pericarp on seed germination. That is why their separation is needed.
By gel filtration (Sephadex G-25 filled column) of pericarp aqueous extracts, we isolated four groups of phenolic substances in terms of their molecular weight. Results are shown in the figure 2.
Figure 2 Separation of phenols (a) and flavonoids (b) of aqueous extracts of sugar beet pericarps using Sephadex G-25 column
Indicators of the ratio of phenolic compounds to flavonoids in the extracts of seed balls and pericarp residues had four maxima in terms of their molecular weight (Tab 1).
Table 1 Distribution of phenols (Fn) and flavonoids (Fl) in the volume of solvent (n = 4)
|V, мл||Pl (Sb)*||Per *||Per / Pl (Sb)|
|Fn *||Fl*||Fn / Fl||Fn||Fl||Fn / Fl||Fn||Fl|
|1||1,6 ± 0,06||0,7 ± 0,03||2,2 ± 0,09||3,6 ± 0,15||1,1 ± 0,04||3,4 ± 0,14||2,3 ± 0,09||1,5 ± 0,06|
|2||0,7 ± 0,03||0,7 ± 0,03||1,0 ± 0,04||18,4 ± 0,74||2,2 ± 0,09||8,4 ± 0,33||28,2 ± 1,13||3,2 ± 0,13|
|3||13,1 ± 0,52||1,1 ± 0,04||12,2 ± 0,49||99,0 ± 3,96||10,7 ± 0,43||9,3 ± 0,37||7,6 ± 0,30||10,0 ± 0,4|
|4||16,4 ± 0,65||2,0 ± 0,08||8,4 ± 0,34||77,8 ± 3,11||7,0 ± 0,28||11,1 ± 0,44||4,8 ± 0,19||3,6 ± 0,14|
|5||13,6 ± 0,54||1,0 ± 0,04||13,4 ± 0,54||35,1 ± 1,40||2,8 ± 0,11||12,4 ± 0,50||2,6 ± 0,10||2,8 ± 0,11|
|6||10,4 ± 0,42||1,7 ± 0,07||6,2 ± 0,25||31,3 ± 1,25||1,7 ± 0,07||18,9 ± 0,76||3,0 ± 0,12||1,0 ± 0,04|
|7||10,6 ± 0,42||2,7 ± 0,11||4,0 ± 0,16||36,9 ± 1,48||2,4 ± 0,10||15,3 ± 0,61||3,5 ± 0,14||0,9 ± 0,04|
|8||20,9 ± 0,84||2,0 ± 0,08||10,4 ± 0,42||41,4 ± 1,66||2,4 ± 0,10||16,9 ± 0,68||2,0 ± 0,08||1,2 ± 0,05|
|9||14,0 ± 0,56||0,7 ± 0,03||19,1 ± 0,76||45,0 ± 1,80||2,8 ± 0,11||15,9 ± 0,64||3,2 ± 0,13||3,8 ± 0,15|
|10||12,1 ± 0,49||0,5 ± 0,02||23,6 ± 0,94||35,7 ± 1,43||1,9 ± 0,08||18,6 ± 0,74||2,9 ± 0,12||3,7 ± 0,15|
|11||5,4 ± 0,22||0,5 ± 0,02||10,2 ± 0,41||22,5 ± 0,90||1,2 ± 0,05||19,5 ± 0,78||4,2 ± 0,17||2,2 ± 0,09|
|12||2,8 ± 0,11||0,7 ± 0,03||3,8 ± 0,15||20,4 ± 0,82||1,9 ± 0,07||10,9 ± 0,44||7,3 ± 0,29||2,5 ± 0,10|
|13||1,9 ± 0,08||0,6 ± 0,02||3,1 ± 0,12||9,4 ± 0,38||1,0 ± 0,04||9,4 ± 0,38||4,9 ± 0,20||1,6 ± 0,06|
|14||6,0 ± 0,24||0,6 ± 0,03||9,3 ± 0,37||9,2 ± 0,37||0,7 ± 0,03||13,0 ± 0,52||1,5 ± 0,06||1,1 ± 0,04|
Note: * Fn – phenolic compounds; Fl – flavonoids; Pl (Sb) – seed balls; Per – pericarp; maximum values marking successive output of a separate fraction of phenolic compounds in the process of gel filtration of extracts are given in bold
The high content of phenols in aqueous extracts of pericarp residues compared to the ones of seed balls was due to better conditions for the dissolution of secondary metabolites in the solvent during the grinding of pericarps in the process of purification of sugar beet seeds.
Study of phytotoxic and bacteriostatic action of endometabolites
The biological activity of the isolated compounds was different in terms of type of action. To illustrate, the eluents of the first volumes (3 ml) of water stimulated the germination of radish seeds. Substances contained in the following volumes of eluents (4-5 ml) had inhibitory action. This indicates that there are both highly active inhibitors and growth promoters among the endometabolites of the sugar beet pericarps (figure 3).
Figure 3 Influence of endometabolites of pericarp beet on germination of radish seeds: a – control treatment; b – 2-3 ml of solvent; c – 4-5 ml; d – 7-8 ml; e – 9-10 ml; f – 11-13 ml; bar – 20 mm; * – різниця достовірна по відношенню до контролю при р < 0.05, significant difference relative to the control p < 0.05
The model of the formation of endogenous and exogenous biochemical barriers, which was developed by us on the basis of empirical data, was confirmed in the course of the experiment. Results are shown in the figure 4.
Figure 4 Effect of aqueous extracts of pericarps on soil microorganisms and propagation of chlorella cells: a, b – results of soil microorganism culture from soil samples; c, d – when sugar beet seed balls are incorporated into the soil; e, f, g – aggregation of chlorella cells when adding seed ball extracts; k(c) – control treatment.
When sugar beet seed residues were introduced into the soil, the number of microorganisms decreased. The inverse relationship was found between the number of colonies formed on agar nutrient medium and the concentration of seed ball residues per unit volume of soil.
Consequently, water-soluble fractions in the process of gradual leaching from pericarp tissues can create a specific biochemical sphere in the soil around the seed that for some time inhibits the development of certain types of microorganisms and fungi that potentially can do harm to young sprouts of sugar beet at the early stages of their development. Sugar beet seed germination was increasing along with increasing soil moisture, with the number of microorganisms’ colonies decreasing. Results are shown in the figure 4 a-d.
The content of biologically active compounds in sugar beet pericarps revealing phytoelicite properties of phytoelicitors (isoflavones, flavanones, dihydroflavonones and their glycosides) and seed germination inhibitors (simple indoles and bisindole alkaloids) allows us to consider the possibility of utilisation of seed residues.
It is known that pericarp makes about 75˗85 % of seed ball (Oehme J. Rüben, 1981). As 1 g of pericarps contains 20 to 40 mg/g phenolic compounds, according to our previous research (Klyachenko et al., 2015), from 1 kg of pericarp residues up to 40 g of phenolic metabolites, indole alkaloids and triterpene saponins can be extracted. In the process of purifying and coating seeds, seed residues will be up to 800 g/kg, consequently, the maximum amount of secondary metabolites that can be extracted from 1 kg of seed residues will be 15-30 g of valuable organic compounds of commercial interest and can be used as natural biologically active substances.
As a result of the conducted studies it was established that in the pericarp of sugar beet fruits there are compounds with a pronounced biologically active effect. In the extracts of sugar beet pericarps, there are phenols, flavonoids and their glycosides that are highly active inhibitors and growth promoters and therefore can be used as natural biologically active substances.
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Himachal Pradesh (India) is well known for its topography and culture throughout India where the climatic conditions range from semi-tropical to semi-artic; having the altitude from 350 meters to 6975 meters above the sea level. These all lead to a wide variation in the living pattern of the hill folks including their costumes and dialects, food pattern, and celebrations; where a wide range of foods (fermented or non-fermented) are prepared and consumed traditionally to adapt to cold climatic conditions (Savitri and Bhalla, 2007). A wide range of fermented foods, both alcoholic (sur, lugdi, ghanti, angoori) and non-alcoholic (sidu, bhatura, seera) have been consumed throughout the state since time immemorial (Bhatia et al., 1977; Thakur et al., 2004; Savitri and Bhalla, 2007; Kanwar et al., 2011; Joshi et al., 2012). Among these, alcoholic beverages are attracting the attention of researchers because of their wide variability, popularity and acceptance among the tribes. However, the production of these beverages is confined to the specific or local areas according to the availability of raw materials such as angoori (grapes) is prepared in Kinnaur; chhang (barley) in Lahulspiti; sur (finger millet) in Kullu, Mandi and Kangra; lugdi (rice) in the Kangra, Kullu and Mandi (Thakur et al., 2004; Senthilkumar, 2009; Kanwar et al., 2011; Joshi et al., 2015). These beverages have an aesthetic appeal as well as a religious and sociocultural value. For instance, sur is offered to local gods like Hurang Narayan and lugdi is sprinkled on the guests to welcome them (Savitri and Bhalla, 2007; Joshi et al., 2015). As per the traditional knowledge available, these beverages possess numerous health benefits such as improving digestion, treatment of stone and jaundice (Das et al., 2012).
Different researchers have already mentioned lugdi in literature as an alcoholic traditional beverage (Bhatia et al., 1977; Thakur et al., 2004; Savitri and Bhalla, 2007; Kanwar et al., 2011) but a detailed study is still lacking which needs to be explored. Lugdi is also known as jhol and is a popular low alcoholic drink in Palampur region of Kangra district. Besides its intoxicating and health properties, it also has socio-cultural value. Hilly areas have witnessed a significant change in alcoholic beverage consumption behaviour; however, this beverage is still popular among local folks. Starting from household, lugdi production has its identity in small-scale industries. Still the uniformity and quality of the product is an issue and scientific interventions are needed to improve the quality and maintain the identity of product for longer time. Therefore, the present study was designed to point out the basic steps of its production along with its analysis with the aim of fulfilling this research gap.
Palampur is a tehsil and city in the Kangra district of Himachal Pradesh, India. It is situated 25.2 Km west to Kangra city and has an area of 429 Km2 including 428.62 Km2 rural area and 0.67 Km2 of the urban area (Anonymous, 2018a). It extends between 32.11°N latitude to 76.53°E longitude. The mean annual temperature of Palampur is 19.1°C, which ranges from a minimum of 9.9°C in January to maximum of 27.1°C in June (Anonymous, 2018b).
A field survey using an open-ended pretested questionnaire was conducted in the Palampur region (Banodoo, Paraur, Bandla, and Paprola) of the district Kangra, Himachal Pradesh, India. The workers in the production unit were interviewed regarding the production (inocula, processing steps), sale, consumption pattern and the believed positive health benefits (trusted positive medical or health benefits) of lugdi. The customers were also explored for the reasons of lugdi consumption.
Procurement and analysis of samples
Freshly prepared lugdi was sampled from different lugdi production units in sterilized bottles of 200 mL capacity and analysed for various quality attributes under laboratory conditions within three days of procurement. Physico-chemical characteristics viz. total soluble solids, total solids, titratable acidity, and pH were analysed as per the standard methods (AOAC, 1984). Reducing sugar was estimated by di nitro salicylic acid method while, total sugars were determined using phenol sulphuric method (Sadasivam and Manickam, 1991). Ethanol was measured colorimetrically by potassium dichromate method (Caputi et al., 1968). Protein content was determined as per the standard procedure proposed by Lowry (Sadasivam and Manickam, 1991) while, amino acids were measured by the ninhydrin method (Lee and Tunekazu, 1966). Fusel alcohols were measured by the method given by Guymon et al. (1961). Total phenols were measured colorimetrically using gallic acid as standard (Singleton and Rossi, 1965), total esters were measured by the method given by Liberty (1961).
Fourier Transform Infrared Spectroscopy (FTIR)
The collected samples of phab and lugdi were analysed qualitatively using FTIR analyser (Shimadzu 8400S FTIR spectrometer, equipped with KBr beam splitter). Phab was ground to a fine powder using lab scale grinder while lugdi was filtered through whatman filter paper and was dried at room temperature (30-35°C). Approximately 5 mg of each sample along with 5 mg KBr was used for analysis and FTIR spectrophotometer was operated at a spectral range of 4000-400 cm-1 with a maximum resolution of -0.85 cm-1. The spectra obtained for the samples were interpreted by following the guidelines given by Stuart (2004).
To conduct the onsite sensory evaluation of lugdi, a local panel of the consumers was constituted. The panel was provided with a sensory proforma describing the terminology for colour, appearance and taste. The samples of the lugdi were served and the members of panel were asked to give their response on the proforma as per the standard procedure (Joshi, 2006).
The information generated from the survey is presented in tabular form or flowsheets while data obtained from the physico-chemical analysis was analyzed using Graph Pad Prism (La Jolla, CA, USA) software. The results are expressed as means ± standard deviation of the respective measures.
Production of lugdi
As per the information obtained in survey, lugdi has been produced at both household and commercial levels. Commercial production of lugdi was limited to the Palampur region of district Kangra, Himachal Pradesh. In this region four small scale lugdi production units were located at Banodoo, Paprola, Paraur and Bandla. General information of the lugdi production process has been provided in Table 1.
Table 1 General information on lugdi preparation in different small scale commercial units of Kangra, Himachal Pradesh (n=4)
|Raw material||Broken rice|
|Utensil used to carry fermentation||Earthen pots|
|Lot preparation||20-35 kg|
|Inoculum used||1-1.5% or 300-500 g/35 kg|
|Temperature for inoculum addition||Lukewarm (28-32°C)|
|Saccharification (duration)||Summer- 2-3 days
Winter- 6-7 days
|Fermentation (duration)||Summer- 2-3 days
Winter- 6-7 days
|Filtration||1st filtration- with locally made sieve by cutting and puncturing plastic cans
2nd filtration- nylon sieve for fine filtration
|Recovery||100-110 litres per 35 kg batch|
|Bottling||In cleaned glass bottles|
|Sale||30-40 INR per 700 mL bottle|
|Storage life||2-3 days in summers; 6-7 days in winters|
|Residue utilization||Animal feed|
Where, n = number of lugdi production units covered under study
The survey revealed that broken rice was used as the main substrate for commercial production of lugdi (Table 1). The reason for the use of broken rice might be the less cost of brokens as compared to head rice. The results of present study were in agreement with Senthilkumar (2009), who reported the use of local rice varieties like Ram juvanae, Totu, Gharsai, Chinnu, Zhinni and Pandpermal in lugdi production.
