Production,Immobilization and Characterization of Fungal Phytase and its Utilization in Food and Feed Industry

Abstract

The phytases hydrolyze the phytic acid to release the bound phosphorus and other vital nutrients in the feed of domestic animals, plummeting the requirement for extra subjection to feed. However, its short half-life owing to less stability and the high production cost due to the unavailability of cheaper sources for phytase production limited its application in developing countries. Also, fewer studies have been reported on phytases usage in humans. The use of phytase as a food additive can overcome the issue of malnutrition and silent hunger in developing countries. Besides this, using phytase in the animal feed not only reduces the cost of the livestock industry but also deals with the concern of phosphorus pollution (eutrophication) up to a level. Therefore, in the current study, a fungal culture, Aspergillus niger NT7, was isolated from the soil sample of the agriculture field and identified as a potential phytase producer. The A. niger NT7 phytase production was done using an economical substrate, wheat bran, an agricultural waste, via solid-state fermentation (SSF). The enhanced phytase production was obtained through optimizing different physiochemical parameters by two approaches; one variable at a time (OVAT) and statistical Response Surface Methodology (RSM). The optimized parameters obtained by the OVAT are the moistening agent (distilled water), inoculum age (3-days) and level (15×107 spores/ml), pH (5.0), temperature (30 °C) and different supplements of biochemical such as nitrogen (ammonium sulphate), sugar (mannitol), phosphorous (potassium dihydrogen phosphate) and detergent (Tween 80) source. The optimization procedures through OVAT (208 ± 0.22 U/gds) enhanced the overall output by around 2.7-fold compared to unoptimized culture conditions (76.34 ± 0.99 U/gds). Since the OVAT does not study the interaction between multiple factors concurrently and the approach is time-consuming and laborious, the parameters for phytase production were sub-optimized. In contrast, through response surface methodology (RSM), various factors significantly affecting phytase production in SSF can be studied concurrently through statistical and mathematical models. Therefore, a statistical RSM approach was used to optimize further six significant parameters identified from the OVAT approach to enhance the additional phytase production. After five days of SSF, when carried out at 35 °C with 5 g of wheat bran, supplemented with 2% mannitol and 0.5% ammonium sulfate maintaining a pH of 4.3, resulting in the improved phytase (521 ± 28.16 U/gds) production. Compared with the unoptimized culture conditions and the OVAT method, the phytase yield (521 ± 28.16 U/gds) after statistical optimization increased by 6.8 and 2.5-times, respectively. The crude phytase exhibited the proficient and persistent discharge of proteins, sugars (>60 h) and principally inorganic phosphorus from the wheat and maize bran. As per our best knowledge, this is the first report that shows maize bran nutritional enhancement and ICP-MS analysis of released minerals using crude enzyme A. niger NT7 preparation. The current work effectively indicates the possible use of A. niger NT7 phytase in feed supplementation to diminish the antinutrient character of phytate molecules. For the application of phytase for humans, the phytase was purified to homogeneity using ammonium sulphate precipitation and anion exchange chromatography. The purified phytase was found to be proteolytic resistant and has optimal activity in acidic pH (5.0) and at a pretty high temperature (50 °C). The physiochemical properties of purified phytase make it a possible option to be utilized as a food additive. The purified phytase was immobilized on chemically synthesized ZnO nanoparticles to improve the stability and other physiochemical properties further. The immobilized phytase on ZnP (Phy-ZnP) has shown improved thermostability, protease resistance, higher optimal temperature (70 °C) and low pH (4.0) compared to free phytase (Phy). Phy-ZnP can be used effectively for up to 10 catalytic cycles. The application of purified phytase on sorghum flour improves its nutritional content in simulated digested conditions. The release of the nutrients (phosphorous, reducing sugar, proteins and free amino acid) were higher after the test gastric phase treatment than the test intestinal phase. The observed release of Zn and Fe metal ions was higher in the stomach phase, but Mn and Ca were more released in the intestinal phase. This is the first study on sorghum flour to show such results. Another study on maize flour for dephytinization has shown that Phy-ZnP dephytinizes maize flour more efficiently than Phy, resulting in improved nutrient release. The current prevailing study efficiently explains the potential application of fungal phytase as feed and food additive.