降酸酵母菌株的构建及其在枇杷酒酿造中的应用研究
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摘要
枇杷酒的发酵多采用酿酒酵母，其利用苹果酸的能力较低，发酵结束后大部分留在成品中。因苹果酸口感酸涩，酒体柔和指数较低，影响枇杷酒的品质。本研究通过克隆粟酒裂殖酵母苹果酸通透酶(mae1)基因，构建整合型质粒，并在产朊假丝酵母中表达，使重组酵母菌株具有降解苹果酸的能力。将降酸酵母菌株投放于发酵后的枇杷原酒进行降酸发酵，消耗酒中部分苹果酸，同时增加枇杷酒部分香气成分，改善枇杷酒的感官品质。
研究结论如下：
1、采用Trizol法提取粟酒裂殖酵母的总RNA，以报道的粟酒裂殖酵母的苹果酸通透酶（mae1）的序列为依据设计引物，分别以基因组和cDNA为模板，克隆得到苹果酸通透酶（mae1）基因。通过双酶切、回收、连接，构建重组表达质粒pSH47-mae1。将pSH47表达质粒上的gal启动子换成PGK1启动子，成功的构建了重组表达质粒pSH47-PG-mae1。将线性化的重组质粒以同源重组的方式整合到产朊假丝酵母的基因组中表达。通过菌液PCR检验和摇瓶筛选，获得重组菌株CU-6。经连续传代实验表明，重组酵母菌株CU-6有良好的传代稳定性。
2、研究了SO_2浓度、糖度、酒精度、接种量、温度5个单因素对重组酵母CU-6降酸的影响，筛选出3个主因素（SO_2浓度、糖度、酒精度）。利用中心组合设计试验的原理设计交互试验，采用响应面分析法优化枇杷酒的降酸工艺条件。通过响应面分析法得到枇杷酒降酸优化工艺：初始SO_2浓度50mg·L~(-1)，酒精度7.8%，残糖量4.3g·L~(-1)。在苹果酸浓度4.5g·L~(-1)，接种量1.5%，发酵温度为24℃的条件下发酵5d，产朊假丝酵母CU-6的在枇杷酒中的理论降酸量可达到1.82g·L~(-1)。实验实际值为1.80±0.02g·L~(-1)，与理论值十分接近。
3、研究6种固定化材料对重组酵母菌株CU-6在枇杷酒的降酸影响。其中2%海藻酸钠＋1.4%SiO_2＋0.6%PVA组合固定化酵母CU-6，降酸效果最好。研究发现固定化后酵母CU-6耐受SO_2和酒精度能力适度提高，在SO_2浓度80mg·L~(-1)和酒精度为11%时，降酸量仍可达到1.6g·L~(-1)。该材料制作的固定化微球连续使用5次以上，酵母的降酸能力仍保持较好。
4、采用固定化酵母CU-6降酸处理后的枇杷酒，感官品质发生一定的变化。早钟和解放钟为原料酿造的枇杷酒经处理后，双乙酰影响较小，色度的影响较大，分别下降了37%和55%，总酸分别下降1.43和1.35g·L~(-1)，柔和指数分别提高1.44和1.37。降酸处理使枇杷酒中的香气成分比例发生了改变，部分醇类物质比例提高，如苯乙醇、柏木醇等，部分酯类物质的比例提高，如丁二酸二乙酯等，部分酮类物质增加较多，如5-羟基-4-辛酮。
5、固定化重组酵母菌株CU-6降酸处理后的枇杷酒，其营养组成未发生明显变化。降酸前后的矿物质含量保留较好。降酸处理后游离氨基酸有不同程度的增加，其中必需氨基酸苏氨酸、缬氨酸、蛋氨酸、异亮氨酸、亮氨酸、苯丙氨酸等明显增加。早钟和解放钟枇杷酒中的抗氧化活性物质含总酚、总黄酮和维生素C。研究发现，两种枇杷酒降酸前后总抗氧化能力均比对照的国产解百纳干红低，但高于国产雷司令干白；羟自由基清除能力均高于国产解百纳干红，但低于国产雷司令干白；对DPPH自由基表现出较好的清除作用，且随着稀释倍数的增加能力逐渐减弱，当将其稀释20倍时DPPH自由基的清除能力均仍能维持在60%以上。
Malic acid cannot be degraded during loquat wine fermentation by Saccharomycescerevisiae and ultimately result in high total acid level in liquor. Malic acid taste sour andastringent, which impact flavor balance index and sensory quality of loquat wine. Malatepermease (mae1) gene of Schizosaccharomyces pombe was cloned, integration plasmid wasconstructed and expressed in Candida utilis in the study. Recombinant yeast strains wasemployed in loquat wine after primary fermentation to deacid, which can degrad malicacid,increase the aroma substrances and improve sensory quality of loquat wine.
Results of the study are as follows,
⑴Total RNA of Schizosaccharomyces pombe was extracted of by Trizol method,primers of mae1was designed based on reported sequence of Schizosaccharomyces pombe.Genome and cDNA as template was employed and Malate permease (mae1) wascloned.Recombinant expression plasmid pSH47-mae1was construct by double enzymedigestion, recovery, connection.Gal promoter pSH47in expression plasmid was replaced byPGK1promoter and recombinant expression plasmid pSH47-PG-mae1was construction.Recombinant plasmid pSH47-PG-mae1was linearized and homologous recombinated intogenome of Candida utilis to express. Recombinant yeast strains CU-6was screened bybacterium liquid PCR inspection and shake flask fermentation.It shows preferable passagestability through continuous subculture experiments.