Phab, crude inocula was used to carry out the fermentation in all the production units covered under the present study. It was not prepared locally or insitu but was procured from the Khampas of Sidhbaddi (Dharamshala) region of Himachal Pradesh. These results are in corroboration with the findings of Thakur et al. (2004); Senthilkumar (2009); Kanwar et al. (2011). In earlier studies, Bhatia et al. (1977); Angmo and Bhalla (2014) have discussed the phab production process in detail.
The detailed process of lugdi production has been provided in figure 1(a, b and c). Depending upon the local demand a batch of 20-35 kg of rice was being cooked in a big vat (Figure 1b) on a traditional wooden chullah or gas stove to a point of softness except the Paprola unit where rice was overcooked till the browning of the outer layer to impart good colour and flavour in the final product. The development of colour and flavour might have been due to the caramelization of sugars as the decomposition of sugars results in the formation of volatile and brown-coloured compounds (Kroh, 1994). The cooked rice was spread in a thin layer and cooled to about 28-32°C. Phab granules were ground to a fine powder and mixed uniformly at the rate of 350 g-500 g per 35 kg of cooked and cooled rice. The prepared mixture was filled in pre-sterilized earthen pots (20 L approx.) with the capacity of 1/2nd-3/4th of total volume, capped tightly with clothes and covered with gunny bags or woollen clothes to provide the required incubation temperature to facilitate the saccharification. The saccharification was allowed for 2-3 days in summers and 6-7 days in winters or until the mixture was converted to a cream-like slurry. After the completion of saccharification, the slurry was transferred (3-4 kg) to sterilised earthen pots (20 L approx.). To the slurry, water was added up to the brim and the mixture was allowed to ferment (2-3 days during summers and 6-7 days in winters). The increased fermentation rate during the summers might have been due to the shortening of lag phase as the lag phase decreases with increase in temperature (Merrit, 1966). During fermentation, the mouth of the pot was left uncovered and the absence of air bubbles on the top of the pot was used as an indicator for completion of fermentation.
After completion of fermentation, the fermented material was filtered to separate lugdi from the spent material. The filtration was a two-step process involving coarse and fine filtration. Coarse filtration was carried out with a large sieve made by cutting plastic cans from one side and engraving holes on the other side (Figure 1c). It was followed by a fine filtration with a fine mesh strainer to get the clear product, which was further filled, into pre-washed glass bottles.
Onsite sensory analysis of the samples revealed that product had a turbid appearance with colour ranging from white to yellowish white (Table 2). The turbid appearance might be due to the presence of unfermented starch in the drink as starch produces a turbid solution (Sandhu and Singh, 2007) whereas the difference in colour might be due to the caramelization of rice as discussed earlier. The flavour of collected samples varied from a sweet alcoholic to sour alcoholic depending on the freshness of the sample i.e. freshly prepared samples had a sweet alcoholic taste whereas stored samples were sour alcoholics (Table 2). The development of sour taste might be due to the growth of lactic acid along with fermentation as the phab contains a mixed microflora i.e. Saccharomyces cerevisiae and Lactobacillus species (Joshi and Sandhu, 2000; Thakur et al., 2004; Joshi, 2016).
As per the survey, it was observed that lugdi was not pasteurized and had a shelf life of 2-3 days in summers and 6-7 days in winters. The reason for not pasteurizing lugdi might be the lack of pasteurization facilities at the production units. It was also observed in the survey that product had a good demand in Palampur region and was sold onsite at a sale price of INR 30-40 per 750 mL bottle. The spent material was used as a feed for the mules (Figure 1c).
Figure 1a Flow diagram of the traditional technology of the lugdi production as per the information gathered from lugdi production units
Figure 1b Pictorial representation of lugdi production process at small-scale commercial units
Figure 1c Pictorial presentation of filtration process and end-use of lugdi
Table 2 Sensory attributes of the collected lugdi samples
|Colour||White to yellowish white|
|Appearance||Turbid with starch settled at the bottom of bottle|
|Aroma||Acido-alcoholic (mixture of acid and alcohol)|
|Taste||Sour with the lingering taste of starch|
Physicochemical characteristics of collected lugdi samples
The collected lugdi samples were analysed for the different physicochemical parameters (Table 3), which revealed a wide variation even among the batches of the product prepared in the same unit. This variation might be due to the non-standardized process and recipe used for lugdi production. Among the collected samples, total solids ranged from 6.3% to 7.8%, total soluble solids ranged from 2.5 to 3.9°B, total sugars were in between 0.48% to 0.72% and reducing sugars was in the range of 0.15% to 0.42%. The results of these parameters were in agreement with the results reported by Senthilkumar (2009). The ethanol content of the product varied from 4.61% to 5.68%. Kanwar et al. (2011) had reported similar ethanol content in lugdi samples collected from LahulSpiti, Himachal Pradesh (India). However, Thakur et al. (2004) had reported a higher ethanol content of 8.5% in lugdi samples collected from Kullu district of Himachal Pradesh (India). Analysis also revealed pH, titratable acidity, protein, total phenols, esters and fusel alcohol in the ranges of 2.82 to 3.55, 0.83 to1.15% as lactic acid, 568.42 to 618.24 mg/100 mL, 49.1 to 100.7 mgGAE/100 g, 85.34 to 108.78 mg/L and 22.48 to 32.94 mg/L, respectively. A pH of 3.21 and titratable acidity of 0.20% has been reported by Tamang and Thapa (2006) in bhattijannr (a fermented rice beverage of north India). Chiang et al. (2006) reported a pH of 3.4 and acidity of 0.86% in tapai (an alcoholic beverage prepared from glutinous rice). Phab might be the major source of phenolics in the lugdi as phab consists of several herbs (as information gained during the survey). Esters are synthesized by yeasts during fermentation via several complex pathways (Saerens et al., 2010). The wide variation in the physicochemical attributes of the collected samples might be due to the non-standardized recipe and local variation in process of the lugdi production.
Table 3 Physico-chemical attributes of collected lugdi samples
|Total solids (%)||6.3-7.8||6.83±0.35|
|Acidity (% as lactic acid)||0.83-1.15||0.97±0.18|
|Free amino acids
|Total phenols (mg/100 mL)||127.56-168.54||148.58±20.51|
|Total esters (mg/100 mL)||85.34-108.78||96.93±11.72|
|Fusel alcohols (mg/l00 mL)||22.48-32.94||27.59±5.23|
FTIR spectrophotometer is among one of the most important analytical tools used to study physiochemical and conformational properties of wide range of samples including food (Stuart, 2004). The infrared spectra of phab and lugdi have been provided in (Figure 2a and 2b). The area of the respective peaks (phab and lugdi) is tabulated in Table 4. FTIR wave numbers are associated with the absorption bands of the compounds present in the foods. The interpretation of the peaks confirmed the presence of numerous nutritional and phytochemical compounds including sugars, amino acids, amides, aliphatic compounds, phenols and alcohols. Panda et al. (2014) reported similar functional compounds on FTIR analysis of bael wine.
Table 4 FTIR spectra of phab and lugdi
|2||521.76||48.691||KBr||1034.84||33.895||Alcohols, C=C-CH2-OH, C-O stretch (phenolics)|
|3||575.77||48.283||NaCl||1254.74||6.156||Alcohols, phenols, Ar-O-H, C-O stretch|
|4||762.87||63.167||Aromatic compounds C-H bend||1463.06||6.841||Alanine, valine|
|5||860.28||40.29||β-D-Sucrose, β-D-glucopyranoside||1547.93||7.554||Protein amide II|
|6||930.68||50.758||Carboxylic acids C–O–H out-of-plane bending||1647.26||8.821||Proteins|
|7||1021.34||171.132||Phenols C-O stretching, O-H aromatic||1739.85||5.625||Carbohydrates, aldehydes and ketones aliphatic aldehyde C=O stretching|
|8||1154.43||73.021||Cellulose||1835.33||3.878||C=O stretching (carbonyl group)|
|9||1458.23||20.931||Alanine, valine||2361.91||10.574||X-H stretching, X is phosphorus or silicon|
|10||1539.25||18.343||Lysine, protein, amide II||2927.08||31.278||O-H stretching (carboxylic acids), aliphatic compounds|
|11||1651.12||21.561||Proteins, amide I||3619.54||7.21||O–H stretching (phenols)|
|12||2362.88||41.079||X-H stretching, X is phosphorus or silicon||3743.96||10.776||O–H stretching (water)|
|13||2927.08||173.138||O-H stretching (carboxylic acids), aliphatic compounds||3848.12||3.638||X-H stretching region|
|14||3342.75||5.326||O–H stretching (water)|
Figure 2a FTIR spectra for phab
Figure 2b FTIR spectra for lugdi
Production and consumption of lugdi is an inherent part of the traditional culture of Palampur, Himachal Pradesh, India. Physicochemical and FTIR analysis of the product confirmed that it is a rich source of nutrients and phytochemicals like amino acids and phenolics, which strengthen its traditional claim of numerous health benefits. However, the availability of the commercial beverages and reluctance of the new generation to get involved in the production of these beverages has put the traditional heritage under threat. It was concluded from the present study that the lugdi production process was not standardized and varied from place to place. Physicochemical analysis has also revealed a wide variation among the composition of the samples. Therefore, there is a need for an intervention by regulatory bodies to standardize the recipe and production process to produce a uniform product in terms of ethanol and other alcohols. This could also help in shielding the rich traditional heritage of the state.
CONFLICT OF INTEREST: There is no conflict of interest among authors.
Acknowledgements: Authors are thankful to the owners and workers of the small-scale lugdi production units located in Banodoo, Paraur, Bandla, and Paprola, Palampur, Himachal Pradesh for sharing their knowledge, allowing working with them and photographing the lugdi production process. Authors are also thankful to local folks for sharing their knowledge on the lugdi production process and believed health benefits.
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Post-harvest losses are one of the major causes of the loss of fresh vegetables during the supply chain (Nunes et al., 2012). Aspergillus niger is one of the major causes of black rot of plain (Prakash et al., 1988). Therefore, control of A. niger during the preservation is very necessary. The widespread use of synthetic fungicides for preserving agricultural products has significant limitations such as handling of hazards, pesticide residues, and risk to health and the environment (Dharini et al., 2014). Current trends in antimicrobial agent research from the natural origin in which plant-derived essential oils are of great interest. In previous studies, lemongrass oil is considered an essential oil for safe and effective natural preservatives which has an effective antibacterial activity (Vazirian et al., 2012). Essential oils are natural products consisting of a complex mixture of volatile molecules (Mahian et al., 2016), which are liquid, soluble in organic solvents and insoluble in water (Bakkali et al., 2008). The evaluation of the antimicrobial activity, the essential oils are often diluted at different concentrations by emulsifying agents (Burt, 2004). Hilbig et al. (2016) reported that emulsifier has the negative effect on the antimicrobial activity of essential oil (Hilbig, 2016), whereas the combination of ethanol and other antimicrobial agents (chitosan, potassium sorbate …) show the antimicrobial activity better than using in single agent (Romanazzi et al., 2007; Karabulut et al., 2005). Besides essential oil, ethanol also showed antimicrobial effect (Gianfranco et al., 2007). However, to achieve good antibacterial efficacy, ethanol is commonly used at high concentrations leading to increased production costs and the risk of fire safety (Ozgur et al., 2005). Ethanol is made up of hydrophilic (-OH) and hydrophobic (CH3CH2-) radicals. Hydrophilic radicals (-OH) to help dissolve the polarizing elements and ions. Short chain CH3CH2– hydrocarbons can attract non-polar molecules. Therefore, the combination of ethanol and essential oil can both increase the dilution effect of the oil in the media and can synergize with the essential oil to enhance the effectiveness of the antimicrobial. Although the combination of ethanol and the essential oil is very promising, very few studies exist on the synergic effect of ethanol and lemongrass oil on fungi. In this study, the antifungal activity of lemongrass oil and ethanol used alone or in combination against A. niger was evaluated by agar disk diffusion method, mycelial growth inhibition and broth dilution method to determinate MIC and MFC. The ultraviolet (UV) absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity. Scanning electron microscopy (SEM) performed to observe the morphology of Aspergillus niger spores.
MATERIAL AND METHODS
Aspergillus niger M1 was obtained from strain collection of Faculty of Food Technology, Ho Chi Minh City University of Food Industry. A. niger was grown in PDA (Potato Dextrose Agar) medium at 30oC for 6 days. Then, the mass was harvested by rinsing plates with PDB (Potato Dextrose Broth). The freshly grown microbial cell at approximately 6 log CFU/mL was used for the evaluation of the antifungal activity.
The essential oil in this study was lemongrass oil (Cymbopogon flexuosus) from Tien Giang province, the City is located at 10°25′N 106°10′E in the southern region of Vietnam. Lemongrass was hand-collected and immediately used to obtain lemongrass oil by steam distillation. Lemongrass oil was stored in glass vials in the absence of light until gas chromatography analysis and to test its antifungal activity. The essential oil was directly analyzed by gas chromatography coupled to mass spectrometry (Agilent GC 7890B GC System, 7010 GC/MS Triple Quad). The used column was an HP-5MS (30 m long, 0.25 mm and 0.25 μm film thickness). The operating conditions were as follows: Helium was used as a carrier gas with a back pressure of 0.8 atm; flow rate of 1.0 mL/min; split 1:20; injection volume 0.2 μL; The injector temperature was 250oC and the oven temperature program started at 60 for 5 min and then increased at the rate of 5oC/min up to 150oC at 5oC/min, and increased from 150oC to 280oC at 10oC/min. The constituents in the essential oils were identified by computer matching of their mass spectral fragmentation patterns with those of compounds in the data bank NIST 98 and Wiley 275 library. Lemongrass oil and ethanol used alone or in combination with or without tween 20 (0.3% v/v) (an emulsifying agent) were used as antifungal agents for the next step.
