Optimization of Upstream Process Parameters for Enhanced Production of Thermostable Milk Clotting Enzyme from Bacillus Subtilis MTCC 10422

Abstract

The process protocol of high titer milk clotting enzyme (MCE) production from Bacillus subtilis was delineated through optimizing different energy sources and growth conditions like incubation period, temperature and pH. The culture broth showed maximum production of MCE at pH 6.0 and temperature 40C after 48 h of incubation with the yield of 191.55 U/mL. Quantitative comparison of MCE activity before and after optimization revealed 7.46-fold increase in MCE production after the process conditions were standardized. The characteristics of MCE viz. broad pH and temperature stability could make it useful as a new source of milk coagulant in dairy processing. PRACTICAL APPLICATIONSEach microbial strain is unique in its molecular, biochemical and metabolic enzyme production properties. These production characteristics would have to offer competitive advantages over the existing products of un-optimized medium. This warrants thorough study of isolated microbial species under variable growth conditions. Fungal MCEs have been widely reported. The Milk Clotting Activity (MCA) and MCA per Proteolytic Activity (PA) index of the bacterium under study are comparable to that of many fungal reports and better than quite a few bacterial MCEs. Bacillus subtilis can be a promising microorganism for MCE production and has a good probability of successful use in cheese production.

Production, purification and characterization of a milk clotting enzyme from Bacillus methanolicus LB-1

“…1E), and it decreased at higher speeds due to increased mechanical shear force that was not conducive to cell growth (Hashem, 2000;Narwal et al, 2017). Proper agitation of culture would facilitate MCA by letting in more air, therefore oxygen, but some other studies presented otherwise different results, that still culture would yield a higher MCA (Hashem, 2000;Narwal et al, 2017;Sun et al, 2014), which was possible due to the differences in the nature of strains. He et al (2012) indicated that the initial pH of growth medium strongly affected enzyme reaction and transport of various components across the cell membrane, thus the MCA of B. methanolicus LB-1.…”

“…1C). Extension of fermentation time accompanied with gradual depletion of nutrients in the medium, preventing normal metabolism of microorganisms and MCA (Narwal et al, 2017). The MCA reached the maximum at 35°C (Fig.…”

“…With the increasing rotation speed, the MCA of B. methanolicus LB-1 reached the maximum at 140 r/min (Fig. 1E), and it decreased at higher speeds due to increased mechanical shear force that was not conducive to cell growth (Hashem, 2000;Narwal et al, 2017). Proper agitation of culture would facilitate MCA by letting in more air, therefore oxygen, but some other studies presented otherwise different results, that still culture would yield a higher MCA (Hashem, 2000;Narwal et al, 2017;Sun et al, 2014), which was possible due to the differences in the nature of strains.…”

Inactivation thermodynamics and iso-kinetic profiling for evaluating operational suitability of milk clotting enzyme immobilized in composite polymer matrix

“…subtilis MTCC 10422 isolated from soil was maintained on modified yeast extract milk agar media (YEMA) supplemented with sucrose, at 4°C by transfer onto a fresh medium after every 2-4 weeks. The enzyme was produced under liquid state fermentation (LSF) conditions in yeast extract milk media (pH 6.0, temperature 40°C) [9]. The bacterial growth was allowed for 48h of incubation.…”

“…Although milk clotting enzymes (MCEs) have many applications in cheese industry and recently authors [9,10] have reported on their production and purification, yet their commercial exploitation can only be realized after immobilization. In the present investigation, attention hasbeen focused on encapsulation of MCE using biopolymers like alginate which has numerous applications in food processing, textile industry and pharmaceutical sector.…”