IMPROVEMENT OF PHYTASE BIOSYNTHESIS BY NEW BACTERIAL ISOLATE, Pediococcus pentosaceus C4/1A VIA CONTINUOUS CULTIVATION

Phytase enzyme is widely distributed in animals, plants and microorganisms which help in the degradation of phytate. Among the microorganisms, members of lactic acid bacteria (LAB) such as Pediococcus pentosaceus were found to produce phytase enzyme. The production of phytase in this study was identified by qualitative and quantitative enzyme assay. Qualitative assay was done by cultivation of the strains on modified MRS agar which contains MOPS and sodium phytate to observe the presence of clear halo zone, which indicates the enzyme production. Quantitative test was carried out to identify the highest phytase producer by cultivation of all the five isolates in modified MRS broth in shake-flask. The highest phytase producer was then selected and identified as Pediococcus pentosaceus C4/1A for conventional screening process based on medium formulation and culture conditions using Erlenmeyer shake-flask. Different nitrogen sources (peptone with casein, meat extract, yeast extract), carbon sources (glucose, maltose, lactose), different concentration of sodium phytate and inoculum size were determined on phytase production. From the study, yeast extract (20 g/L), glucose (15 g/L), sodium phytate (5 g/L) and inoculum size (5% (v/v)) gave effect on the phytase production by P. pentosaceus C4/1A. Further optimization by using Response Surface Methodology (RSM) was carried out on phytase production and cell biomass. The standardized protocol for phytase production was carried out in large scale using 2L stirred tank bioreactor. Formulated were used to obtain high phytase activity. Continuous cultivation was then conducted for mass production and to determine the productivity of microbial biomass and production of phytase. Different range of dilution rate (D) was applied for 48 h to study the productivity of the cell and phytase. Secondary data based on phytase production, cell concentration and substrate consumption in small scale and large scale cultivation was developed and compared.

Phytase enzyme is widely distributed in animals, plants and microorganisms which help in the degradation of phytate. Among the microorganisms, members of lactic acid bacteria (LAB) such as Pediococcus pentosaceus were found to produce phytase enzyme. The production of phytase in this study was identified by qualitative and quantitative enzyme assay. Qualitative assay was done by cultivation of the strains on modified MRS agar which contains MOPS and sodium phytate to observe the presence of clear halo zone, which indicates the enzyme production. Quantitative test was carried out to identify the highest phytase producer by cultivation of all the five isolates in modified MRS broth in shake-flask. The highest phytase producer was then selected and identified as Pediococcus pentosaceus C4/1A for conventional screening process based on medium formulation and culture conditions using Erlenmeyer shake-flask. Different nitrogen sources (peptone with casein, meat extract, yeast extract), carbon sources (glucose, maltose, lactose), different concentration of sodium phytate and inoculum size were determined on phytase production. From the study, yeast extract (20 g/L), glucose (15 g/L), sodium phytate (5 g/L) and inoculum size (5% (v/v)) gave effect on the phytase production by P. pentosaceus C4/1A. Further optimization by using Response Surface Methodology (RSM) was carried out on phytase production and cell biomass. The standardized protocol for phytase production was carried out in large scale using 2L stirred tank bioreactor. Formulated medium with glucose (16.2 g/L), yeast extract (17.2 g/L), sodium phytate (11.8 g/L) and inoculum size (10% (v/v)) were used to obtain high phytase activity. Continuous cultivation was then conducted for mass production and to determine the productivity of microbial biomass and production of phytase. Different range of dilution rate (D) was applied for 48 h to study the productivity of the cell and phytase. Secondary data based on phytase production, cell concentration and substrate consumption in small scale and large scale cultivation was developed and compared.