Evaluation of antifungal activity of lemongrass oil and ethanol used alone or in combination against A. niger
The antifungal activity of lemongrass oil and ethanol was carried out according to Lieu et al. (2018a) with slight modifications. Briefly, the A. niger suspensions were spread over the surface of PDA (Potato Dextrose Agar) plates (at final concentration 6 log CFU/mL approximately) and allowed to dry in 5 min. Lemongrass oil (10; 20; 40; 80 and 100 µL/mL) and ethanol (10%; 20%; 40%; 80% and 100% v/v), used alone or in combination with or without tween 20 0.3% v/v (an emulsifying agent) were spotted on PDA agar (15 µL), that containing A. niger spores and tween 20 were used as controls. The plates were incubated at 30oC in 24 hours. After 24h incubated, Petri dishes were examined by inhibition zone.
Determination of the antifungal effect of lemongrass oil and ethanol on mycelial growth
The effect of emulsifying agents to antifungal activity of lemongrass oil on mycelial growth was carried out as assay previously described (Boubaker et al., 2016) with slight modifications. Briefly, PDA supplemented individually with antifungal agents such as lemongrass oil; ethanol and the combination of lemongrass oil with ethanol. The mixture was poured into Petri plates. Afterward, the Petri plates were incubated with A. niger by spore culture at the middle of the Petri plates. The agar Petri plates were then incubated at 30oC in seven days. The control consisted of PDA medium supplemented with emulsifying agents without lemongrass oil. The antifungal activity was expressed in terms of percentage of mycelial growth inhibition (MGI) and calculated according to the following formula:
MGI (%) = (C-T)/C x 100%
Where C and T represent mycelial growth diameter in control and lemongrass oil treated Petri plates, respectively
Determination of MFC Using Broth Dilution Method
Minimum fungicidal concentration (MFC) of lemongrass oil and ethanol were carried out according to the previous description (Lieu et al., 2018a). A range of lemongrass oil (100÷1,000 µL/L in tween 20 0.3% v/v) and ethanol (10,000÷100,000 µL/L) concentrations use alone or in combination were prepared in PDB (Potato Dextrose broth) medium. Each flask was inoculated with 6 log CFU/mL of the A. niger spores. Flasks containing only tween 20 (without lemongrass oil) were used as the control. The flasks were incubated at 30oC in an orbital shaking incubator (100 rpm) for 48h. One mL of culture was taken from each flask (where growth was not observed) for serial dilution to make the inoculum of 6 log CFU/mL and inoculated on DRBC (Dichloran Rose Bengal Chloramphenicol) agar plates at 30oC in three days
UV absorption and conductivity determination
The experiments were conducted based on a previously published method (Suxia et al., 2015) with slight modifications (adding the equation). Briefly, the spore of A. niger diluted to the test concentration by optical density (at final concentration 6 log CFU/mL approximately) and separated into several flasks. The lemongrass oil and ethanol used alone or in combination at MFCs were added to each flask, except to the control and incubated at 30oC. During incubation time, 15 mL sample was removed from the flasks at 0, 2, 4, 6, 8, 19, 12, 14 and 16 hours of incubation. The samples were immediately filtered using 0.22 µm syringe filters to remove bacteria and recorded by spectrophotometer at 260 nm and by the conductivity meter. The effect of antifungal agents to the leakage of cytoplasmic contents was evaluated by the following equation:
δOD: delta values of UV absorption
ODt: OD value at t time
OD0: Initial OD value
δconductivity: delta values of electrical conductivity
Ct: Electrical conductivity value at t time
C0: Electrical initial conductivity value.
The treated and untreated samples after 8h of incubation were observed through the Scanning Electron Microscope (SEM) to evaluate the effect of antifungal agents to the spore of A. niger.
The data were subjected to analysis of variance (ANOVA) using SigmaPlot 11 followed by Student-Newman-Keuls t-test to compare means, with a significance level of 5% when the significant difference between treatments was noted. All tests were performed in triplicate and the data expressed as means ± standard deviation.
The chemical composition of the lemongrass oil
Major components of lemongrass oil were confirmed and listed in Table 1. β-citral was identified as the main compound with the highest peak area percentage (41.2%). α-citral (39.80%) was the second major compound detected in the lemongrass oil, followed by Neryl acetate (8.1%); Caryophyllene (1.5%), Linalool (1.5%), Caryophyllene oxide (1.1%). Other compounds such as Verbenol, Carveol, Eucalyptol … were found to be at the trace level.
Table 1 Major components of lemongrass oil
*Percent of the peak area of the evaporated organic compound
Antifungal activity of essential oils and ethanol in the agar diffusion method
The antifungal activity of lemongrass oil and ethanol were shown in Figure 1 and 2. The antifungal zone depends on the concentration of antifungal agents and their combination. The diameter of the antifungal zone of lemongrass oil, ethanol, and their combination was 4.67÷21 mm; 4.67÷10 mm and 4÷29 mm respectively (Figure 1).
Figure 1 The impact of lemongrass oil and ethanol used alone (a) or in combination (b) against A. niger (LO: lemongrass oil; E: ethanol; TW: tween 20)
Figure 2 The diameter of the antifungal zone of ethanol 90% v/v (a), lemongrass oil 50 µL/mL in tween 20 (b) and the combination of lemongrass oil (50 µL/mL) and ethanol (100 µL/mL) in tween 20 (c).
In case of alone treatments, lemongrass oil (in tween 20) showed more antimicrobial effect than ethanol with the minimum inhibitory concentration (MIC) was 5 μL/mL, while the MIC values of ethanol was 500 μL/mL (50% v/v) which was 100 times higher than the lemongrass oil. The result in case of combined treatments showed that the antimicrobial activity of the lemongrass oil in tween 20 was no significant different (p>0.05) with the lemongrass oil (without tween 20) in ethanol (Figure 1). The inhibition zone diameter of the combination of lemongrass oil (in tween 20) with ethanol (10%) were higher than these lemongrass oils and ethanol at the same concentration, but the MIC value of the combination of lemongrass oil with ethanol (10%) was not different compared to lemongrass oil that using alone. However, the combination of lemongrass oil in tween 20 with ethanol (20% v/v) show the best result (Figure 1, 2). The MIC value of this combination was at 2.5 μL/mL.
Effects of lemongrass oil and ethanol on Mycelial Growth
The effects of lemongrass oil and ethanol on A. niger mycelial growth are shown in Figure 3. The results showed that, after five days of incubation, the colony diameter of A. niger in control sample was 90 mm while in the treatment samples, the colony diameter of A. niger was decreased significantly through the MGI (%) values. In case of alone treatment, the MGIs (%) of ethanol at 10,000; 20,000 and 50,000 ppm were 1.11%; 63.33% and 50.33%. The MGIs (%) of lemongrass oil (in tween 20 0.3% v/v) and the combination of lemongrass oil with ethanol 10,000 ppm or 20,000 ppm at 150 and 300 ppm of concentration were 67.22% and 83.89%; 58.33% and 73.89%; 62.22% and 82.22% respectively (Figure 3).
Figure 3 Effect of lemongrass oil and ethanol used alone or in combination on mycelial growth of A. niger (LO: lemongrass oil; E: ethanol; TW: tween 20)
The combination of lemongrass oil (in tween 20 0.3% v/v) with ethanol 20,000 ppm showed the best result that the MGI (%) was 100%. The results showed that the MGIs (%) of lemongrass oil were significantly higher than ethanol. The combination of lemongrass oil (in tween 20) with ethanol is necessary to enhance the antifungal activity.
Antifungal activity of essential oils in the liquid phase
According to the results obtained (Table 2), the MFC values of lemongrass oil and ethanol has the same result as that observed in the agar diffusion test. In case of alone treatments, the MFC values of lemongrass oil (in tween 20 0.3% v/v) was 250 ppm which has 200 times lower than ethanol that needs to increase the concentration to 50,000 ppm to reach the same result (Table 2).
Table 2 MFCs value of lemongrass oil and ethanol
|Antifungal agents||MFC (ppm)|
|Lemongrass oil + ethanol 1%||280|
|Lemongrass oil + ethanol 2%||250|
|Lemongrass oil + tween 0.3%||250|
|Lemongrass oil + tween 0.3% + ethanol 1%||180|
|Lemongrass oil + tween 0.15% + ethanol 2%||150|
The pure lemongrass oil, which uses alone without tween 20 or ethanol has the lowest antifungal activity with the MFC value was 450 ppm. In case of combined treatments showed that the MFCs values of lemongrass oil (in tween 20 0.3% v/v) with ethanol (10,000 ppm or 20,000 ppm) were 180 ppm and 150 ppm respectively which was significantly lower than in case of alone treatments. The results showed that ethanol and tween 20 enhanced the antifungal activity of lemongrass oil at low concentration in which the combination of lemongrass oil (in tween 20) with ethanol showed the best results.
The effect of lemongrass oil and ethanol on bacterial cell membrane integrity
The UV absorption and conductivity of A. niger culture supernatants are shown in Figure 4. The results obtained indicate that the UV absorption and electrical conductivity values increased quickly after 16h incubated in the antimicrobial agents (Figure 4). The delta values of UV absorption and electrical conductivity of lemongrass oil 250 ppm (in tween 20); ethanol 50,000 ppm; the combination of lemongrass oil (in tween 20) 150 ppm with ethanol 20,000 ppm were 0.289 and 0.378; 0.235 and 0.290; 0.278 and 0.369 respectively, at 16h incubation and both absorption and conductivity were stable thereafter (Figure 4a; 4c). The delta values of UV absorption and electrical conductivity of the control group were not increased during the experiment (Figure 4b).
Figure 4 Effects of lemongrass and ethanol on UV absorption at 260 nm (a and b) and electrical conductivity (c) of A. niger; a. the antifungal agents without A. niger; b and c. the antifungal agents with A. niger
The morphological changes of the A. niger were observed through SEM to evaluate the effect of the antifungal agents on the A. niger. Figure 5b, 5c shows that the morphological changes of the A. niger spores due to significant wrinkles and distortion in the samples treated by lemongrass oil (in tween 20) or lemongrass oil (in tween 20) combining with ethanol while the A. niger spores in ethanol show slight wrinkles (Figure 5a). The results showed that, after 8h incubation, the morphology of the A. niger spores in the lemongrass oil (250 ppm) in tween 20 sample was wrinkles and distortion (Figure 5b). The similar result also observed in the lemongrass oil (150 ppm) in tween 20 combined with ethanol (20% v/v) (Figure 5c).
Control of A. niger during the preservation is necessary because of their effect caused to post-harvest losses (Prakash et al., 1988; Lieu et al., 2018b). Plain essential oils are gaining interest in their antimicrobial activity. Due to the components of essential oil depending on the variety of plant, the part of the plant considered, harvesting seasons, storage conditions, the concentration of essential oil… as well as the type of tested microorganism (Burt et al., 2004; Tajkarimi et al., 2010, Tyagi et al., 2010; Lieu et al., 2018a), which make it difficult to compare results across laboratories. It is well-known that essential oil affects bacterial cells by many different mechanisms such as the following: disrupt the phospholipid bilayer of the cell membrane; break down the phospholipid membrane which leading to structural breaking, affecting the integrity of cell membranes and changing the permeability of H+ and K+ ions (Dinesh et al., 2013; Dharini et al., 2014). The lemongrass oil with monoterpenes is formed by α-citral and β-citral that the main antimicrobial activity, which showed an inhibitory effect and causing distortion of cytoplasmic membranes of Candida albicans (Tyagi et al., 2010). Similarly, the antimicrobial action of ethanol, which able to penetrate the cell wall, causing protein degradation, lipid dissolution, and finally cellular breakage (Weber et al., 1996).
Figure 5 Scanning Electron Microscope of A. niger spores treated with lemongrass and ethanol. a. A. niger spores were grown in the presence of ethanol 50,000 ppm; b. A. niger spores grown in the presence of lemongrass oil (in tween 20) 250 ppm; c. A. niger spores grown in the presence of the combination of lemongrass oil (in tween 20) 150 ppm and ethanol 20,000 ppm.
In previous studies, ethanol (20% v/v) showed the ability to reduce the spore growth of Botrytis cinerea compared to the control (Ozgur et al., 2005) as well as to reduce the grape damage caused by Botrytis cinerea, while the combination of ethanol and chitosan is necessary to control fungi effectively (Gianfranco et al., 2007). In the present study indicates that ethanol shows the antifungal activity against A. niger at 500 µL/mL in the agar diffusion test and 50,000 ppm in MFC test which has 100 times and 200 times higher than lemongrass oil (5µL/mL and 250 ppm respectively) (Figure 1 and Table 2). These suggest that lemongrass oil was more effective at inhibiting A. niger than ethanol. The differences in the antimicrobial concentration of essential oil in agar diffusion tests and broth dilution assays were reported in previous studies (Boubaker et al., 2016; Lieu et al., 2018a). The differences are due to the type of media, the ability to dilute the essential oil of emulsifiers. Verica et al. (2014) reported that the agar diffusion method is not considered an ideal method for essential oil dilution, because of their volatile and poorly soluble components. This makes the essential oil in the agar diffusion method is required high concentration than broth dilution method to have the equivalent antimicrobial effect (Figure 1 and Table 2). In the MFC assays, pure lemongrass oil showed a lowest antifungal activity (Table 2). Water is not an effective method for dispersing the essential oil due to the insoluble phenolic compounds, leading to a decrease in the antimicrobial activity of essential oil (Laird, 2011). Therefore, the emulsifying agent is necessary to enhance the antifungal activity.
