Breeding Of High-yield Acid Pectinase Strain

Pectinase plays an important role in improving the juice yield,accelerating clarification,and improving the quality of fruit wine brewing.The fruit wine industry of China is in a stage of rapid development,while relying on imports of pectinase in fruit wine industry not only increases production costs,but also makes Chinese fruit wine production subject to technical constraints.Therefore,the development of an acidic pectinase for fruit wine with independent intellectual property rights is an urgent problem to be solved in the current fruit wine industry.In this study,Aspergillus terreus gxy12-2 was selected as the starter strain in this study.Subjected to UV and nitrosoguanidine mutagenesis,two positive-acting mutant strains A.terreus ZH2-4 and A.terreus ZH2-11 were obtained.The pectinase produced by the mutagenized strains A.terreus ZH2-4 and A.terreus ZH2-11 was vacuum freeze-concentrated and then applied to the brewing of low alcohol strawberry,loquat,watermelon and ice wine to study its influence on the quality of the fruit wine.The main contents and conclusions of this study are as followded:1.One UV mutagenesis and two nitrosoguanidine mutagenesis were performed on strain A.terreus gxy12-2 and finally a high-yield mutant strain of pectinase A.terreus ZH2-4 and A.terreus ZH2-11 was obtained.The gxy12-2 was first subjected to UV mutagenesis and then A.terreus strain C73D-2 was obtained The activity of the strain C73D-2 was increased by 34.9%compared with the original strain,and then the cellulase specific activity and PL specific activity were 1.28 and 1.1 times than that of the original strain,respectively.After chemical mutation of C73D-2,strain ZH1-4 was obtained.Its enzyme activity was increased by 21.3%,then the specific activity and PL specific activity were 1.09 times and 1.15 times higher than that of the original strain,respectively.The PG specific activity of ZH1-4 was higher than that of other mutation strains.Subsequently,two excellent mutagenized strains ZH2-4 and ZH2-11 were obtained after chemical mutation of ZH1-4.The enzyme activity of ZH2-4 was increased by 145.08%,and the specific enzyme activity was increased by 239.24% compared with the original strain gxy12-2.The PG specific activity,the cellulase specific activity,the PL specific activity and the PE specific activity of ZH2-4 were 1.28,1.62,1.38,1.23 times then that of the strain gxy12-2,respectively.Besides The enzyme activity of ZH2-11 wasincreased by 102.17%,and the specific activity was increased by 177.8% compared with that of the original strain gxy12-2.In the activity of the monomer enzyme,The PG specific activity,the cellulase specific activity,the PL specific activity and the PE specific activity of ZH2-11 were 1.27,1.68,1.38,and 1.57 times higher than those of strain gxy12-2,respectively.After genetic stability tests,the mutant strains ZH2-4 and ZH2-11 all had relatively stable genetic properties of pectinase.2.The enzymatic properties of pectinase produced by A.terreus ZH2-4 and A.terreus ZH2-11 were tested.(1)The best optimum reaction temperature of pectinase produced by A.terreus ZH2-4 and A.terreus ZH2-11 is 55 °C and 60 °C,respectively,and the enzyme activity was maintained at 50% or more after being treated at a high temperature of 70 °C.(2)The best optimum pH values for the pectinase produced by A.terreus ZH2-4 and A.terreus ZH2-11 are all 4 and belong to acidic pectinase,all have strong acid resistance,among them pectinase produced by A.terreus ZH2-11 is more tolerant to acids.(3)After vacuum freeze-concentration,the enzymatic activities of the concentrates of A.terreus ZH2-4 and A.terreus ZH2-11 increased by 320.56% and 387.34%,respectively,but their specific activity decreased by 30% and 20%.3.Commercial pectinase and pectinase produced by A.terreus ZH2-4 and A.terreus ZH2-11 are used in the brewing of low alcohol strawberry wine,watermelon wine and loquat wine.At the end of the alcoholic fermentation,the volatile acids of all fruit wines were less than 1.2g/L.Compared to commercial pectinase EX,the volatile acid of three wines treated by pectinase produced by A.terreus ZH2-4 and A.terreus ZH2-11 pectinase are slightly reduced.The effect of ZH2-4 pectinase on color,juice yield,and self-flowing juice rate in the three fruit wines was consistent with commercial enzyme,and the color of strawberry wine after ZH2-4 pectinase treatment was significantly higher than that of commercial enzyme treatment.Pectinase produced by ZH2-4 has outstanding performance in increasing total phenol content.In watermelon and strawberry wine,the total phenol content is higher than that of the same type of wine after commercial enzyme treatment.The content of methanol after ZH2-4 pectinase treatment was lower than commercial enzyme treatment.