Isolation, characterization and optimization of extracellular phytase producing Bacillus amyloliquefaciens AUPPB02

Lentils (Lens culinaris Medik.) are one of the important pulse crops in the world, because of their nutritional quality. They are rich sources of carbohydrates, proteins, iron, dietary fibres, vitamins, minerals and high energetic value. Unfortunately, the acceptability of lentils as staple food is limited, due to presence of various antinutritional factors. One of them is phytic acid (myo-inositol hexakisdihydrogen phosphate), which forms complexes with other nutrients, such as metallic ions (Ca, Mg, Fe, Cu etc.), amino acids, proteins, lipids and vitamins, rendering them unavailable to plants via lowering their solubility and bioavailability. The phytic acid (PA) can only be degraded by the enzyme, phytases (myo-inositol hexakisphosphate phosphohydrolase enzyme), which break it into to myo-inositol and phosphoric acid. The present study gives information about PA, Iron (Fe) and protein present in lentils. It also elaborates isolation, characterization and optimization of phytase producing PA degrading bacteria. Wet food processing methods (soaking, germination and fermentation) were employed to minimise the inhibitory effect of PA, as well asto increase Fe and protein in samples. The contents of PA (Wade Method), iron (Bathophenathroline method) and protein (Bradford’s method)were estimated by their respective standard curves, using double beam UV/VIS spectrophotometer (Systronic, 119) at 500 nm, 535 nm, 595 nm, respectively. Among all the wet processes, fermentation supported best for degrading PA (62.35 %) at 72 h than 48 h and 24 h. At this fermentative stage of 72 h, as a result of PA degradation, maximum increment of Fe (245%) and protein (42%) were observed. A total of 11 PPB (AUPPB01-AUPPB011) were isolated from fermented lentil sample and screened out. Among them AUPPB02 exhibited a halozone of 19 mm, having 89.47% hydrolysis efficiency and 1.68 U/ml of enzyme producing capacity. On the basis of morphological, physiological and genotypical characteristics, the isolate was identified as Bacillus amyloliquefaciens AUPPB02 (Accession no.- OR187307). The optimized conditions for phytase production were 48h of incubation time, 5.0 pH, 37 °C temperature, 1% inoculum size, 1% of lactose as carbon and 1% of peptone as nitrogen source. The strain showed increment of 21.33% phytase production in optimised media (1.76± 0.10 U/ml) than in pre-optimised media (1.45±0.12 U/ml).