The Milk-clotting Eenzyme From Bacillus Subtilis PNG27 And Its Application In Cheese Processing

The domestic cheese industry is on the rise,the calf rennet can not meet the needs of cheese production because of high prices,low yield,the against of animal protection organizations and other factors,and the milk-clotting enzyme from microbial which has become a hotspot in the study of calf rennet enzyme substitutes,has the advantage of low cost,easy to quantify the production and high activity.But the milk-clotting enzyme from microbial has strong protein hydrolysis,and there is a risk of excessive hydrolysis of the protein to produce bitter substances.During the study of antimicrobial substances,we discovered accidentally that the Bacillus subtilis PNG27 which was deposited in this laboratory would produce a substance to coagulate the milk.The substance was identified as milk-clotting enzyme.In this paper,the culture formula and fermentation conditions of the production of the milk-clotting enzyme were optimized,and the purefied enzyme was obtained by separation and purification.The enzymatic properties and the application in the cheese were studied.The aim of this study is to provide a theoretical basis for the application of the milk-clotting enzyme from Bacillus subtilis in the production of cheese,especially in yak cheese production.The main findings are as follows:1.Optimization of fermentation for the ppriduction of the milk-clotting enzyme by Bacillus subtilis PNG27.Bran concentration as well as incubation time and temperature were identified as key medium components and fermentation conditions by one-factor-at-a-time and Plackett-Burman experimental designs.A Box-Behnken response surface methodology was used to determine the optimal medium composition and fermentation conditions as 42.22 g/L bran.40 g/L sucrose.10 g/L NaCL.4%inoculation amount?pH 7 and incubation at 33.12 ? for 41.0 h.The activite of the milk-clotting enzyme from Bacillus subtilis was 222.34 ± 4.99 U/mL.2.Separation and Purification of the milk-clotting enzyme produced by Bacillus subtilis PNG27.The enzyme was purified by graded precipitation with ethanol,DEAE-cellulose ion exchange column chromatography and Sephadex G-100 molecular sieve chromatography.The purified enzyme was named BY-1.The specific activity of the enzyme reached 794.30 U/mg,increased by 8 times,the purification factor was 8.29 times and the final recovery was 4.25%.SDS-PAGE analysis showed that the molecular weight of BY-1 was about 42 kDa.3.Determination on the Enzymatic Properties of BY-1.In this paper,the enzymatic properties of BY-1 were studied systematically,including the effects of thermal stability,pH stability as well as the temperature,pH,metal ion and protease inhibitor on enzyme activity.The results showed that BY-1 had stable enzyme activity at 20 to 50?,and had stable enzyme activity in the range of pH(5-9).Mn2+,Mg2+ and Ca2+ have a strong effect on the enzymatic activity,in contrast,K+,Zn2+ and Cu2+ have an inhibitory effect on it.BY-1 has a stronger substrate specificity for ?-casein and yak milk protein.Therefore,BY-1 has a broader prospect in yak milk cheese industry.4.Effect of factors in cheese processing on cheese curd characteristics.Take the OD value of whey as an indicator,the effects of the addition amount of fermentation agent,the amount of CaCl2 and the total activity of milk-clotting enzyme on the curd effect of cheese were studied.When the addition amount of fermenting agent was 0.3 g/L and the total activity was 80 U,the OD value of the two kinds of milk-clotting enzyme was the smallest.When calf rennet was used,the OD value was smallest when the amount of Ca2+was 0.02%and pH was 5.7,when BY-1 was used,the OD value was smallest when the amount of CaCl2 was 0.01%and pH was 5.9.5.Physical and chemical indicators and flavor evaluation of cheese during ripening.The water content,fat content,total nitrogen content,water soluble nitrogen(WSN),TCA-nitrogen(TCA-N),PTA-nitrogen(PTA-N),protein nitrogen(PN),casein nitrogen(CN)and polypeptide nitrogen(PPN)were determined according to the national standard.The results showed that BY-1 had higher degradation activity of protein,The protein content of yark cheese is higher than that of cow cheese.Electronic nose can be clearly distinguish the cheese flavor between the four kinds.The result of SPME-GC-MS showed that BY-1 produced the most flavor of cheese and the sensory evaluation showed that the yak cheese produced by BY-1 was the most acceptable and had no obvious bitterness.