【作者】 朱凤妹；

【导师】 刘长江； 李军；

【作者基本信息】 沈阳农业大学 ， 食品科学， 2008， 博士

【摘要】 β-葡萄糖苷酶（β-glucosidase,EC.3.2.1.21）全称β-D-葡萄糖苷水解酶,是纤维素酶系的一个组成部分。它能酶解键合态的风味前体物质,释放出香气成分。本实验对高产β-葡萄糖苷酶菌株黑曲霉（Aspergillus.niger）和米曲霉（Aspergillus.oryzae）进行原生质体融合育种、发酵条件优化、β-葡萄糖菅酶分离及动力学性质进行研究,结合Kramer感官评定法和气相色谱-质谱（GC-MS）法对其在葡萄酒增香中的作用进行研究。主要研究内容和结果如下:1.对米曲霉3.481、米曲霉3.483、米曲霉L、黑曲霉3.316、黑曲霉3.4523、黑曲霉L1、黑曲霉L2菌株进行初筛,以β-葡萄糖苷酶活力为筛选指标进行比较,确定黑曲霉3.316和米曲霉3.481为出发菌株。。2.优化黑曲霉3.316原生质体和米曲霉3.481原生质体形成和再生的条件。黑曲霉3.316原生质体形成和再生的最适条件:菌龄为对数生长中期60h,0.6M氯化钠/0.01M磷酸缓冲溶液为渗透压稳定剂,0.2%L-半胱氨酸为脱壁促进剂,1.0%的纤维素酶和蜗牛酶（1:2,V/V）30℃水浴酶解4h。米曲霉3.481原生质体形成和再生的最适条件:菌龄为对数生长早期48h,0.6M氯化钠/0.01M磷酸缓冲溶液为渗透压稳定剂,0.1%L-半胱氨酸为脱壁促进剂,1.5%纤维素酶和蜗牛酶（1:1,V/V）30℃水浴酶解3h。3.黑曲霉3.316原生质体紫外灭活条件:紫外灯20W,垂直距离15cm,磁力搅拌3min灭活,原生质体存活再生率为0。米曲霉3.481原生质体热灭活条件:60℃水浴30min灭活,原生质体存活再生率为0。采用35%聚乙二醇（PEG）4000做为促融合剂,0.1mmol/LCaCl₂,30℃,融合30min。融合子经过初筛、复筛、再复筛得到一株产酶活力高、遗传稳定的菌株。4.通过单因素试验、Box-Benhnken中心组合试验和响应面分析试验对融合菌株的发酵培养基进行优化,确定其最佳培养基条件为:麸皮+淀粉浓度9.19%、硫酸铵浓度0.59%、pH4.78、CaCl₂浓度0.02%、KCl浓度0.04%。通过正交交互试验确定融合菌株产β-葡萄糖苷酶的发酵条件:装液量20mL/250mL,接种量12%,发酵温度27℃,摇床转速180r/min。最佳发酵时间:7d。5.对β-葡萄糖苷酶进行分离纯化。利用饱和度为60%～90%的硫酸铵对β-葡萄糖苷酶进行盐析,去除大部分杂蛋白。硫酸铵盐析浓缩后酶液经过DE-22纤维素离子交换柱层析,纯化倍数为1.73倍,比活力为3.71U/mg;交联葡聚糖Sephadex G-150凝胶柱层析,纯化倍数达138.85倍,比活力为297.14U/mg,经PAGE电泳检测为1条谱带,达到电泳纯。SDS-PAGE电泳测定其亚基分子量为125kD左右。6.对纯化后β-葡萄糖菅酶的酶学进行研究。结果表明:β-葡萄糖苷酶的最适温度65℃,最适pH5.4,在pH3.0～6.6条件下较稳定,热稳定性较好。Na⁺、Ca²⁺、Mg²⁺、K⁺、EDTA对β-葡萄糖苷酶活力有明显抑制作用,Fe³⁺对酶活力有促进作用。β-葡萄糖苷酶的动力学方程为:1/V=0.0201×1/[S]+0.5809,Vmax=1.7215μmol/min,米氏常数Km=0.03 5mmol/L。7.采用Amberlite XAD-2大孔吸附树脂吸附葡萄酒中游离态芳香物质,戊烷:乙醚（1:1,V/V）做为溶剂具有较好的洗脱效果。利用β-葡萄糖苷酶酶解葡萄酒中分离出的键合态物质,将键合态呈香物质酶解前后进行HPLC分析。结果显示:键合态物质在酶解后出现了鼠李糖、木糖、葡萄糖。并且键合态物质在酶解后出现葡萄酒特有的香气。将葡萄酒中游离态芳香物质和键合态呈香物质酶解得到的芳香物质经过GC-MS分析。结果表明:键合态的芳香物质经过β-葡萄糖苷酶酶解后释放出许多芳香物质,如甲酸异戊酯、苯乙烷、3-戊醇、正己醇、异戊酸、乙酸异戊酯、丁内酯、丁醇、2-甲基-丁酸、α-蒎烯、己酸、苦杏仁油、苄醇、苯乙醛、己酸乙酯、苯乙醇、乙酸-2-苯乙酯、辛酸乙酯、丁二酸二乙酯、丙酸-2-苯乙酯、月桂酸乙酯、邻苯二甲酸二丁酯等,这些物质有典型的香气,对葡萄酒的香味有一定的增强作用。8.L₉（3⁴）正交试验设计通过Kramer感官评定,确定葡萄酒酶解增香综合影响因素的顺序为:酶解温度＞酶量＞酶解时间。酶解最佳条件为:酶解温度45℃、酶解时间90min、加酶量8mL/100mL葡萄酒。利用水蒸气蒸馏β-葡萄糖苷酶酶解的葡萄酒和对照未处理的葡萄酒,经GC-MS分析发现酶解后出现更多的香气成分,主要包括:甲酸异戊酯、3-戊醇、糠醛、异戊酸、2-甲基-丁酸、3-羟基-丁酸乙酯、己酸、己酸乙酯、苄醇、辛酸、辛酸乙酯、月桂酸乙酯等。更多还原

【Abstract】 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.更多还原