Due to hydrophobic properties, high volatility and flavoring properties of essential oil which could affect the organoleptic quality of food, the essential oil needs to dilute to required concentrations to ensure sufficient antimicrobial effect without affecting the organoleptic properties of the food. In previous studies, the essential oil was usually diluted in emulsifier such as tween 80, tween 20, xanthan gum (Boubaker et al., 2016; Lieu et al., 2018a,b) or other antimicrobial agents (Ghellai et al., 2015) to enhance the solubility of essential oil and reduce the amount of essential oil needed to use. The combination of essential oil 0.5% (v/v) with acetic acid 0.25% (v/v) and lactic acid 0.25% (v/v) showed the antimicrobial effect was equivalent to essential oil 1% (v/v) (Ghellai et al., 2015). However, the combination of essential oil (in the emulsifying agent) and ethanol is poorly reported. The solubility of lemongrass in tween 20 is better than ethanol (data not showed). The antifungal activity of the combination of lemongrass oil (in tween 20) and ethanol was improved significantly (Figure 1, 2, 3, 4). This finding suggests that ethanol has a synergistic effect which enhances the antimicrobial activity of lemongrass oil. Meanwhile, tween 20 helps to disperse the essential oil in the water phase and increasing the antimicrobial activity. The combination of lemongrass oil and ethanol showed the antimicrobial activity increase when increasing the ethanol concentration (over 20% v/v) (data not showed). However, the use of ethanol at high concentration in agricultural product preservation would raise costs and the risk of un-safety (Ozgur et al., 2005). In the present study, the combination of lemongrass oil, tween 20 (0.3% v/v) and ethanol showed the best result which not only minimizes the amount of essential oil but also ensures the antimicrobial activity.
The use of UV absorption assay, an electrical conductivity test, and SEM observation are considered an effective way to evaluate the antifungal effect of antifungal agents. In the previous study, the SEM results showed that, at a concentration of 0.5 µL/mL, lemongrass caused swelling of the cell wall and much of the cell contents in many bacteria were lost when increasing to 2 µL/mL (Jareerat et al., 2011). The antimicrobial components of lemongrass oil can cause yeast deformation or bacterial membrane deformation, leading to leakage of cellular contents (Tyagi et al., 2010; Jareerat et al., 2011). In the present study, the initial absorbance values and electrical conductivity of the samples treated with antimicrobial agents are much different, this is due to the antimicrobial agents caused different absorbance. However, the Figure 4 shows that the UV absorption values and electrical conductivity values in the samples treated by antimicrobial agents that without A. niger were almost unchanged at the point of time (0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h and 16h). These results indicated that the concentration of antimicrobial agents does not affect the changes in absorbance values and electrical conductivity of the culture medium during the experiment. It is interesting to note that, the delta value and SEM result in the combination of lemongrass oil (150 ppm) in tween 20 0.3% v/v and ethanol 20,000 ppm were no significant difference compared to lemongrass oil 250 ppm in tween 20 (0.3% v/v) which 40% higher than that lemongrass oil in the combination treatment. These data suggest that ethanol has a synergic effect which enhances the antifungal activity of lemongrass oil. This combination is necessary to ensure the antimicrobial effect as well as reduce the amount of used essential oil.
The results indicated that the MIC and MFC values of lemongrass oil (in tween 20 0.3% v/v) were 5 μL/mL and 250 ppm respectively that 100 and 200 times lower than ethanol. The antifungal effect of the combination of ethanol and lemongrass oil against A. niger showed better results than used alone. The combination of lemongrass oil (in tween 20) and ethanol (20% v/v) with the inhibition zone and MGI (%) value was at 2.5 μL/mL and 100% respectively that showed the best result. The UV absorption and electrical conductivity values increased quickly during incubated in the antifungal agents, whereas there were almost unchanged in the control samples at the point of time. The SEM results show that the morphological changes of the A. niger spores due to ethanol (50,000 ppm) treatment showed slight wrinkles whereas, in the samples treated by lemongrass oil or lemongrass oil combining with ethanol, A. niger spores were significant wrinkles and distortion. The results showed that ethanol and tween 20 enhanced the antifungal activity of lemongrass oil at low concentration in which the combination of lemongrass oil (in tween 20) and ethanol showed the best results. This combination is necessary to ensure the antimicrobial effect as well as reduce the amount of used essential oil.
Bakkali, F., S. Averbeck, D. Averbeck, M. Idaomar. “Biological effects of essential oils – A review.” Food and Chemical Toxicology 46, 2008: 446–475. https://doi.org/10.1016/j.fct.2007.09.106
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The thermostable DNA polymerases, like other DNA polymerases (E.C 18.104.22.168), catalyze template directed synthesis of DNA from nucleotide triphosphates. Commercial preparations of DNA polymerases have a variety of applications in DNA manipulations in vitro, such as sequencing, labeling, cDNA synthesis etc. (Ishino and Ishino, 2014).
These enzymes are commercially produced in convenient, high-yielding mesophilic hosts such as E. coli, by the use of recombinant DNA technology. DNA polymerases such as Taq polymerase which have an optimal temperature of ~80°C are extensively used in PCR. DNA polymerases from moderate thermophiles have uses in molecular diagnostic techniques such as Loop Mediated Isothermal Amplification (LAMP). It is a low cost alternative to PCR that can be applied in low and middle-income countries for screening /diagnosis of infectious diseases (Mori et al., 2013). LAMP has been observed to be less sensitive to inhibitors in clinical samples when compared to PCR and successful detection of pathogens from minimally processed samples such as heat-treated blood has been reported (Curtis et al., 2008: Sattabongkot et al., 2014). Further, they can be applied for in vitro DNA manipulation techniques in the molecular biology, where a higher temperature reaction is more suitable.The objectives of this project were to clone and over-express the DNA polymerase ǀ (DNAP-ǀ) gene from a thermophilic bacterium, with a view to subsequent scale up.
MATERIALs AND METHODS
The Bacillus licheniformis strain NWMF1 was cultured in LB medium at 55°C with constant shaking. Plasmid DNA isolation was by alkaline SDS method (Sambrook et al., 2012). DNA fragments were purified from agarose gels using Wizard SV gel purification system (Promega USA). All DNA ligations were carried out using Liga-Fast Rapid DNA Ligation System according to manufacturers‘ protocol. Sanger sequencing was outsourced (Macrogen, Korea). DNA and translated amino acid sequences were analyzed by nucleotide and protein BLAST tools using online sequence databases available at NCBI.
Isolation and identification of thermophilic bacteria
The thermophilic bacterium was isolated from soil near a hot water spring at Nelumwewa, Polonnaruwa District, Sri Lanka, by enrichment for growth at high temperature (~60°C). The isolated bacterium was characterized morphologically and biochemically (Grams reaction and catalase test). Molecular identification was based on 16s RNA gene sequencing (using 27F and 534R universal primer pair). The 16s rRNA analysis and biochemical characteristics of bacteria were compared to confirm the bacterial species as Bacillus licheniformis strain NWMF1
Cloning of B. licheniformis DNA polymerase-1 gene
Multiple sequence alignment of open reading frames (ORF) of B. licheniformis strain NWMF1, DNAP-ǀ genes were carried out to identify conserved sequences for designing of the PCR primers. NcoI and BamHI restriction enzyme recognition sequences were added to the 3’ region of the forward and reverse primers respectively. Further, the His-tag purification sequence (CAC)6 was added to the reverse primer. The Ompa (Outer Membrane Protein-A) signal sequence was added to the forward primer. The forward primer, DNAP-1F (5’- GCA TGA CCA TG GGT ATG AAA AAG ACA GCT ATC GCG ATT GCA GTG GCA CTG GCT GGT TTC GCT ACC GTT GCG CAA GCT ATG ACT GAA AAA AAA TTA GTA TT-3’) and the reverse primer DNAP-1R (5’-GCA TGA GGA TCC CTA CAC CACCACCACCACCAC TTT TGC ATC GTA CCA TGAA-3’) were custom-synthesized.
Bacterial genomic DNA was isolated according to the method of Dubnau (1982). The isolated B. licheniformis strain NWMF1 was PCR- amplified from genomic DNA, gel-purified and cloned into PGEMR-T easy vector (Promega) and transformed into E.coli JM109 high efficiency competent cells. Recombinant plasmid DNA was isolated and confirmed by sequencing.
The recombinant plasmid was digested with Nco1 and BamHI restriction enzymes to remove the cloned the DNAP-ǀ gene and subsequently gel-purified. It was then cloned into the NcoI/BamHI site of pET 28a+ expression vector and transformed into E. coli BL-21(DE3)pLysS high efficient competent cells, compatible with kanamycin selection. Selected recombinants were screened by colony PCR and confirmed by sequence analysis as previously stated. Additionally, Interpro (EMBL-EBI) protein analyses were performed to detect conserved domains in the translated protein sequence. Structural and functional features of the protein were obtained from Predictprotein online prediction tool, UniProt protein database and Protein Data Bank (PDB).
Purification and analysis of expressed recombinant DNAP-ǀ protein
Extracellular DNA Polymerase-ǀ production from recombinant E. coli BL-21(DE3)pLysS cells were induced by IPTG. The supernatant of the LB broth was separated and over expressed DNA Polymerase-ǀ were purified by using MagneHis protein purification system (Promega, USA) according to the manufacturer’s instructions.
The activity of recombinant, purified DNAP-ǀ was demonstrated by using a modified protocol of a previously optimized 30-cycle PCR program to amplify the alkaline protease gene from B. licheniformis (Wanigasekara et al., 2016). In this protocol, the extension step was carried out at 62°C and1ml of enzyme (out of a total of 60 ml of His-tag purified enzyme extract) was added in each cycle at the primer annealing step, in order to accommodate the moderate thermostability of the recombinant DNAP-ǀ.
Protein concentration was measured by the bicinchoninic acid assay (Smith et al., 1985) with BSA as the standard. Protein samples were prepared for SDS-PAGE as described in Tang et al., (2001) and analyzed according to the method of Laemmli (1970).
RESULTS AND DISCUSSION
The selected bacterium showed optimum growth at 55°C. The Gram positive, motile, catalase positive, spore forming, rod shaped bacterium was identified as Bacillus licheniformis and the strain was designated as NWMF-1.
The PCR amplified DNAP-ǀ gene from B. licheniformis NWMF-1 was observed between 2500 bp and 3000 bp (Figure 1).
Figure 1 Gel electrophoresis photographs of PCR amplified DNAP-ǀ gene from Bacillus licheniformis strain NWMF1. Lane 1: Negative control, Lane 2: PCR amplified DNAP-ǀ gene, Lane 3: 1kb DNA ladder (Promega).
Sequence analysis revealed that the complete coding sequence was 2640bp. The sequence has been submitted to the NCBI data base and is available under the accession number: MF536412.1. Nucleotide BLAST (NCBI) of the complete nucleotide sequence obtained for B. licheniformis NWMF-1 DNAP-ǀ gene showed 99% identity with DNA polymerase-ǀ gene from B. licheniformis DSM13 = ATCC 14580 (AE017333.1) and B. licheniformis BL1202 (CP017247.1).
PCR-amplification of the alkaline protease gene (1140 bp) from B. licheniformis (NCBI data base accession number: MF496035.1) using His-tag purified recombinant DNAP-ǀ revealed a DNA fragment of expected size during electrophoresis on a 0.8% agarose gel. This indicated that the recombinant DNAP-ǀ enzyme was active (Figure 2).
Figure 2 Gel photograph of PCR-amplified alkaline protease gene using recombinant DNAP-ǀ enzyme. Lane 1: negative control (No enzyme), Lane 2: positive control (amplified using GoTaq polymerase), Lane 3: 1kb ladder, Lanes 4 and 5: amplified products using recombinant DNAP-ǀ enzyme.
SDS-PAGE of His-tag purified recombinant DNAP-ǀ revealed a single protein band between 75-100kDa (Figure 3). The size of the protein was approximately 92 KDa, as obtained by the EXPASy – ProtParam tool.
Figure 3 SDS PAGE gel photograph for His-tag purified DNAP-ǀ. Lane 1: DNAP-ǀ. Lane 2: Broad ranged protein ladder.
The complete amino acid sequence obtained for B. licheniformis NWMF-1 DNAP-ǀ consisted of 879 amino acids and it showed 100% identity with Bacillus licheniformis strain NWMF1 DNA polymerase-ǀ protein sequences from UniProt (EMBL-EBI).
Structural features of the DNAP-ǀ was obtained using ProteinPredict online tool (Yachdavet al, 2014). The active site of the mature peptide contains 24 amino acid residues (aspartic acid; positions 11, 61, 112, 113, 136, 138, 833, histidine; positions 685, 832, serine; position 620, threonine; positions 614, 616, 793, glutamate; position 110, arginine; position 618, 705, 792, glutamine; position 615, 800, leucine; position 619, valine; position 831, lysine; position 709, tyrosine; position 717 and asparagine; position 796). The conserved 5’ to 3’ exonuclease domain (PIN_53EXO) of family A DNA polymerases was also observed in B.licheniformis NWMF-1 DNAP-ǀ. The predicted secondary structure consists of 36.2% loop, 55.4% helix and 8.4% strand (Rost et al, 2004). The predicted protein contains a single disulphide bond.
A moderately thermostable bacterium isolated from soil near a hot water spring was identified as Bacillus licheniformis. The strain was designated as NWMF-1. The DNA polymerase-ǀ gene from Bacillus licheniformis strain NWMF1 was over expressed in an E. coli expression system. The His-tag purified DNAP-ǀ showed polymerase activity while carrying the histidine tag and is expected to improve when the tag is cleaved. The cloned DNAP-ǀ has potential for application in loop mediated isothermal amplification (LAMP) and other molecular biology techniques, especially in developing countries.
Acknowledgments: MSc vote Department of Biochemistry and Molecular Biology, Faculty of medicine, University of Colombo.
