Studies On The Properties Of β-glucosidase From Aspergillus Sp. Obtained By Protoplast Fusion And Aroma-Enhancing Of Grape Wine

β-glucosidase(EC.3.2.1.21),which is calledβ-D-glucoside glucohydrolase,belongs to the cellulase family.The bound aromatic compounds,also named flavour precursor,are enzymatically hydrolyzed byβ-glucosidase,at the same time,the odour is released.Many flavour precursors exist in grape wine.The potential aromatic compounds are liberated by enzymolysis ofβ-glucosidase.Therefore,the aroma of wine is enhanced and the quality of grape wine is improved.It would be very available to study the aroma-enhancing usingβ-glucosidase during the winemaking process.In this paper,the protoplasts of Aspergillus.niger and Aspergillus.oryzae high producingβ-glucosidase were prepared,formed, regenerated and fused for screening strain.The fermentation conditions were investigated with orthogonal test and response surface analysis.The purification processes and kinetic properties ofβ-glucosidase were discussed.In addition,on the basis of the kinetic properties,the application ofβ-glucosidase for enhancing aroma in wine was also studied using Kramer sensory evaluation and GC-MS analysis.The main content and results are as followed:1.In this study,7 Aspergillus strains selected,named Aspergillus.oryzae 3.481, Aspergillus.oryzae 3.483,Aspergillus.oryzae L,Aspergillus.niger 3.316,Aspergillus.niger 3.4523,Aspergillus.niger L1,Aspergillus.niger L2 were preliminarily selected.And the enzyme activity was as selected target for comparisons with natural selection. Aspergillus.niger 3.316 and Aspergillus.oryzae 3.481 were as original strain for next study.2.The optimum conditions of formation and regeneration of Aspergillus.oryzae protoplast and Aspergillus.niger protoplast were studied.The optimum condition of Aspergillus.niger protolast is,the cell age is in logarithm metaphase of growth 60h,1% cellulose and snail enzyme(1:2,V/V),the time of promoting cell wall degradation is 4 hours, the temperature of promoting cell wall degradation is 30℃,0.2%L-cysteine act as promoter of detaching cell wall,NaCl(0.6M)in phosphate buffer(0.01 M)act as osmotic stabilizer.The optimum condition of Aspergillus.oryzae protolast is,the cell age is in logarithm early stage of growth 48h,1.5%cellulose and snail enzyme(1:1,V/V),the time of promoting cell wall degradation is 3 hours,the temperature of promoting cell wall degradation is 30℃,0.1% L-cysteine act as promoter of detaching cell wall,NaCl(0.6M)in phosphate buffer(0.01M)act as osmotic stabilizer.3.The purified protoplast of Aspergillus.oryzae and Aspergillus.niger were inactivated, respectively.The inactivation conditions of Aspergillus.niger protolast are:the ultraviolet lamp(20W,15cm),magnetic stirring 3min,the viability rate is zero.The inactivation conditions of Aspergillus.oryzae protolast are,water bath at 60℃for 30 min,the viability rate is zero.35%PEG4000 is acting as promoting fusion agent,0.01mmol/LCaCl₂,30℃, 30min.A strain which is genetically stable and highly producedβ-glucosidase is attained by primary screening and secondary screening.4.The culture medium of fusion strains were optimized by means of single-factor test, Box-Benhnken central test and response surface analysis test.The optimum medium were determined,which were as follow:bran+starch of 9.19%,ammonium sulphate of 0.59%, pH4.78,CaCl₂ of 0.02%,KCl of 0.04%.The optimum fermentation conditions were obtained by orthogonal test,the optimum conditions were,rotational rate of 180r/min,fermentation temperature of 27℃with inoculum amount of 12%and medium volume of 20mL/250mL.The best fermentation time was 7days.5.The separation ofβ-glucosidase was studied.The crude enzyme was purified by ammonium sulphate(saturation 60%～90%)to remove other proteins.The enzyme solution after precipitation was purified by DE-52 ion exchange and Sephadex G-150 gel filtration chromatography,the extent of purification was 1.73-fold and 138.85-fold,respectively,and the specific activity were 3.71U/mg and 297.14U/mg,respectively.Electrophoretic pureβ-glucosidase with one electrophoresis band was attained by polyacrylamide gel electrophoresis(PAGE).The molecular mass of the subunits was estimated to be 125kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE).6.Properties of purifiedβ-glucosidase were discussed.The results showed:optimum pH forβ-gulcoside activity was determined to be 5.4,and optimum temperature to be 65℃. Purifiedβ-gulcoside showed relatively high stability against pH and temperature.This enzyme was stable in the pH range of 3.0～6.6.β-gulcoside was highly activated by Fe³⁺and inhibited by Na⁺,Ca²⁺,Mg²⁺,K⁺,EDTA.The K_m and V_maxvalues of this enzyme against salicin as substrate were 0.035mmol/L and 1.7215μmol/min.7.The extrcation was carried out by adsorbing free aromatic compounds in wine on a non-ionic resin,Amberlite XAD-2,and then elution with various selective solvents.At last, pentane and ether(1:1,V/V)were as fine eluent.The bound compounds in wine were enzymatically hydrolyzed byβ-glucosidase to release aglycones.The bound fractions after enzymolysis yielded rhamnose,xylose and glucose through HPLC analysis. Free aromatic compounds and bound compounds after enzymolysis were analyzed by GC-MS.The results were as followed:many aromatic compounds were released,such as 3-methyl-1-Butanol formate,Ethylbenzene,3-Pentanol,1-Hexanol,3-methyl-Butanoic acid, 3-methyl-1-Butanol acetate,Butyrolactone,1-Butanol,2-methyl-Butanoic acid, 1S-.alpha.-Pinene,Hexanoic acid,Benzaldehyde,Benzyl Alcohol,Benzeneacetaldehyde, Hexanoic acid ethyl ester,Phenylethyl Alcohol,Acetic acid,2-phenylethyl ester,Octanoic acid ethyl ester,Butanedioic acid diethyl ester,Prppanoic acid 2-phenylethyl ester,and so on.8.The factors which were influencing aroma-enhancing of wine were investigated through Kramer sensory evaluation and L₉(3⁴)orthogonal test,the orders were as followed: temperature＞enzyme volume＞time.The optimum enzymolysis condition was that: temperature of 45℃,enzymolysis time of 90min with enzyme amount of 8mL/100mL grape wine.The enzymolysis hydrolyzed wine,which was extracted by steam distillation extraction, was analyzed by GC-MS.And the control wine sample was also analyzed.The aromatic compounds were as followed:3-methyl-l-Butanol formate,3-Pentanol,Furfural, 3-methyl-Butanoic acid,2-methyl-Butanoic acid,3-hydroxy-Butanoic acid ethyl ester, Hexanoic acid,Hexanoic acid ethyl ester,Benzyl Alcohol,Octanoic Acid,Octanoic acid ethyl ester,Dodecanoic acid,ethyl ester and so on.