Study On Factors Of Producing The Milk Clotting Enzyme From Mucor Pusillus And Enzyme Characterization

The milk clotting enzyme is an important one of the industrial enzymes widely applied in food, medicine industry and other field. In cheese-making, it plays a important role of condensing milk and improving flavor. According to different sources, the rennet can be divided into three categories: rennet enzyme from animal, plant and microbe. Of all the rennet from different way, rennet produced from microbe has the best foreground to apply to dairy industry. Therefore, based on some research results to mention above, with Mucor pusillus AS 3.3445 as object, this paper studied the condition of enzyme-producing, enzyme properties by solid state fermentation and feasibility to serve for practical production. Results summarized as following:1. After separation and rejuvenation, the stain AS 3.3445 was inoculated into bran medium to culture. Through observing the laws of producing enzyme, it was found that the strain get highest enzyme activity after four day. And we discussed the effect of the water volume and extraction time on the enzyme activity obtained by water extract method. The results showed that the fermentation medium extracted by 80mL distilled water 15h can almost get the all rennet, as same as the way can improve the previous enzyme activity increased by 23 percent.2. The optimization of fermentation media of the stain was researched. The effect of factors (car -bon,nitrogen,yeast powder,Mg²⁺ and K₂HPO₄.3H₂O etc.) on the milk clotting enzyme production were investigated based on the single factor test. It was found that the enzyme activity was aggrandiz -ed while added something to bran medium respectively 0.8 % fructose, 0.5 % milk, 0.4 % K₂HPO₄. -3H₂O,0.2% Mg²⁺ and 0.2%yeast powder.3. The cultivation conditions of the strain were studied and the results followed: the fittest parti -cle size is between 20-40 purpose, the fittest inoculation percentage is 10%, the fittest fermentat -ion temperature is 28℃, the fittest solid-liquid ratio is 1:1.2, the fittest medium quantity of flask is 20g/250mL4. Based on the single factor tests, three ingredients' (fructose X₁,milk X₂,solid-liquid ratio X₃) of medium was optimized with Response Surface Analysis. The functional relationship between the main factors and response value (milk clotting enzyme activity, Y) was established, and the Regress -ion equation was obtained. The regression equation was:Y = 14573.80+533.50X₁+354.38X₂+2138.63X₃+516.75X,X₂+908.75 X₁X₃-72.00X₂X₃ -1553.65X₁²-1484.40X₂²-2239.40X₃²by analyzing the regression equation, the optimal value of the factors were obtained: fructose 1.04%, milk 0.58%, solid-liquid ratio 1:1.02. The activity of the milk clotting enzyme reached 14733Su/g.5. The process for producing rennet on solid-state fermentation medium was researched. During fermentation, the change trend of milk clotting enzyme activity in fermented production had a certain similarity with proteolytic activity. Both of them showed highest activity after four days, and the ratio between rennet and proteolytic activity got the largest that is 21.75. In the process of fermen -tation, the pH value in the medium remain nearly unchanged, while the moisture and reducing sugar have the trend to decrease.6. The properties of milk clotting enzyme from Mucor pusillus AS3.3445 was studied. The resistance of enzyme to acid was stronger than alkaline. Along with the acidity increasing, the enzyme activity was up in pH5.4-7.8. The enzyme showed highest activity at 60℃, and stability under 50℃and the pH5-8.