PROCESS OPTIMIZATION FOR THE PRODUCTION OF MICROBIAL PHYTASE AND ITS APPLICATION IN FEED INDUSTRY

Abstract

Phytase breaks the phytic acid molecule and liberates inorganic phosphate and myo inositol esters. It also releases other biomolecules attached to the phytate and helps to overcome the anti-nutritive effect. Due to the lack of certain features of an ideal phytase, its application on a commercial level is limited. As a feed additive, the demand for phytase is continuously increasing in the market of feed enzymes. Phytase increases the bioavailability of nutrients and enhances the nutritional value of feed. It decreases the cost of livestock feed and load on phosphate reserves. Further, phytase is also involved in reducing the level of phosphorus, and eutrophication in water bodies, thus mitigating environmental issues. In the current investigation, among the screening of various microbes, Penicillium oxalicum PBG30 was selected as the most potent source of phytase. This fungus was isolated from a rotten orange sample. For the cost-effective production of phytase, the solid-state fermentation method was applied. In the SSF method, wheat bran was used as a substrate due to its high nutritional value and cost effectiveness. For the enhancement of the phytase level, optimization was performed using traditional and statistical approaches. One variable at a time (OVAT), a traditional method, is used to study various parameters like the amount of wheat bran, moistening media, substrate and moisture ratio, incubation temperature, incubation days, and pH. The production of phytase applying OVAT method reached 200.407 ± 6.01 U/g DMR with 10 g wheat bran, mixed with moistening media (0.5% Urea, 0.1% MgSO₄.7H₂O, 0.1% KCl and 0.1% FeSO₄.7H₂O) of pH 7.0 in a ratio of 1:2, having incubation of 5 days at 30°C. The OVAT approach leads to a 2.4-fold increase in the phytase production. However, the main drawback of OVAT is that it examines factors one by one, and not the interaction among the factors, making the process costly and time-consuming, hence, statistical optimization was conducted. The statistical optimization includes Plackett-Burman design (PBD) and Response Surface Methodology (RSM). PBD screened the critical variables essential for the production, while RSM analyzed the interaction among the variables and provided the exact amount of that variable required for the production. The four significant variables, viz., magnesium sulphate, pH, incubation days and Tween-80 were identified by PBD during the study that v was further examined by RSM. Magnesium sulphate (0.75%), pH (7.0), incubation days (5), and Tween-80 (3.5%) were the parameters that influenced the phytase production majorly and enhanced the phytase production by 4.4-fold. The highest production (373.32 ± 3.28 U/g DMR) was obtained when 5 g wheat bran supplied with moistening media (0.5% ammonium sulphate, 0.01% FeSO₄, 3.5% Tween 80 and 0.75% MgSO₄) of pH 7.0 in a ratio of 1:2 having incubation of 5 days at 30°C. For sustainable production, phytase is produced in trays on a large scale. The amount of phytase was recorded as the highest in the trays, resulting in a 5.6-fold increase in its production level. After statistical optimization, the production level of phytase enhanced up to 5.6-fold. Besides phytase, P. oxalicum PBG30 also produces cellulase (51.06 U/g DMR), amylase (86.20 U/g DMR), xylanase (18.05 U/g DMR) and lipase (7.05 U/g DMR). The phytase was partially purified by ammonium sulphate precipitation and dialysis methods and exhibited a 4.9-fold purification with a 55.31% yield. The biochemical characterization was done to determine the properties of phytase isolated from P. oxalicum PBG30. The phytase was optimally active at 70°C and pH 3.0. P. oxalicum PBG30 phytase showed broad substrate specificity with various substrates which was found maximum with sodium phytate. Phytase is positively affected by organic solvents such as ethanol, methanol, isopropanol, butanol, DMSO, and acetone. Tweens and Triton-X-100 considerably increased the phytase activity, while SDS, EDTA, ß-ME, DTT, and sodium molybdate decreased the activity. The impact of different metal ions was also studied and varied with the microbial species. P. oxalicum PBG30 phytase is found thermostable with a t1/2 value of 1 h, stable in pH 3.0 and pH 5.0 and protease resistant. The Kₘ and Vₘₐₓ values were measured as 4.42 mM and 909.1 U/ml, respectively. Phytase showed a long storage life at 4°C and -20°C. P. oxalicum PBG30 phytase was immobilized with the calcium alginate method and exhibited reusability up to 5 cycles. Phytase efficiently degrades phytic acid and secretes inorganic phosphate and other nutritional by-products. The phytase was supplied in fish feed that shows maximum liberation of inorganic phosphate (33.986 mg/g of feed), reducing sugars (134.4 mg/g of feed), and soluble protein (115.52 mg/g of feed) with the addition of 200 U of phytase in 0.5 g of feed mixed with 20 ml buffer and incubated at 37°C for 6 h. In another experiment, phytase was added into experimentally prepared fish feed with different doses, and the phytic acid hydrolysis was observed. The maximum reduction of phytic acid was 46.6% occurred with a 1500 FTU/kg phytase dose. Moreover, the effect of phytase was studied in the hydrolysis of insoluble phytate-metal and phytate-protein and found efficient in releasing inorganic phosphate with time. In addition, vanadium inhibits vi the phytase activity and enhances the peroxidase activity, thus allowing the phytase to perform as haloperoxidase. The present study depicts the cost-effective production of phytase and its utilization as a feed additive in the feed industry.