THE QUALITY OF HONEYS INFLUENCED BY THE TRADITIONAL HEATING METHOD
AUTHORSLynda Haouam, Hélène Dailly, Etienne Bruneau, Ali Tahar
In this study, three parameters (hydroxymethylfurfural, diastase activity and invertase activity) were used to evaluate the quality of twenty samples of multifloral honey from northern Algeria heated by a traditional method (conventional heating in a water bath at 100 °C) for five treatment periods (2, 4, 6, 8 and 16 min). The assay was carried out with honey samples whose initial HMF, diastase activity and invertase activity values were within the recommended food authority limit. HMF was determined by HPLC, diastase was measured with Phadebas and invertase was determined using the Siegenthler method. During heating, it was observed an increase in the HMF related to an increase in temperature, but still below the international standard limit (40 mg.kg-1). Diastase activity and invertase activity also decreases during heating. However, invertase is more heat-sensitive and heating time than diastase and HMF, and it's an important parameter to determine if honey has been submitted to heating. Therefore to liquefy honey we can use this method but with a low temperature and a short time because time has a deep impact on the quality of heating treatment of honey.
KEYWORDShoney, traditional heating, hydroxymethylfurfural, diastase activity, invertase activity, Algeria
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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