Isolation And Identification Of Dominant Strains Of Fruit Enzymes After Natural Fermentation And Study On Efficacy Characteristics Of Their Metabolites

The enzyme is derived from Japan and Taiwan and is a fermented beverage made from fruits and vegetables.However,the fermentation process,metabolites and efficacy characteristics of the enzyme still require in-depth research.In this study,several typical fruits are used as raw materials,the dominant bacteria in the enzyme are screened out through natural fermentation,and the isolated strains are identified using the methods of morphology and molecular biology;the content of total phenol in the enzyme liquid is determined using the Fulin-Phenol method,and the content of flavone in the enzyme liquid is determined using the aluminum trichloride colorimetric method,the content of ascorbic acid is calculated based on the absorbance of the enzyme liquid before and after alkali treatment,and the antioxidant activity of the enzyme liquid is determined using reducing power,DPPH clearance rate and hydroxyl radical scavenging rate;the zymogram in the enzyme liquid is analyzed using the method specified by the national standard,the organic acid species and content in the enzyme liquid are determined using the high performance liquid phase method,and the correlation between enzyme sensory evaluation index and metabolites is analyzed;use Escherichia coli,Salmonella and Staphylococcus aureus as indicator bacteria to determine the inhibition zone size of the enzyme liquid against pathogenic bacteria using the filter paper diffusion method,the inhibition temperature stability and acid-base stability of the enzyme liquid is detected by using Escherichia coli as an indicator bacterium,and the relationship between metabolites and bacteriostatic activity is explored through correlation analysis.The main conclusions are as follows:1.After nine common fruits are naturally fermented for 6 months,the dominant bacteria are identified using the methods of morphology and molecular biology.A total of 12 strains are isolated.Torulaspora debrueckii is detected in all of the enzymes.Two species of Torulaspora debrueckii and Acetobacter are detected in the pear enzyme,and Zygosaccharomyces rouxii is detected in the dragon fruit enzyme.Among the 12 kinds of bacteria isolated,B1,M,Y1,HI,L1,L2,Q1,C,P1,G are Torulaspora debrueckii,H2 is Zygosaccharomyces rouxii and L3 is Acetobacter.No mould or pathogenic bacteria are detected during the fermentation of the enzymes.2.The changes of antioxidant components before and after fermentation are compared,and the correlation between active ingredients and antioxidant capacity of enzymes is studied.It's found that except for the dragon fruit enzyme,the content of antioxidant components in other samples decreases after fermentation,down 25%-55%.Among them,dragon fruit enzyme has the highest antioxidant content,with the total phenol of 0.61±0.02 mg/mL,flavone of 0.52±0.01 mg/mL and ascorbic acid of 1.34±0.02 mg/mL.The antioxidant activity test shows that the dragon fruit enzyme has the strongest antioxidant ability,with the DPPH clearance rate and hydroxyl radical scavenging rate of 95.91%and 104%respectively.The reducing power test results of each sample show a dose effect(P>0.05).It's found that there is significant positive correlation between total phenol content and hydroxyl radical scavenging rate(r=0.800,P<0.01)based on the correlation analysis.3.The biologically active enzymes and organic acids in the enzyme samples are tested and the enzyme sensory evaluation criteria are established.A total of ten components including amylase,protease,SOD enzyme,tartaric acid,L-malic acid,lactic acid,acetic acid,citric acid,D-malic acid,and succinic acid are detected in all the samples.The dragon fruit enzyme has the highest amylase activity(4.39±0.02 U/ml),the mango enzyme has the highest SOD enzyme activity(11.56±0.02 U/mL),and the grapefruit enzyme has the highest protease activity(0.79±0.01 U/ml).The organic acids in the enzymes are mainly malic acid and acetic acid,and the content of lactic acid and succinic acid is relatively low.Some samples are detected to be free of tartaric acid and citric acid.Comprehensive evaluation is carried out based on the four aspects of color,taste,form and smell,so as to construct the regression equation:total score = 27.651 + 2.569 x form + 0.898 x fruit + 0.695 × color(R2 = 0.984).The weight of the three selected indicators is recalculated and the new score is finally determined as form(45 points)+ fruit smell(25 points)+ color(30 points).According to the analysis of correlation between sensory quality and metabolites,the citric acid is significantly positively correlated with and color(r=0.923,P<0.01)and significantly positively correlated with form and refreshing taste(r=0.734,P<0.05,r= 0.681,P<0.05),while the protease is significantly positively correlated with color(r=0.751,P<0.05).4.The bacteriostatic experiment shows that each enzyme sample has inhibitory efifect on the three pathogenic bacteria.The orange enzyme has the best inhibition effect on Escherichia coli and Staphylococcus aureus,with the inhibition zone diameter reaching 21.33±3.79 mm and 21.67±2.52 mm respectively,the pineapple enzyme has the best inhibition effect on Salmonella,with the inhibition zone diameter reaching 17.33±1.53 mm,and some enzyme samples are detected to be free of bacteriostatic activity.Bacteriostatic activity stability of the enzymes has relatively poor acid-base stability.The bacteriostatic effect is significantly declined in case the pH is alkaline.The bacteriostatic activity has relatively good thermal stability,and becomes strongest at about 80?.The analysis of correlation between metabolites and bacteriostatic characteristics shows that protease is significantly positively correlated with the inhibition zone of Escherichia coli(r=0.685,P<0.05).