| The blackberry,which is rich in nutrition,anthocyanins,polyphenol compound vitamins and a variety of minerals,is the third generation of special berries.It plays roles in antioxidation,hyperlipidemia and anti-arrhythmic,etc.Blackberry wine is the product of yeast fermentation of blackberry juice.It keeps its nutrients and functional ingredients to the maximum extent.The aroma and flavor of blackberry wine is due to the interaction among aroma compounds and those compounds contain fruity and glycoside aroma precursors.Glycosidases such as beta-glucosidases can hydrolyze glycoside aroma precursors to release volatile aglycone,thus improve the flavor of the wine.The beta-glycosidase can hydrolyze glycosidic bond between aglycone and glycosyl,which produce aromatic substances and glucose.At present,most of the studies on glycosidase flavoured wine are focus on the effects of beta-glucosidase on wine aroma substances.Limited researches base on the influence of the yeast strains,heat treatment and ultrahigh pressure processing on the blackberry wine aroma.In this study,four strains of Lactobacillus which can generate beta-glycosidase with strong enzyme activity were selected.Due to adverse factors such as enzyme activity mainly exist in the cell pellets,low-density liquid fermentation,it is easy to get natural beta-glucosidase.Three Lactobacilli beta-glucosidase genes were selected for molecular cloning by using the high homology of biological information and the recombinant enzyme Lb0241 with a beta-glucosidase enzyme activity was obtained.Based on enzymology characteristics,its effects on blackberry wine aroma substances were studied.It provides a theoretical basis for the development of new blackberry wine.The study results are as follows:1.The screening,identification and initial enzyme localization of Lactobacillus that can produce beta-glucosidase.The esculin glycoside display tablet method was used to select 4 strains of high yield of beta-glucosidase Lactobacillus CM3,Hsb,FL12 and T61.With biochemical experiments and 16S rDNA test,identified the strains were Lactobacillus pentosus,Lactobacillus plantarum and Lactobacillus brevis,respectively.With initial enzyme localization,the enzyme activity was observed in complete bacteria,cell disrupter and cell pellet of the three strains CM3,Hsb and FL12.Strain T61 was stunted under the condition of liquid culture and thus could not to expand training.Finally,4 homologous beta-glucosidases were obtained using molecular cloning technology.2.The cloning,expression and purification of recombinant of beta-glucosidase.Through bioinformatics homologous comparison,a variety of known source of Lactobacilli beta-glucosidase genes were selected for designing primers.From CM3,FL12 and T61 genome,three beta-glucosidase genes were successfully cloned and named LplaG,Lb0241,Lb367,respectively.The obtained gene fragments were inserted into pET30a vector and then transferred recombinant plasmids into E.coli BL21(DE3)for expression,respectively.Large amounts of target proteins were obtained by auto-induction in high-density shaking cultures.Through SDS gel electrophoresis analysis,observed that the three kinds of recombinant enzyme could soluble expression in E.coli system.Unfortunately,only recombinant enzyme Lb0241 possessed beta-glucosidase activity.50 mL auto-induction culture produced 61.99 μg/mL of pure recombinant enzyme Lb024 with Ni2+-NTA affinity chromatography column purification.3.The determination of the biochemical characteristics of the recombinant beta-glucosidase.pNP-β-D-Glc was used as the substrate and the biochemical characteristics of the recombinant beta-glucosidase Lb0241 were investigated.The results showed that the optimal pH of recombinant Lb0241 was 5.5;the optimum reaction temperature is 30℃;The glucosidase catalyzed reactions do not require metal ions as coenzymes,in which the enzyme activity of Lb0241 was enhanced by Ni2+ and K+,while the enzyme activity was decreased by Fe3+,Fe2+and Mg2+.All five chemical reagents(including urea,SDS,imidazole,ethanol and 2-ME)could reduce the enzyme activity of Lb0241.The inhibition of SDS was the most obvious,and the enzyme tolerated ethanol.The Km value of Lb0241 was 0.84 mmol/L forpNP-β-D-Glc;Vmax value was 0.693 mmol/L/min;Kcat was 0.018 s-1.The catalytic reaction of recombinant Lb0241 was monitored in real time with seven kinds of 2-chloro-4-nitrobenzene-glycosides(pNP-glycosides)as substrates.The results showed that the recombinant enzyme Lb0241 could hydrolyze pNP-β-D-Glc and pNP-β-D-Gal.4.Effect of recombinant beta-glucosidase Lb0241 on aroma components of blackberry wineFirstly,blackberry glycoside aroma precursors were extracted by SPE column,then recombinant Lb024 was uesed hydrolyze aroma precursors and blackberry wine.The SPME-CG-MS technology was used to detect and identify the volatile compounds of different examples.The results demonstrated that:1.after the enzymatic treatment to the glycoside aroma precursor,the detected substances increased from 9 to 13.2.after the blackberry wine was enzymatically digested,the detected substances increased from 17 to 21.Although there was a reduction in some substances,the aroma after enzyme treatment was higher than control sample,indicating that the recombinant Lb0241 had a significant aroma-increasing effect. |
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