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WANIGASEKARA, W. M. J. M. B., JAYASENA, S. M. T., WITHARANA, A. W. C. P., MATHEW, C. P. D. W., WIJESUNDERA, W. S. S. 2016. Cloning of Alkaline Protease Gene from Thermophilic Bacillus licheniformis into an E.Coli Expression System.(Proceding of the work of the 3rd Edition of the Young Researchers in Biosciences International Symposium) Cluj-Napoca: Romania, 54. https://www.researchgate.net/publication/312492199_Cloning_of_DNA_polymerase_gene_from_thermophilic_Bacillus_licheniformis_into_an_Ecoli_expression_system
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The genus Salmonella (S), an Enterobacteriaceae member, is facultative aerobic intracellular bacteria that capable of causing varieties of illnesses in a wide range of hosts. Salmonella has been recognized as the leading cause of foodborne diseases in human, leading to 1.3 billion cases of gastroenteritis, 16 million cases of typhoid infection, and about 3 million deaths around the globe every year (Bhunia, 2007). A variety of food items have been incriminated in human Salmonellosis outbreaks, especially those derived from animals such as beef, pork, poultry, and eggs. Nevertheless, poultry and poultry products are the most associated foods with Salmonella outbreaks in humans (EFSA and ECDC, 2013; Antunes et al., 2016).
Most of Salmonella food poisoning outbreaks are caused by Salmonella enterica subspecies enterica, though, over 2,500 Salmonella serovars have been identified and new serovars are designated frequently (Hassan et al., 2015). The pathogenicity of Salmonella is managed by several factors established by virulence genes that enable the pathogen to express its virulence in the host and eventually produce the characteristic symptoms of the disease, besides, antibiotic resistance-associated genes. The invasion gene invA is unique for the genus Salmonella, so it represents a suitable DNA target in diagnostic approaches. invA is situated in Salmonella pathogenicity islands coding to produce certain proteins, which are accountable for the invasion of the pathogen into the host cells (Valdez et al., 2009). Plasmid-encoded fimbria (pef) locus assists the bacteria to adhere to the intestinal epithelial cells (Friedrich et al., 1993), and spv is another plasmid-located virulence gene, which suppresses host innate immune system to bacterial infection (Yang et al., 2016). Salmonella outer proteins (sop A-E) encoded with sop gene are responsible for the pathogen penetration through cell membrane deformities and rearrangement of the host cell cytoskeletons (Borges et al., 2013), besides hilA, which is considered a hyper invasive locus (Lostroh et al., 2000). While, the virulence gene stn arbitrates the production of enterotoxins and was found to be linked to causing acute gastroenteritis in infected hosts (Zou et al., 2012).
In addition to virulence, the emergence of antibiotic resistant strains of Salmonella has become a significant public health risk. It was found that the improper application of antibiotics in livestock production for preventive and therapeutic purposes, as well as growth promotion is a noteworthy factor in appearance of antibiotic resistant bacteria in animals and poultry. These resistant bacteria which is subsequently transferred to human through food chain is challenging the efforts of serving safe food for consumers (Antunes et al., 2016).
Factors related to the antibiotic resistance and virulence of Salmonella may be situated on chromosomes, plasmids, integrons and transposon. Integrons are genetic elements that play an important role in the dissemination of resistance genes between bacteria owing to the associated conjugative plasmids. There are two main groups of integrons: mobile integrons and chromosomal integrons (Cambray et al., 2010). Based on the sequence of the encoded integrases, five different classes of mobile integrons have been identified. Even though, only classes 1, 2, and 3 have been reported in the spread of multidrug-resistance phenotypes, all 5 classes have been associated with antibiotic-resistance determinants (Siriken et al., 2015). Furthermore, several non integron-related resistance genes have also been reported in Salmonella; such as quinolones resistance determinants (qnrA, qnrB, qnrS, aac(6′)-Ib-cr, qepA) and β-lactams resistance-related genes (blaTEM, blaCTX, and blaCMY-2) (Robicsek et al., 2006; Wiesner et al., 2016). Accordingly, the identification of virulence genes, antibiotic resistance genes and integrons in Salmonella isolates from retail chicken became a very crucial approach for risk assessment of such pathogen in this food item.
Therefore, the present study was conducted to estimate the prevalence and antibiotic susceptibility/resistance of Salmonella species from retail chicken meat (CM) and pooled giblets (PG) in Beni-Suef governorate, Egypt, beside serological identification of the isolates. As well as, molecular identification of virulence genes, β-lactams and quinolones resistance-associated determinants and integrons (classes 1, 2, and 3) using Polymerase Chain Reaction (PCR).
MATERIALS AND METHODS
A total of 50 broiler chicken carcasses (around 40 days age at time of slaughtering) were randomly collected from retail poultry outlets in Beni-Suef governorate, Egypt during 2016 – 2017. All carcasses were purchased in fresh state directly after slaughtering in the retail market. Each carcass was represented by a specimen from muscle as chicken meat (CM) and another from gizzard, liver, and heart as pooled giblets (PG), with a total of 100 samples (50 CM and 50 PG). The collected samples were identified and wrapped separately in sterile polyethylene bags to be directly transferred without delay in an icebox to the laboratory for further preparation and examination.
Isolation and identification of Salmonella
Isolation and morphological and biochemical identification of Salmonella spp. from CM and PG was done according to the standard protocol of ISO 6579 (2002). Briefly, 25 g specimen was aseptically removed from each CM and PG of each carcass, and then homogenized with 225 mL of 0.1 % sterile buffered peptone water (Biolife; Italy). Afterwards, the homogenate was incubated at 36 ± 1 °C for 16-20 h. Then 0.1 and one mL of the pre-enrichment broth were inoculated into 10 mL of Rappaport-Vassiliadis (RV) broth (Biolife; Italy) and 10 mL of Müller-Kaufmann Tetrathionate (MKT) broth (Biolife; Italy), respectively. The enrichment broths were further incubated at 41.5 ± 0.5 °C (for RV) and 36 ± 1 °C (for MKT) during 18-24 h. A loopful from each broth after incubation was streaked onto each of Salmonella-Shigella (SS) and Xylose Lysine Desoxycholate (XLD) agar plates and incubated at 36 ± 1 °C for 18-24 h. Colorless colonies with black centers on SS and slightly transparent red colonies with black center on XLD agar plates were suspected as Salmonella and selected for further identification procedures. Suspected colonies of Salmonella were identified morphologically by Gram`s staining, and biochemically by oxidase, indole, methyl red, voges proskauer, citrate utilization, triple sugar iron (TSI), and urease tests. All morphologically and biochemically confirmed Salmonella isolates were consequently identified by serology based on somatic (O) and flagellar (H) antigens by slide agglutination using commercial antisera (SISIN, Berlin) following the Kauffman-White scheme (Popoff et al., 2004). See the schematic protocol in Figure 1.
Figure 1 Schematic protocol of detailed procedures of Salmonella isolation, identification and antibiotic resistance testing during the study. MDR (multidrug resistant).
Antibiotic sensitivity/resistance testing
All serologically identified Salmonella enterica subspecies enterica serovars were tested for their antibiotic susceptibility pattern by disc diffusion technique according to the Clinical and Laboratory Standards Institute, CLSI (2018). Commercial discs of antibiotic (Oxoid, UK) soaked with cefotaxime (30 μg), ampicillin (10 μg), ciprofloxacin (5 μg), ceftazidime (30 μg), amikacin (30 μg), piperacillin-tazobactam (100/10 μg), amoxicillin-clavulanic acid (20/10 μg), nalidixic acid (30 μg), aztreonam (30 μg), and tetracycline (30 μg) (Oxoid, UK) were used. Multidrug resistant isolate (MDR) is defined as that isolate resist three or more antibiotics belonging to different antibiotic categories.
Molecular detection of Salmonella virulence genes and integrons using PCR
MDR Salmonella serovars were molecularly identified for the presence of 6 virulence genes (hilA, stn, pef, invA, sopB and spvC), 8 antibiotic resistance determinants, out of them, 5 are linked to quinolones resistance (qnrA, qnrB, qnrS, aac(6`)-Ib-cr and qepA) and the rest three antibiotic resistance genes are related to beta-lactams resistance (blaTEM, blaCMY-2 and blaCTX), in addition to identification of integrons classes 1,2 and 3. Genomic DNA was extracted from overnight bacterial cultures using Qiagen DNA extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. Specific primers obtained from Metabion (Germany) for each target gene were used for DNA amplification using uniplex PCR. The sequences of primers and sizes of amplified segments are listed in Table 1. Primers were utilized in a 25 µL reaction tube containing 12.5 µL of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µL of each primer of 20 pmol concentrations, 6 µL of DNA template, and 4.5 µL of nuclease free water. The reactions were performed in an Applied Biosystem 2720 thermal cycler. Briefly, initial denaturation step was done at 94 °C for 5 min, then followed by 35 cycles of 94 °C for 45 sec, afterwards, 40 sec of annealing was applied according to the temperatures showed in Table 1. Subsequently, an extension step at 72 °C for 45 sec and a final extension step at 72 °C for 10 min were conducted. The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. 20 µL of the PCR products were loaded in each gel slot. The fragment sizes were determined using Gelpilot 100 bp and 100 bp plus DNA Ladders (Qiagen, Germany, GmbH) and Gene ruler 100 bp ladder (Thermo Scientific, Germany). Afterwards, the gel was photographed by a gel documentation system (Alpha Innotech, Biometra).
Table 1 Primers sequences of target genes, amplicon sizes and annealing temperatures.
|Primers sequences||Amplified segment (bp)||Resistance /virulence *||Annealing temperature
|qnrA||ATTTCTCACGCCAGGATTTG||516||(R) Quinolones||53||Robicsek et al. (2006)|
|aac(6′)-Ib-cr||CCCGCTTTCTCGTAGCA||113||(R) Quinolones||53||Lunn et al. (2010)|
|qepA||CGTGTTGCTGGAGTTCTTC||403||(R) Quinolones||50||Cattoir et al. (2008)|
|54||Colom et al. (2003)|
|54||Pérez-Pérez and Hanson (2002)|
|60||Archambault et al. (2006)|
Disseminate the resistance
|50||Kashif et al. (2013)|
Disseminate the resistance
Disseminate the resistance
|hilA||CATGGCTGGTCAGTTGGAG||150||(V)||59||Yang et al. (2014)|
|Stn||TTGTGTCGCTATCACTGGCAACC||617||(V)||59||Murugkar et al. (2003)|
|invA||GTGAAATTATCGCCACGTTCGGGCAA||284||(V)||54||Oliveira et al. (2003)|
|sopB||TCAGAAGRCGTCTAACCACTC||517||(V)||58||Huehn et al. (2010)|
* The role of the target gene; either antibiotic resistance or virulence activity. (R) means that the target gene is responsible for antibiotic resistance, while (V) means that the target gene is responsible for specific virulence activity of the strain.
Prevalence and serotyping of Salmonella spp. in CM and PG
According to the results of morphological and biochemical identification of Salmonella isolates, it was found that out of 50 CM samples, 36 samples harbored Salmonella (72%), while out of 50 PG samples, 32 contained Salmonella (64%) (data not shown). The serotyping of 36 and 32 Salmonella isolates from CM and PG revealed that S. Infantis represented 52.8 and 50% in CM and PG, respectively. While S. Kentucky was identified with incidences of 36.1 and 25% of Salmonella isolates from CM and PG, respectively. Each of S. Ferruch and S. Kottbus represented 6.25% of PG isolates, conversely, it was failed to find them in CM. Although, S. Colindale represented 5.55% of CM Salmonella isolates, it was none in PG. On the other hand, S. Virchow represented 5.55 and 12.5% in CM and PG, respectively (Table 2).
Table 2 Distribution of Salmonella serovars in examined chicken meat and pooled giblets.
|Serovars (antigenic formula)||Chicken meat (n=50)||Pooled giblets (n=50)||Total (n=100)|
|No. (%*) (%**)||No. (%*) (%**)||No. (%*) (%**)|
|S. Infantis (O: 6,7,14; H1: r; H2: 1,5)||19 (38) (52.8)||16 (32) (50)||35 (35) (51.47)|
|S. Ferruch (O: 8; H1: e, h; H2: 1,5)||0 (0) (0)||2 (4) (6.25)||2 (2) (2.94)|
|S. Kentucky (O: 8, 20; H1: i; H2: Z6)||13 (26) (36.1)||8 (16) (25)||21 (21) (30.88)|
|S. Kottbus (O: 6, 8; H1: e, h; H2: 1,5)||0 (0) (0)||2 (4) (6.25)||2 (2) (2.94)|
|S. Virchow (O: 6,7,14; H1: r; H2: 1,2)||2 (4) (5.55)||4 (8) (12.5)||6 (6) (8.82)|
|S. Colindale (O: 6,7; H1: r; H2: 1,7)||2 (4) (5.55)||0 (0) (0)||2 (2) (2.94)|
|Total Salmonella isolates, no. (%)||36 (72)||32 (64)||68 (68)|
Where %* represents the percentage in relation to the number of examined samples, while %** represents the percentage in relation to the number of Salmonella isolates.
Antibiotic resistance/susceptibility of Salmonella serovars
The results illustrated in Table 3 show the antibiotic resistance/susceptibility of Salmonella serovars (n= 68) isolated from CM and PG. High rates of resistance were explored by the serotyped Salmonella isolates, it was evident that the highest rate of resistance was against nalidixic acid, when all isolates showed resistance against it (100%), followed by tetracycline (89.7%), cefotaxime (67.5%), and then 64.7% of the isolates were resistant to both ciprofloxacin and ampicillin, followed by ceftazidime (45.6%), and amikacin (35.3%) comes after, then amoxicillin-clavulanic acid (29.4%), while the lowest resistance was against each of piperacillin-tazobactam and aztreonam (23.5%). All isolates of S. Virchow (n=6) were resistant to all antibiotics investigated during the study expect piperacillin-tazobactam and aztreonam. Likewise, the two isolates of S. Kottbus (100%) isolated from PG were resistant to all tested antibiotics. Additionally, high levels of resistance were found in S. Kentucky, followed by S. Infantis and then S. Ferruch and S. Colindale.
Interestingly, 64.7% (44 out of 68) of the isolates were resistant to ciprofloxacin, which is considered the drug of choice against Salmonella spp. in animals and humans, whereas, the other 24 isolates (35.3%) showed intermediate resistance, thus, none of the isolates exhibited any sensitivity to ciprofloxacin. Therefore, we report in the present study the emergence of ciprofloxacin-resistant isolates of S. Kentucky (100%), S. Virchow (100%), S. Kottbus (100%), and S. Infantis (42.8%) from CM and PG in Egypt (Table 3).