INTRODUCTION
Phytate known as myo-inositol hexakisphosphate, is one of the major storage form of phosphate in plants seeds (Suhairin et al., 2010). Phytate acts as anti-nutritional factor and reduces the bioavailability of phosphorus to monogastric animals (Lopez et al., 2002). Feeds given to poultry animals were initially supplemented with inorganic phosphate, due to deficiency of phytase in gastrointestinal tract (Feil, 2001). Many studies have been conducted on hydrolysis of phosphorus and reduce the excretion of excess phytate-phosphorus into the manure. Several bacterial strains were studied for the production of phytase to reduce the content of phosphorus in the environment. Those bacterial strains were Lactobacillus, Enterobacter, Bacillus sp., Escherichia coli and Mitsuokella jalaludinii (Quan et al., 2001).
In the year 1962, International Minerals and Chemicals (IMC) made an effort to develop commercial phytase. Thousands of microorganisms were tested for their ability to produce phytase. However, after many attempt made by IMC, finally an isolate was identified to produce phytase and able to hydrolyse phytate (Lei et al., 2013). The valuable isolate was Aspergillus niger PhyA and phytase was first commercialized in 1990s after characterization by Irving & Cosgrove (1971). However, fungus was no longer applied in animal feed as it contains toxin which might be harmful to animals. In this research, Pediococcus pentosaceus was chosen since it is probiotic and able to produce different types of enzymes such as phytase (Zamudio et al., 2001), and βgalactosidase (Semjonovs & Zikmanis, 2008). Phytase producing microorganisms are found mostly from soil especially bacteria which has fast growth (Tseng et al., 2000) and the rumen (Yanke et al., 1998).
Nowadays the use of enzyme in feeds and the demand for phytase are increasing rapidly in the global market. Improvement in the digestibility of nutrient leads to higher needs of feed utilization. India is in high demand of phytase usage since they have the largest population of cattle in the world which was estimated around hundred million. The demand for phytase was expected to increase in the coming years. Besides that, the usage of phytase as feed enzyme was rapidly growing in China after the high increase in the year of 2004 (Sareen Sarah, 2015). Researches nowadays were being carried out on LAB which is also known as probiotics where it has the ability to improve the growth of poultry and beneficial to the host organisms. Hence, LAB has the potential to eliminate pathogens and prevent from infections (Mozzi et al., 2010). It is known as "Generally Regarded as Safe" (GRAS) microorganisms that can be applied in animal feed.
In literature, extracellular production of phytase by native Bacillus subtilis was assayed using chemical assay and plate assay. Plate assay was performed on specific medium and chemical assay was carried out using ferrous sulphate molybdenum blue method (Shamna et al., 2012). In spite, most of the studies conducted have used these two methods to analyze and quantify the production of phytase. Optimization is the most important step in fermentation of industrial bioprocess to enhance the enzyme production by the desired bacterial strain (S. Y. Lee & Kim, 2015). Nowadays, conventional method is not only being used in many studies but also statistical optimization using Response Surface Methodology (RSM) which was introduced by Box & Wilson (1951) is used and helps the researcher to design the experiment, study the effect of parameters used towards the responses in the experiment. Highest yield production can also be achieved through RSM (Kharel et al., 2002;Raissi, 2009). It provides several experimental runs to conduct the fermentation in correct order and helps to improve the production of desired product.
Continuous cultivation is a large scale production of desired product. It is a technique used by most of the researchers and being applied in biotechnology industries.
Continuous cultivation process has potential to increase the productivity of the desired product compared to other methods especially microbial enzymes. Culture in continuous cultivation retains at steady state throughout the fermentation period, hence, maximum cell biomass and products can be achieved at this state. Most of the industrial enzymes such as single cell protein and vinegar production are produced by continuous cultivation which is up to 100 m 3 capacity and there are also enzymes which produced in a scale of hundred litres or less (Waites et al., 2009).
The focus of this study was on improvement of medium formulation and culture conditions for mass production of phytase by Pediococcus pentosaceus C4/1A in submerged fermentation. Hence, the specific objectives of the study were; 1) To identify and select the highest phytase producing LAB 2) To optimize medium formulation and culture conditions for phytase production by the selected strain of LAB using conventional and statistical approach of response surface methodology. 3) To evaluate continuous cultivation of the selected strain of LAB for mass production of phytase using 2L stirred tank bioreactor.