Production of Milk-Clotting Protease from Bacillus subtilis

Abstract

An indigenous Bacillus subtilis strain isolated from soil was found to be a potent milk-clotting protease (mcp) producer. Production optimized using response surface methodology (RSM) yielded 1,190 U/ml of enzyme in medium containing 6% fructose, 1% casein, 0.3% NH4NO3, 10 mM CaCl2, pH 6.0 and inoculated with 3% inoculum and incubated at 250 rpm for 72 h. Solid-state fermentation resulted in 1,080 and 952.3 U/gds of milk-clotting protease using soybean meal and rice bran, respectively, with higher proteolytic values of 18.97 and 9.1 IU/gds. Production in a biphasic system using an overlay of RSM-optimized medium on solid layer of 6% fructose and 1% casein with 1.5% agar resulted in significant enzyme production. Maximum mcp was obtained using a biphasic system where solid: liquid ratio of 3.0 resulted in a final yield of 1,276.65 U/ml with a yield index of 1.80 as compared to static liquid culture. However, significant increase or difference was noted as compared to yield obtained after RSM. This is the first report on the use of RSM for production of mcp from a bacterial species.

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“…A similar response curve in Fig. 3 shows that protease activity varied as a function of shaker speed in an interactive effect along with skimmed milk, which corroborated the results reported by Dutt et al 25 .…”

“…for soy flour, which has also been reported as an inducer for alkaline protease from Conidiobolus coronatus 24 as well. Casein, a component of skimmed milk, has also been reported as an inducer of this enzyme 25 . Although shaker speed was grouped among the insignificant variables, we used it as one of the major factors for further study, due to its importance in terms of oxygen and nutrient transfer into the liquid medium, especially for the growth of aerobic bacteria like Bacillus spp 26 .…”

Identification and Characterization of Psychrotolerant Sporeformers Associated with Fluid Milk Production and Processing

“…Food spoilage due to nonspore-forming psychrotolerant bacteria generally occurs due to inadequate heating or postpasteurization contamination, which can be eliminated by corrections in pasteurization protocols and improved sanitation (22). Conversely, Gram-positive psychrotolerant sporeformers have the potential to survive conventional pasteurization regimens, such as high-temperature short-time (HTST) and low-temperature long-time (LTLT) pasteurization, and can grow during refrigerated storage; some of these produce proteases (1,26), resulting in off-flavors and curdling in the final product.…”

Statistical Optimization of Culture Conditions for Milk-Clotting Enzyme Production by Bacillus Amyloliquefaciens Using Wheat Bran-An Agro-Industry Waste

“…Xylanase from bacteria, such as B. pumilus, are being applied in textile processing [8]. Numerous bacteria belonging to Bacillus such as B. licheniformis, B. subtilis and B. subtilis natto have been suggested as promising microbial rennet producers [9][10][11][12].Wheat bran, an agro-industrial residue, a cheap source of energy has high potential in the area of fermentation for the production of enzymes. Wheat bran contains cellulose material, starch, crude protein, trace elements and other certain ingredients, which can be used as carbon and nitrogen sources to promote the growth of microorganisms and enzyme production.…”

“…Xylanase from bacteria, such as B. pumilus, are being applied in textile processing [8]. Numerous bacteria belonging to Bacillus such as B. licheniformis, B. subtilis and B. subtilis natto have been suggested as promising microbial rennet producers [9][10][11][12].…”