Table 3 Antibiotic resistance of isolated Salmonella serovars from chicken meat and pooled giblets.
Number of resistant serovars (resistant %)
|Chicken meat (36)||11 (30.6)||24 (66.7)||23 (63.9)||10 (27.8)||9 (25)||29 (80.6)||17 (47.2)||8 (22.2)||36 (100)||33 (91.7)|
|S. Infantis (19)||5 (26.3)||9 (47.4)||6 (31.6)||0 (0)||3 (15.8)||15 (78.9)||3 (15.8)||2 (10.5)||19 (100)||18 (94.7)|
|S. Kentucky (13)||4 (30.8)||13 (100)||13 (100)||8 (61.5)||6 (46.15)||12 (92.3)||12 (92.3)||6 (46.2)||13 (100)||13 (100)|
|S. Virchow (2)||2 (100)||2 (100)||2 (100)||2 (100)||0 (0)||2 (100)||2 (100)||0 (0)||2 (100)||2 (100)|
|S. Colindale (2)||0 (0)||0 (0)||2 (100)||0 (0)||0 (0)||0 (0)||0 (0)||0 (0)||2 (100)||0 (0)|
|Pooled giblets (32)||13 (46.6)||20 (62.5)||21 (65.6)||10 (31.3)||7 (21.9)||23 (71.9)||14 (43.8)||8 (25)||32 (100)||28 (87.5)|
|S. Infantis (16)||4 (25)||6 (37.5)||5 (31.3)||0 (0)||2 (12.5)||11 (68.8)||2 (12.5)||2 (12.5)||16 (100)||14 (87.5)|
|S. Ferruch (2)||0 (0)||0 (0)||2 (100)||0 (0)||0 (0)||0 (0)||0 (0)||0 (0)||2 (100)||0 (0)|
|S. Kentucky (8)||3 (37.5)||8 (100)||8 (100)||4 (50)||3 (37.5)||6 (75)||6 (75)||4 (50)||8 (100)||8 (100)|
|S. Kottbus (2)||2 (100)||2 (100)||2 (100)||2 (100)||2 (100)||2 (100)||2 (100)||2 (100)||2 (100)||2 (100)|
|S. Virchow (4)||4 (100)||4 (100)||4 (100)||4 (100)||0 (0)||4 (100)||4 (100)||0 (0)||4 (100)||4 (100)|
|Total sensitive (%)||36 (52.9)||0 (0)||13 (19.1)||32 (47.1)||24 (35.3)||14 (20.6)||27 (39.7)||39 (57.3)||0 (0)||7 (10.3)|
|Total intermediate (%)||8 (11.8)||24 (35.3)||11 (16.2)||16 (23.5)||28 (41.2)||2 (2.9)||10 (14.7)||13 (19.2)||0 (0)||0 (0)|
|Total resistant (%)||24 (35.3)||44 (64.7)||44 (64.7)||20 (29.4)||16 (23.5)||52 (67.5)||31 (45.6)||16 (23.5)||68 (100)||61 (89.7)|
AK (amikacin 30 µg), CIP (ciprofloxacin 5 µg), AMP (ampicillin 10 µg), AMC (amoxicillin-clavulanic acid 20/10 µg), TZP (piperacillin-tazobactam 100/10 µg), CTX (cefotaxime 30 µg), CAZ (ceftazidime 30 µg), ATM (aztreonam 30 µg), NA (nalidixic acid 30 µg), and TE (tetracycline 30 µg).
Integron profile and antibiotic resistance genes
The data illustrated in Table 4 clarify the antibiotic resistance pattern, integron profile (classes 1, 2 & 3) and antibiotic resistance-associated genes in 13 isolates which were selected from MDR Salmonella serovars isolated from CM and PG. The selected 13 isolates showed variable degrees of MDR which ranged from 0.2 (resistant to 2/10 antibiotics) to one (resistant to 10/10 antibiotics). Integron class 1 was detected in 100% of the isolates, while integron class 3 existed in 92.3% of the isolates. Interestingly, the only isolate that had not integron 3 showed the lowest MDR pattern (0.2). On the contrary, integron class 2 was absent in all isolates (0%).
Regarding plasmid-mediated quinolone resistance (PMQR) genes, it was surprising that the three most significant qnr genes (qnrA, qnrB and qnrS) were absent in all molecularly identified isolates, and aac(6′)-Ib-cr and qepA either. However, 53.8% and 100% of the isolates were found resistant to ciprofloxacin and nalidixic acid, respectively (Table 4).
Concerning β-lactams resistance-related genes, blaTEM, blaCMY-2 and blaCTX were found in 100%, 30.7% and 53.8% of the isolates, respectively (Table 4).
Table 4 Resistance pattern, integron classes (1, 2 and 3) and antibiotic resistance genes among MDR Salmonella serovars isolated from chicken meat (CM) and pooled giblets (PG).
|No.||Serovar (Origin)||Resistance pattern||MDR
|Integron profile||Antibiotic resistance genes|
|1||Kentucky (CM)||AK, CIP, AMP, AMC, TZP, CTX, CAZ, ATM, NA, TE||1||+||–||+||–||+||+||–|
|2||Kentucky (PG)||CIP, AMP, CTX, CAZ, NA, TE||0.6||+||–||+||–||+||+||–|
|3||Kentucky (CM)||CIP, AMP, AMC, TZP, CTX, CAZ, NA, TE||0.8||+||–||+||–||+||+||–|
|4||Infantis (CM)||CTX, NA, TE||0.3||+||–||+||–||+||+||–|
|5||Virchow (CM)||AK, CIP, AMP, AMC, CTX, CAZ, NA, TE||0.8||+||–||+||–||+||–||+|
|6||Kentucky (CM)||CIP, AMP, NA, TE||0.4||+||–||+||–||+||–||+|
|7||Infantis (PG)||AK, NA, TE, CTX||0.4||+||–||+||–||+||–||+|
|8||Infantis (CM)||NA, TE, CTX||0.3||+||–||+||–||+||–||+|
|9||Infantis (CM)||CIP, AMP, TZP, CTX, CAZ, ATM, NA, TE||0.8||+||–||+||–||+||–||–|
|10||Infantis (CM)||CIP, CTX, NA, TE||0.4||+||–||+||–||+||–||+|
|11||Infantis (PG)||AK, AMP, NA, TE||0.4||+||–||+||–||+||–||+|
|12||Infantis (CM)||AK, AMP, CTX, NA, TE||0.5||+||–||+||–||+||–||+|
|13||Infantis (PG)||NA, TE||0.2||+||–||–||–||+||–||–|
AK (amikacin 30 µg), CIP (ciprofloxacin 5 µg), AMP (ampicillin 10 µg), AMC (amoxicillin-clavulanic acid 20/10 µg), TZP (piperacillin-tazobactam 100/10 µg), CTX (cefotaxime 30 µg), CAZ (ceftazidime 30 µg), ATM (aztreonam 30 µg), NA (nalidixic acid 30 µg), and TE (tetracycline 30 µg). CM: chicken meat, PG: pooled giblets. MDR ratio (multiple drug resistance ratio), for instance, MDR 0.6 means that this strain was resistance to 6 out of 10 antibiotics tested (6/10=0.6). PMQR genes, plasmid-mediated quinolone resistance genes (qnrA, qnrB, qnrS, aac(6′)-Ib-cr and qepA).
Molecular identification of virulence-associated genes
The results of molecular identification of virulence genes in 13 MDR isolates of Salmonella were illustrated in Table 5. The data showed that each of the invasion gene invA, the hyper invasive locus hilA and sopB gene were detected in all isolates (100%). While, stn gene was found 94.7%. Conversely, the plasmid-encoded fimbria (pef) locus was not detected in any of the identified isolates, as well as the other plasmid-located virulence gene spvC was distinguished in only one out of 13 isolates.
Table 5 Virulence genes among MDR Salmonella serovars isolated from chicken meat (CM) and pooled giblets (PG).
|No.||Serovar (Origin)||Virulence genes|
The surprisingly higher rates of Salmonella spp. in the present study than previous reports (Ammar et al., 2016; Gharieb et al., 2015) could be attributed to the slaughter of live birds inside the poultry retail markets with absence of veterinary supervision and without even a minimum hygienic measure during different stages of carcass preparation, processing, and handling. Additionally, cross contamination from workers, equipment and utensils used during carcass preparation could be very important source of contamination (Antunes et al., 2016).
Although, S. Typhimurium has not been detected in the present study, it was determined as a predominant serovar in poultry meat in some previous studies in other areas in Egypt such as Gharieb et al. (2015). It could be attributed to the difference in the location of sample collection in the current study, as to the best of our knowledge, this is the first study to emphasize the prevalence of Salmonella serovars in retail poultry meat in Beni-Suef, Egypt. Alternatively, S. Infantis and S. Kentucky were reported as predominant Salmonella serovars in poultry meat in the present study. Incidentally, S. Infantis is capable of triggering septicemia and death in both children and adults (Fonseca, 2006). As well as, the European Food Safety Authority and European Centre for Disease Prevention and Control (EFSA and ECDC, 2015) denoted that S. Infantis is the second most predominant serovar in broiler meat and the fourth most dominant one in human non-typhoidal salmonellosis in Europe. Regarding S. Kentucky, Weill et al. (2006) detected 197 S. Kentucky isolates in French travelers during 2000 through 2005, among them 17 ciprofloxacin-resistant strains were detected in 16 patients got the infection during or instantly after travel to Egypt (10 patients), Kenya, Tanzania, and Sudan. Consistent with the current result, Weill et al. (2006) reported that poultry is the main animal reservoir of S. Kentucky. Concerning to S. Virchow, the third highly reported serovar in this study, however it causes mild infection in humans, it produces severe illness in immunocompromised persons; therefore, the European Union has given it a priority for control of entry the food chain (Arnold, 2010).
Resistance to ciprofloxacin has been reported exceptionally in non-typhoidal Salmonella isolates and only in S. Typhimurium, S. Choleraesuis, and S. Schwarzengrund (Weill et al., 2006). Fascinatingly, we report in the present study the emergence of ciprofloxacin-resistant isolates of S. Kentucky (100%), S. Virchow (100%), S. Kottbus (100%), and S. Infantis (42.8%) from CM and PG in Egypt, which is considered the drug of choice against Salmonella infection in animals and humans (CLSI, 2018). Conferring the last report of EFSA and ECDC (2016) on antibiotic resistance in zoonotic and indicator bacteria from humans, animals and food, S. Infantis significantly contributed to the overall numbers of MDR Salmonellae in Europe, when isolates from broilers showed resistance to third generation cephalosporins and great resistance to ciprofloxacin. Therefore, a high worry still leftovers for the public health significance of S. Infantis. It was proposed that the routine practice of using antimicrobials in food animal and poultry production is engaged in the emergence of antibiotic resistant bacterial strains and are subsequently transferred to human beings through the food chain (Stürenburg and Mack, 2003; Threlfall, 2002).
As regard to integrons, in accordance with the present study, the previous report of Siriken (2015) concluded that class 1 is the most widely spread and clinically reported integron in MDR Salmonellae. Worthy mentioning that integron containing isolates are more antibiotic resistant than those lacking integrons (Fluit AC, Schmitz, 2004), alike this study. Quinolones are very significant antibiotic substances for overcoming bacterial infections in both animals and humans, thus quinolone resistance is considered a noteworthy public health risk. The high quinolones-resistance rate (ciprofloxacin and nalidixic acid) of Salmonella serovars reported in the present study, despite absence of quinolones resistance-related genes indicates that other determinants could be encountered. A similar scenario was reported by Myšková and Íšková (2017). This could be explained considering the concepts of Jacoby (2005) who suggested that quinolone resistance is mostly attributed to mutation in chromosomes that modify the antibiotic target enzymes, DNA gyrase (gyrA and gyrB) and DNA topoisomerase IV (parC and parE) or trigger the efflux systems. Moreover, according to Piddock (1999), a single point mutation in gyrA can arbitrate resistance to the nonfluorinated quinolone (100% of the identified isolates in the present study showed resistance to nalidixic acid) and reduce susceptibility to fluoroquinolones (53.8% of the identified isolates in the present study showed resistance to ciprofloxacin), while mutation in the gyrB, parC and parE is rare in Salmonella (Eaves et al., 2004). In addition to quinolones resistance, the antibiotic resistance to expanded spectrum cephalosporines, which are strongly recommended for the treatment of salmonellosis, is determined mainly by the existence of extended spectrum β-lactamases (ESBL) and plasmid-mediated AmpC β-lactamases (PABL) genes of which blaTEM, blaCTX and blaCMY-2 are the most common (Kang et al., 2013). This explains the high level of antibiotic resistance to that group in the present study, as 61.5, 23.0, 23.0, 76.9, 38.4, and 15.3% of the isolates were resistant to ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, cefotaxime, ceftazidime and aztreonam from β-lactams group, respectively.
In addition to the antibiotic resistance, the existence of invA, hilA, sopB and stn virulence genes in almost all MDR Salmonella isolates indicates the high pathogenicity of these isolates to animal, poultry and humans. Since invA and hilA genes are responsible for penetration of Salmonella bacterium into the host cells (Valdez et al., 2009; Lostroh et al., 2000). While sopB, in addition to its role in host cell membrane invasion, is also responsible for rearrangement of the host cell cytoskeletons (Borges et al., 2013). As well as, stn gene is in charge of production of enterotoxins and is linked to triggering acute gastroenteritis in infected hosts (Zou et al., 2012). Consequently, the retail chicken meat marketed in Egypt constitutes high public health risks to consumers. Thus, it should be faced with a high level of care and consideration by the legal authorities.
