The Research Of Chymosin Strains Selection, Chymosin Characterization And Industrial Application

Chymosin is an important enzyme which made the milk coagulation in the production of cheese,and it has an important role for the form of cheese texture and flavor. Chymosin is an important enzyme, one of the industrial enzymes widely applied in food,medicine industry and other field.According to different sources,the rennet can be divided into four categories:rennet enzume from animal,plant,microbe and genetic engineering.Because of the merits,such as wide source, short production period, and low cost,microbe has become the most promising source of chymosin.The research taked Bacillus subtilis JAASB as the initial strain.throuth some mutation methods obtained a bacteria named JAASB-14, then fermented with liquid fermentation.This paper studied the condition of enzyme-producing, the optimum medium and enzyme properties.At last using50L scale-up fermentation which simulting the industrial conditions,and made chedder cheese by milk clotting enzyme which produced by Bacillus subtilis,and studied the composition of chedder cheese.The main contents of the study were as follows:l.The research obtained3strains of bacteria from20strains of bacteria which conserved from a lab.Through the curve of chymosin obtained a ideal bacteria,this bacteria as the initial strain. Effect of mutation was determined by preliminary screening based on the ratio between milk-clotting zone and colony diameter, and secondary screening based on liquid fermentation. The starting strain Bacillus subtilis JAASB producing chymosin was mutated by UV and DES, three mutant strains with good genetic stability and high chymosin activity were obtained. The chymosin activity produced by JAASB-3JAASB-5and JAASB-14was1142.6SU/mL、1263.2SU/mL、1333.3SU/mL, respectively.Comparing with the starting strain, chymosin activity produced by JAASB-3JAASB-5and JAASB-14was increased by52%、68%and78%,respectively.2.Based on the basic medium, Through the single factor experiment and the response surface method the research got the most suitable medium:corn starch1.5%,beans power2.0%,、NaCl0.6%、MgSO4·7H2O0.5%、 Na2HPO4·12H2O0.4%。. Through the single factor experiment study of the culture conditions and medium of nutrition conditions.The optimum culture condition text showed:the initial pH natual(The culture medium is neutral), the amount of inoculation is5%, rate of rotation280r/min.incubation time is24h.3.Characterization of chymosin produced by Bacillus subtilis was studied.The results showed that the optimum temperature of chymosin was55℃, and the optimum coagulating pH was5.0.The clotting activity was more stable at pH6.0. The enzyme almost lost activity by retaining after60min at65℃. The metal ions Mn2+、Li+、Fe2+、Na+、K+、Mg2+、Ca2+increased the clotting activity and Mg2+、Ca2+showed the greatest effect, while Cu2+inhibited the clotting activity. As enzyme modifiers, β-mercaptoethanol had no clear effect on milk-clotting activity, while EDTA inhibited the clotting activity. As protein inhibitors, σ-phenantroline strongly inhibited the clotting activity, so the enzyme is a metalloproteinase. Experimental results showed that chymosin produced by Bacillus subtilis had the similar characteristics to the commercial chymosin.4. The research used50L scale-up fermentation which using the optimized medium,after18h the clotting activity reached2677SU/m L.Cheddar cheese was maded using chymosin produced by Bacillus subtilis,and it was compared with commercial chymosin cheese. In the test the changese of diferent period of physicochemical indexes, water-soluble nitrogen and texture were determined.The results showed that there is no difference between cheddar cheese made by rennet products by Bacillus subtilis and commercial chymosin.The results of sensory evaluation show that cheddar cheese made by rennet products by Bacillus subtilis is less then commercial chymosin in the flavor.