In conclusion, the high rate of Salmonellae in the present study is attributed to the slaughter of live birds without veterinary supervision inside low hygienic poultry retail markets. S. Infantis and S. Kentucky are the top among the mostly isolated Salmonella enterica serovars from poultry meat in Egypt. The emergence of ciprofloxacin-resistant isolates of S. Kentucky, S. Virchow, S. Kottbus, and S. Infantis from CM and PG was reported for the first time in Egypt. Isolates with class 1 integron showed a high level of MDR. Class 2 was absent in all isolates. The determined high rate of quinolones-resistance of Salmonella serovars, despite absence of quinolones resistance-related genes, indicates that other genetic factors could be incriminated. The high level of resistance to β-lactams is attributed to the high incidence of β-lactams resistance-related genes (blaTEM, blaCMY-2 and blaCTX). Eventually, the existence of high incidence of virulence genes within MDR Salmonella serovars gives us an alarm and should be faced with a great worry because consumers could be under a great public health risk.
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L. plantarum is one of the most well studied lactobacilli colonizing plants (Hammes and Hertel, 2006). Presence of L. plantarum in must at detectable levels depended on the origin of the samples. In grapes from Greece vineyards L. plantarum dominated during the middle and final stages of fermentation even in the samples in which initial populations of lactic acid bacteria (LAB) were below the detection limit (Nisitou et al., 2015). In samples from South Africa wines L. plantarum was the dominant species in grape juice, but could not be detected during the later stages of wine production (du Plessis et al., 2004). In samples from Bordeaux vineyards (France) L. plantarum were revealed on grape on the day of harvest at a concentration of 2 cells/ml but after they could not be found anymore. In samples from another French wine-producing region – Cognac, L. plantarum were isolated on the first day of wine fermentation at a concentration of 104 cells/ml, but as in the previous case, the population after decreased below the detectable level (Lafon-Lafourcade et al., 1983). Some L. plantarum strains from grape must together with Oenococcus oeni were proposed for development of starter cultures for the controlled process of malolactic fermentation improving special organoleptic characteristics of certain wines (Bauer and Dicks, 2004; Bravo-Ferrada et al., 2013; Testa et al., 2014).
L. plantarum from fermenting plant material were characterized by strong antagonistic activity (Ben Omar et al., 2008; Knoll et al., 2008; Singh and Ramesh, 2008; Piasecka-Jozwiak et al., 2013). Antagonistic properties of LAB are widely used in food fermentation (Ben Omar et al., 2008; Singh and Ramesh, 2008; Çon and Karasu, 2009), probiotics (Martins et al., 2013) and become promising for plant protection (Visser et al., 1986; Trias et al., 2008; Hoda et al., 2011; Lutz et al., 2012). It could be hypothesized that being the representatives of normal plant microbiota lactobacilli (Hammes and Hertel, 2006) could colonize plants and protect them from phytopathogens. Adhesion of L. plantarum is well studied on a model of epithelial cells (Tallon et al., 2006; Velez et al., 2007). But there is scarce information about the attachment of lactobacilli to plant surfaces. Reina et al. (2002) compared adhesion of bacteria from several genera to fresh cucumber fruit surfaces. Salmonella typhimurium and Staphylococcus aureus surprisingly adsorb to cucumber surfaces at higher levels than L. plantarum. Also the reported levels of adhesion of L. plantarum were lower for dewaxed fruits (Reina et al., 2002). Tests on ability to attach and to form biofilms on plant surfaces should be carried out with the strains intended for plant protection or fermentation of food products of plant origin.
Strains of L. plantarum were characterized by their high diversity within the species that can be revealed by Random Amplification of Polymorphic DNA (RAPD)-PCR typing (Ben Omar et al., 2008; Pisano et al., 2011; Bravo-Ferrada et al., 2013). The results of RAPD-analysis may be used for development of primers for detection of L. plantarum strains (Galanis et al., 2015), to track the survival of lactobacilli during their consumption as probiotics (Mahenthiralingam et al., 2009) or to find out the ways of dissemination of lactobacilli during globalization processes (Song et al., 2016). Analysis of genetic diversity of L. plantarum strains from France and Ukraine will allow to establish whether RAPD-analysis could be helpful to reveal the geographical origin of the strains from two distant regions of the Europe. These data could be implemented in development of methods tracking the dissemination of starter or probiotic cultures important for food industry, medicine or plant protection. Moreover, investigations of possible association between the useful technological properties, such as biofilm formation, and RAPD-profiles are necessary to find out the possibility of rapid evaluation of the strains perspective for plant protection or food fermentation.
The aim of the present work was to study the diversity of L. plantarum strains originated from France and Ukraine comparing their RAPD-PCR profiles and ability to form biofilms.
MATERIAL AND METHODS
Thirteen strains of L. plantarum isolated in France from grape must and seventeen L. plantarum strains isolated in Ukraine from must and pickles were included in the experimental set up (Tab 1).
Strains were stored at –80ºC in a Collection of Bacterial Cultures of Odessa National I.I. Mechnikov University (ONU). MRS medium (de Man et al., 1960) and standard conditions (37 ºC, 24-48 h) were used for cultivation.
Phytopathogenic strain Agrobacterium tumefaciens pJZ labeled with GFP was kindly provided by Dr. Clay Fuqua (USA) and Dr. Igor Golovlev (Sweden). Agrobacteria were cultivated in LB broth (Bertani, 1951) at 28 ºC and stored at –80ºC in 30% glycerol.
Table 1 Origin of the strains used in this study
|Strain||Source of isolation||Country of isolation|
|L. plantarum ONU 12, ONU 311, ONU 312, ONU 313, ONU 333, ONU 335, ONU 337, ONU 339, ONU 340, ONU 342, ONU 345, ONU 348, ONU 349, ONU 350||Grape must||Ukraine|
|L. plantarum ONU 351, ONU 352, ONU 353, ONU 354, ONU 355, ONU 356, ONU 357, ONU 359, ONU 362, ONU 363, ONU 364, ONU 365, ONU 471||Grape must||France|
|L. plantarum ONU 472, ONU 475||Sauerkraut||Ukraine|
|L. plantarum ONU 476||Fermented pickled tomatoes||Ukraine|
|L. plantarum UCM B-2709, UCM B-2694||Reference strains, Ukrainian Collection of Microorganisms||Ukraine|
Seeds of garden cress Lepidium sativum L. var. Azhur were sterilized with 25% of H2O2 for 60 sec and washed three times with sterile distillated water (SDW). After, seeds were brought into sterile Petri dishes with wet filter paper and left for three days at 20-24 °C to germinate. Seedlings of garden cress were used in fast screening of L. plantarum strains for ability of attachment and biofilm formation.
Seeds of tomatoes Lycopersicon esculentum Mill. var. Ballada were sterilized as described above and left to germinate for 7 days. Being a classical model of studying crown gall pathogenesis, tomato plants were used in experiments with competitive adhesion and biofilm formation of L. plantarum and plant pathogen Agrobacterium tumefaciens pJZ.
Attachment and biofilm formation
LAB cultures were cultivated overnight in MRS broth at 37°C till the concentration of 108 CFU/ml measured with spectrophometer SmartSpec Plus (Bio-Rad, USA).
Attachment of bacteria to Lepidium sativum L. plants was studied by incubation of sterile root fragments with LAB cultures for 1 h. After incubation, roots were washed two times in SDW, triturated in 10 mM HEPES, pH 7.5, and the resulted suspensions were inoculated on MRS plates and incubated 48 h at 37 °C (Brisset et al., 1991). Totally 10 root fragments from each independent experiment were plated on LB agar to prove sterility and checked after 24 h of incubation at 37°C for presence of bacterial or fungal growth.
Seedlings and L. plantarum cultures were also placed into the sterile plastic wells and incubated at 37°C overnight to allow biofilm formation. After, the seedlings were treated with 96% ethanol for 15 min to fix the biofilms and stained with 0.1% acridine orange for 5 min. Biofilms were observed on roots, stems, leaves and seed coat shells using a Primo Star PC, Carl Zeiss microscope, with a total magnification x600 and photographed with Olympus DCM (3.0 M pixels) camera. Three independent experiments for each strain were carried out, 10 images in each experiment per strain were analyzed.
Competitive adhesion and biofilm formation
Competition between L. plantarum and plant pathogen on a step of attachment and biofilm formation was studied on an example of Agrobacterium tumefaciens pJZ carrying gfp gene encoding green fluorescent protein kindly provided by Dr. Clay Fuqua (USA) and Dr. Igor Golovlev (Sweden). L. plantarum strains with the best abilities to biofilm formation were included in the experimental set up.
As pathogenic agrobacteria preferentially penetrate plants via root system (Burr and Otten, 1999), roots of tomato seedlings as a model of surface of host plant were used. Three variants of experiment were carried out: (1) simultaneous inoculation of roots with lactobacilli and agrobacteria, (2) inoculation with lactobacilli with addition of agrobacteria after Lactobacillus biofilms have been formed, (3) inoculation with agrobacteria with subsequent treatment of their biofilms with lactobacilli. In case of competition experiments, 1 x 108 CFU/ml of each strain (overnight cultures) were simultaneously added to tomato seedlings in sterile 48 well polystyrol plates and left for 24 h of incubation at 28°C. In the second variant, L. plantarum strains were added together with 1 ml of MRS medium (100 µl of overnight culture), and after 24 h of incubation the medium was substituted for 1 ml of LB with 100 µl of overnight agrobacterial culture followed by the next 24 h of incubation. In the third variant, agrobacteria in LB medium were added to roots, and after 24 h substituted for lactobacilli left for the next 24 h of incubation.
After incubation, seedlings (20-24 in each variant) were analyzed by fluorescent microscopy performed by the method of Barahona et al. (2010) with some modifications. Roots were excised, stained with 0.1% crystal violet for 40 s, transferred on glass slides and analyzed with optical Carl Zeiss epiﬂuorescence microscope system with 20x planachromat objective and Olympus DCM camera. Images of the biofilms on plant roots surfaces were obtained with BP490 filter set, a 505 nm dichroic ﬁlter and 530 nm long-pass emitter (EO530).
DNA from the tested L. plantarum strains was isolated with the kit “DNA sorb” (Amplisens, Russia) and amplified with the primer M13 (Ben Omar et al., 2008) in 35 cycles of 94°C for 1 min, 45°C for 1 min and 72°C for 2 min (with pre-heating at 94°C for 3 min and post-elongation time for 5 min at 72°C). A thermocycler “MyCycler” was used (BioRad, USA). The products were separated by agarose electrophoresis (1% of agarose in Tris-acetate buffer), and the sizes of amplicons were measured using the GelAnalyzer2010 program.
The phylogenetic tree was constructed using the program Mega5 (Tamura et al., 2007, 2011) basing on the method of maximum likelihood (ML) (Tamura et al., 2011) and the Jukes-Cantor model (Jukes and Cantor, 1969). The statistical significance of the order of branching of the tree has been estimated using bootstrap analysis by constructing of the 1000 alternative replicas, or trees.
PCR amplification of plnA gene
Amplification was carried out according to Ben Omar et al. (2008) with primers to plnA gene described in Diep et al. (1996) and Remiger et al. (1996). Detection of products was carried out as described above.
Attachment and biofilm formation
All strains exhibited the ability to attach to Lepidium sativum seedling surfaces and to form biofilms. The intensity of biofilm formation was higher on shoots, leaves and seed coat shells (Fig1, D, E, F, Tab 2). In case of inoculation with all of the tested strains the biofilms were well-formed with microcolonies embedded in developed matrix layer.
Figure 1 Biofilms of L. plantarum strains on Lepidium sativum: A – L. plantarum ONU 335 on a root (individual attached cells without formation of microcolonies); B – L. plantarum ONU 345 on a root (individual well-formed microcolonies); C – L. plantarum ONU 333 on a root (well-formed biofilm with gaps in the structure); D – L. plantarum ONU 12 on a seed coat shell (well-formed biofilm with microcolonies embedded in developed matrix); E – L. plantarum ONU 12 on a leaf (well-formed biofilm with microcolonies embedded in developed matrix); F – L. plantarum ONU 333 on a shoot (well-formed biofilm with microcolonies embedded in developed matrix).
Opposite, on roots the level of biofilm formation was lower and depended on a strain (Fig 1, A, B, C, Tab 2).
Table 2 Amount of Lactobacillus plantarum strains (%) with different characteristics of biofilms on roots of Lepidium sativum L.
|Origin of the strains||Characteristics of biofilms|
|on shoots, leaves and seed coat shells||on roots|
|well-formed with microcolonies embedded in developed matrix layer||well-formed biofilm with gaps in the structure||individual well-formed microcolonies||individual attached cells without microcolonies|
|amount of the strains (%)||name of the strains||amount of the strains (%)||name of the strains||amount of the strains (%)||name of the strains||amount of the strains (%)||name of the strains|
|France||100%||ONU 351, 352, 353, 354, 355, 356, 357, 359, 362, 363, 364, 365, 471||7.6%||ONU 355||77.0%||ONU 352, 353, 354, 356, 357, 359, 362, 363, 364, 471||15.4%||ONU 351, 365|
|Ukraine||100%||ONU 12, 311, 312, 313, 333, 335, 337, 339, 340, 342, 345, 348, 349, 350, 472, 475, 476||11.7%||ONU 12, 333||47.1%||ONU 311, 312, 313, 340, 342, 345, 349, 350||41.2%||ONU 335, 337, 339, 348, 472, 475, 476|
|Reference strains||100%||UCM B-2709, UCM B-2694||–||–||100%||UCM B-2709, B-2694||–||–|
The majority of French isolates had tendency to form separated well-formed microcolonies without the developed matrix layer (Tab 2). Individual well-structured microcolonies or only individual attached cells were observed in almost equal numbers of Ukrainian isolates. Only three strains among tested – L. plantarum ONU 12, ONU 333 and ONU 355, covered roots with well-formed biofilms, but opposite to upper plant parts, biofilm maturation was not regular and gaps in the structure were observed.
As all the tested strains formed biofilms of equal level on upper parts of the plants, selection of strains with adhesive characteristics perspective for plant protection would rather include screening for attachment and biofilm formation on roots that on shoots, leaves or seeds. According to this reason, further experiments were carried out on plant roots. The level of initial attachment of bacterial cells to roots did not coincide with the ability of isolates to subsequent formation of biofilms (Tab 3).
Table 3 Ability of Lactobacillus plantarum strains to attach to roots of Lepidium sativum L.
|Strain||Amount of attached cells, CFU/cm2||Strain||Amount of attached cells,
|L. plantarum ONU 351||(2.1 ± 0.3) x 103||L. plantarum ONU 12||(6.5 ± 0.2) x 104|
|L. plantarum ONU 352||(3.5 ± 0.1) x 104||L. plantarum ONU 311||(3.4 ± 0.1) x 104|
|L. plantarum ONU 353||(2.6 ± 0.5) x 104||L. plantarum ONU 312||(2.8 ± 0.2) x 104|
|L. plantarum ONU 354||(4.1 ± 0.2) x 104||L. plantarum ONU 313||(3.6 ± 0.3) x 104|
|L. plantarum ONU 355||(7.8 ± 0.2) x 104||L. plantarum ONU 333||(5.4 ± 0.4) x 104|
|L. plantarum ONU 356||(2.8 ± 0.3) x 104||L. plantarum ONU 335||(4.3 ± 0.2) x 103|
|L. plantarum ONU 357||(3.5 ± 0.7) x 104||L. plantarum ONU 337||(2.8 ± 0.5) x 103|
|L. plantarum ONU 359||(4.9 ± 0.4) x 104||L. plantarum ONU 339||(3.2 ± 0.3) x 103|
|L. plantarum ONU 362||(5.1 ± 0.5) x 104||L. plantarum ONU 340||(4.1 ± 0.2) x 104|
|L. plantarum ONU 363||(3.2 ± 0.2) x 104||L. plantarum ONU 342||(3.5 ± 0.2) x 104|
|L. plantarum ONU 364||(3.1 ± 0.1) x 104||L. plantarum ONU 345||(3.6 ± 0.7) x 104|
|L. plantarum ONU 365||(1.5 ± 0.4) x 104||L. plantarum ONU 348||(2.7 ± 0.3) x 104|
|L. plantarum ONU 471||(3.7 ± 0.3) x 104||L. plantarum ONU 349||(4.5 ± 0.6) x 104|
|Reference strains||L. plantarum ONU 350||(1.6 ± 0.4) x 104|
|L. plantarum UCM B-2709||(3.7 ± 0.3) x 104||L. plantarum ONU 472||(3.5 ± 0.5) x 104|
|L. plantarum UCM B-2694||(3.1 ± 0.1) x 104||L. plantarum ONU 475||(2.9 ± 0.2) x 104|
|L. plantarum ONU 476||(4.8 ± 0.5) x 103|
Level of attachment varied from (7.8 ± 0.2) x 104 to (2.1 ± 0.3) x 103 CFU/cm2 and depended on a strain.
From nine strains forming slight biofilms, only five strains were characterized by low adhesion to garden cress roots, with amount of attached cells from 2.1 x 103 to 4.8 x 103 CFU/cm2. Others demonstrated the same level of attachment as the strains that formed microcolonies and well-developed biofilms (Tab 3).
Gene plnA was found in 28.1% of all studied isolates (L. plantarum ONU 333, 342, 345, 348, 349, 350, 355, 362, 364). All plnA possessing strains except ONU 348 were characterized by formation of well-developed microcolonies and biofilms. Strains L. plantarum ONU 333 and ONU 355 with the highest level of biofilm formation were plnA-positive.
Treatment of biofilms of A. tumefaciens pJZ on roots of tomato seedlings with suspensions of lactobacilli (L. plantarum ONU 12, ONU 333 and ONU 355) resulted in complete disruption of the pathogen biofilms. No A. tumefaciens pJZ cells were found on roots (Fig 3).
If agrobacteria were added to already formed biofilms of L. plantarum ONU 355, some cells of pathogens could integrate into the biofilm in 25% of tested samples (Fig 3). But treatment with suspensions of L. plantarum ONU 12 and L. plantarum ONU 333 completely eliminated the pathogen (Fig 3).
Figure 3 Competitive adhesion of L. plantarum and A. tumefaciens pJZ: A – negative control (inoculation with L. plantarum 12); B – positive control (inoculation with A. tumefaciens pJZ, developed biofilm of fluorescent GFP-expressing cells of the pathogen covers all the root surface, pointed with arrows); C – treatment of A. tumefaciens pJZ biofilms with L. plantarum ONU 355 (no fluorescent cells of the pathogen); D – addition of A. tumefaciens pJZ to biofilm of L. plantarum ONU 355 (fluorescent cells of the pathogen on root surface, pointed with arrows); E – addition of A. tumefaciens pJZ to biofilm of L. plantarum ONU 12 (no fluorescent cells of the pathogen); F – simultaneous inoculation with L. plantarum ONU 333 and A. tumefaciens pJZ (no fluorescent cells of the pathogen)
Simultaneous inoculation of test plant roots with lactobacilli and agrobacteria also demonstrated the protective effect of L. plantarum (Fig 3). In case of the strains L. plantarum ONU 12 and ONU 355 complete elimination of agrobacteria was observed. A. tumefaciens pJZ formed mixed biofilms with L. plantarum ONU 333 in 16.7%, and 83.3% root samples inoculated with this strain remained free from the pathogen.
All studied strains of L. plantarum after the construction of a phylogenetic tree have been referred to three clusters A, B and C (Fig 4).
The majority of strains has been included in the cluster A, which was divided into five subclusters (A1 – A5). Most of the strains belonged to the subcluster A1 (Fig 4). No association with the geographical origin of the strains was observed in this cluster. Thus, the subcluster A1.1 included four strains from Ukraine – ONU 339, 313, 311 and 349, and three strains from France – ONU 359, 354 and 356. Reference strains (L. plantarum UCM B2709 and L. plantarum UCM B2694) were also found in this subcluster. A similar situation was observed for the subcluster A1.2, which included strains ONU 353, 362, 364, 471 (France), and ONU 476, 333 (Ukraine) (Fig 4).
Most of the internal nodes of subcluster A1 of the phylogenetic tree were confirmed by the statistical significance of the order of branching at the level 57-65%.
The subcluster A4 was the second large subcluster by the number of classified strains. As in the previous case, the formation of groups did not reflect the geographical origins of the strains (Fig 4). The statistical significance of the order of branching for nodes of this subcluster was slightly smaller – 28-46%. The significance of the branching <50% indicates that these strains would belong to different subspecies and increasing of the sample will form separate clusters.
The high value of statistical significance during the bootstrap analysis was noted for the subcluster A5 containing strains L. plantarum ONU 12 (Ukraine) and ONU 355 (France). A distinctive feature of this cluster was the ability of the strains to form well developed biofilms (see Tab 2).
The subclusters A2 and A3 included two strains (Fig 4), and the statistical significance of branching order for them was minimal (4-6%).
As the result of the study of the phylogenetic tree topology, two separate clusters – B and C were identified (Fig 4). Significance of the order of branching internal nodes – 51% and 33% respectively, was the reason of their separation from all other strains. These clusters contained the strains isolated only in Ukraine.
Presented results show the high capability of the studied L. plantarum strains to attach to shoots and leaves of Lepidium sativum L. seedlings and strain-dependent ability to attach to roots. These data are in agreement with the hypothesis of Hammes and Hertel (2006) who suggested that owing to their high potential to form extracellular matrix, lactobacilli could be the components of the biofilms on plant surfaces or even be able to initiate biofilm formation (Hammes and Hertel, 2006). This hypothesis was based on the investigation of Morris et al. (1997) who revealed that 10-66% of the total cultivable microorganisms from biofilms on plant surfaces are represented by Gram-positive bacteria. Biofilms, the same as in case of this study, were found on all parts of the leaves – margin, base, upper and lower surfaces. In nature, biofilms on leaves are composed of exopolymer matrix and various microbial morphotypes – bacteria, filamentous fungi and yeasts (Morris et al., 1997), whereas in the studied case lactobacilli were able to form a monospecies biofilm in absence of all other microorganisms.
Figure 4 The phylogenetic tree of the tested L. plantarum strains built on the results of RAPD-PCR using the method of maximum likelihood and the Jukes-Cantor model. Numerals show a statistically significant branching order (in %) determined by a bootstrap analysis for 1000 alternative replicas
The lower levels of biofilm formation on roots coincide with the scarce evidences about the occurrence of LAB in rhizosphere (Hammes and Hertel, 2006). The same concerns soil (Fhoula et al., 2013), although some authors described soil as a common source for the isolation of LAB including lactobacilli (Chen et al., 2005; Yanagida et al., 2006). L. plantarum was found in rhizosphere of olive trees and desert truffles in Tunisia (Fhoula et al., 2013) and in rhizosphere of Hibiscus esculentum in Nigeria (Oyeyiola et al., 2013). Our investigations showed the possibility of L. plantarum to attach to roots of seedlings on example of Lepidium sativum. The capability to form biofilms on roots was lower than in case of shoots and leaves. Whereas the ability to form biofilms on the upper parts of the plants was equally high for all studied L. plantarum strains, biofilm formation on the roots appeared strain-specific.
In experiments of Calasso et al. (2013) strain DC400 of L. plantarum cultivated on a medium supplemented with chemically synthesized PlnA significantly increased the capability to form biofilms on polystyrene pegs and to attach to Caco-2 human colon carcinoma cell line. Such results together with the investigation of exoproteome of the studied strain enabled authors to conclude that the adhesion and biofilm formation were mediated by the peptide pheromone PlnA (Calasso et al., 2013). In our experiments, indeed, only one strain with plnA in its genome (L. plantarum ONU 348) exhibited low level of biofilm formation. Other plnA possessing strains formed microcolonies or biofilms with developed matrix. These results indicate that strains carrying plnA also had the better ability to form biofilms on plant root surfaces. L. plantarum ONU 12 possessing no plnA gene was an exclusion – bacteria of this isolate formed well-developed biofilms despite of the absence of the gene. Describing the succession of LAB in fermenting cucumbers, Singh and Ramesh (2008) pointed out the time-dependent emergence of PlnA producers during the late stages of fermentation. Together with pediococci, PlnA producers were prevalent in population of bacteriocinogenic strains (Singh and Ramesh, 2008). In our study the percentage of potential PlnA producers among the strains constituted 28.1%.
Lactobacilli could interfere the formation of biofilms by plant pathogen on an example of A. tumefaciens pJZ. The effect was strain dependent and resulted in complete elimination of pathogens from tomato roots in 75.0 – 100% of tested samples depending on a method of inoculation. Antagonistic action of lactobacilli against some phytopathogenic bacteria – Xanthomonas campestris, Pseudomonas syringae, Erwinia carotovora, have been demonstrated by well-diffusion method and on test plants by estimation of disease symptoms (Visser et al., 1986; Trias et al., 2008; Dalirsaber et al., 2012). We found out that inhibition of the pathogen could occur already on a step of attachment and biofilm formation – the first step of disease pathogenesis. Lactobacilli could compete with the phytopathogen, protect plant surface and disrupt mature biofilms of the pathogen.
The results of RAPD-PCR analysis indicated that most of the studied L. plantarum strains from two different geographical regions – France and Ukraine, were similar to each other and formed a monophyletic group. However, the presence of clusters B and C indicated the existing paraphilia within the studied L. plantarum.
The high value of statistical significance during the boot strap analysis was noted for the subcluster A5 containing strains L. plantarum ONU 12 (Ukraine) and ONU 355 (France). A distinctive feature of this cluster was the high ability of the strains to form biofilms (see Tab 2).
Modern investigations have elucidated that L. plantarum strains cluster together according to their particular food niche (Siezen et al., 2010). In case of L. plantarum from ovine milk and cheese, analysis based on RAPD-PCR resulted in seven clusters showing the diversity between the isolates from the two mentioned sources. Moreover, the differences between the geographical regions of isolation could be clearly seen (Oneca et al., 2003). In case of L. plantarum from plants, isolates from maize fermented product potopoto could be divided into the two main groups (Ben Omar et al., 2008). Strains from must of grape gathered in one region of Italy appeared closely related (Spano et al., 2002). At the opposite, a significant genetic diversity of L. plantarum strains from Patagonian red wines was revealed (Bravo-Ferrada et al., 2013). When strains from two regions of grapevine cultivation in Greece were studied, most of the L. plantarum vineyard populations showed a high degree of genetic similarity. Two RAPD-PCR patterns were common between the studied regions, but no clustering according to the zone of origin was revealed (Nisitou et al., 2015). The same was observed in our study – the strains from two distant geographical zones could not be clearly clustered basing on the place of origin. Only clusters B and C with the fewest number of strains contained isolates from Ukraine only.
L. plantarum isolated from products of plant origin (grape must and pickles) exhibited ability to attach to Lepidium sativum seedlings and form biofilms. On roots isolates from France preferentially formed separated microcolonies without developed matrix layer, whereas almost half of Ukrainian isolates exhibited the attachment of individual cells without formation of microcolonies. Ability of the strains to attach to plant roots and to form biofilms did not coincide. Comparison of RAPD profiles and abilities to form biofilms showed no association with the geographical origin of the L. plantarum strains isolated in France and Ukraine. One subcluster – A5 – included two strains with the high level of biofilm formation: L. plantarum ONU 12 (Ukraine) and ONU 355 (France). Since RAPD-typing could not find certain differences between strains from the two geographically distant zones, further investigations should be carried out to select a method of typing the isolates of L. plantarum to trace their origin. Lactobacilli could eliminate biofilms of the phytopathogen A. tumefaciens pJZ from tomato roots. Such antagonistic effect against pathogenic bacteria as well as ability to form developed biofilms on plant surfaces need further detailed investigation for possible practical implementation in plant protection and food industry.
Acknowledgments: This work was supported by a bilateral French-Ukrainian project “Dnipro” (2011-2012, 2015-2016) granted by the Ministry of Foreign and European Affairs of France and the Ministry of Education and Science of Ukraine, and by the individual grant for Andrii Merlich (Campus-France 2013-2017) supported by the French Embassy in Ukraine.
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