⑵Effect on degradation of malic acid of recombinant yeast strains CU-6byconcentration of sulfur dioxide, brix, alcoholicity, inoculation, fermentation temperaturewere study.Concentration of sulfur dioxide, brix, alcoholicity were selected as main effectfactors. Response surface methodology (RSM) was applied to optimize deacidification inloquat wine by recombinant yeast strain. The degradation of malic acid was selected asresponse value and the mathematical model was established by Box-Benhnken centralcomposite design. The optimal fermentation conditions were: sulfur dioxide50mg·L~(-1),alcoholicity7.8%, brix4.3g·L~(-1). Under conditions of concentration of malic acid4.5g·L~(-1),inoculation1.5%, fermentation temperature24℃,1.82g·L~(-1)malic acid in loquat wine wasdegraded by Candida utilis CU-6within5days theoretically. With the optimal approch, the deacidification of loquat wine reaehed1.80±0.02g·L~(-1).
⑶Six kinds of immobilized materials were compared and deacidification ability ofrecombinant yeast strains CU-6applied in loquat wine was study. Group2%SOA+1.4%SO_2+0.6%PVA as immobilized material showed optimal deacidificationability.Tolerance capacity on sulfur dioxide and alcohol of recombinant yeast strains CU-6were increased reasonably by immobilization. The deacidification of loquat wine by CU-6immobilization reaehed1.60g·L~(-1)with concentration of sulfur dioxide80mg·L~(-1)andalcoholicity11%. Deacidification ability of recombinant yeast strains CU-6remains wellafter continuous employ for more than five times.
⑷Sensory quality of loquat wine were changed by immobilized recombinant yeaststrains CU-6after deacidification. After deacidification treatment, diacetyl and tanninchanged faintly, chroma were decreased37%and55%, total acid were decreased1.43g·L~(-1)and1.35g·L~(-1), flavor balance index were increased1.44and1.37in zaozhong andJiefangzhong loquat wine. Component of Aroma components in zaozhong andjiefangzhong loquat wine were varied. The proportion of partial ethanol such asphenethanol,cedrol were increased，partial esters such as diethyl succinate and partialketones such as5-hydroxy-4-octanone was increased via deacidification treatment.
⑸Nutrients in Zaozhong and Jiefangzhong loquat wine were changes faintly afterdeacidification treatmen by recombinant yeast strains CU-6. Content of minerals wereremained preferably and free amino acids were increased in different degrees afterdeacidification. Essential amino acid including methionine, threonine, valine, isoleucine,leucine, phenylalanine were increased significantly. Antioxidants in Zaozhong andJiefangzhong loquat wine contain total phenolics, total flavonoids and vitamin C. Totalantioxidant capacity of loquat wine before and after deacidification was lower thanindigenous Cabernet dry red, but higher than indigenous Riesling white; Hydroxyl radicalscavenging ability of loquat wine before and after deacidification was higher thanindigenous Cabernet dry red, but lower than indigenous Riesling white; loquat wine beforeand after deacidification showed commendable DPPH scavenging capacity,which wasweakened gradually with dilution increasing. It still maintained more than60%when itdiluted20times.
研究结论如下：
1、采用Trizol法提取粟酒裂殖酵母的总RNA，以报道的粟酒裂殖酵母的苹果酸通透酶（mae1）的序列为依据设计引物，分别以基因组和cDNA为模板，克隆得到苹果酸通透酶（mae1）基因。通过双酶切、回收、连接，构建重组表达质粒pSH47-mae1。将pSH47表达质粒上的gal启动子换成PGK1启动子，成功的构建了重组表达质粒pSH47-PG-mae1。将线性化的重组质粒以同源重组的方式整合到产朊假丝酵母的基因组中表达。通过菌液PCR检验和摇瓶筛选，获得重组菌株CU-6。经连续传代实验表明，重组酵母菌株CU-6有良好的传代稳定性。
2、研究了SO_2浓度、糖度、酒精度、接种量、温度5个单因素对重组酵母CU-6降酸的影响，筛选出3个主因素（SO_2浓度、糖度、酒精度）。利用中心组合设计试验的原理设计交互试验，采用响应面分析法优化枇杷酒的降酸工艺条件。通过响应面分析法得到枇杷酒降酸优化工艺：初始SO_2浓度50mg·L~(-1)，酒精度7.8%，残糖量4.3g·L~(-1)。在苹果酸浓度4.5g·L~(-1)，接种量1.5%，发酵温度为24℃的条件下发酵5d，产朊假丝酵母CU-6的在枇杷酒中的理论降酸量可达到1.82g·L~(-1)。实验实际值为1.80±0.02g·L~(-1)，与理论值十分接近。
3、研究6种固定化材料对重组酵母菌株CU-6在枇杷酒的降酸影响。其中2%海藻酸钠＋1.4%SiO_2＋0.6%PVA组合固定化酵母CU-6，降酸效果最好。研究发现固定化后酵母CU-6耐受SO_2和酒精度能力适度提高，在SO_2浓度80mg·L~(-1)和酒精度为11%时，降酸量仍可达到1.6g·L~(-1)。该材料制作的固定化微球连续使用5次以上，酵母的降酸能力仍保持较好。
4、采用固定化酵母CU-6降酸处理后的枇杷酒，感官品质发生一定的变化。早钟和解放钟为原料酿造的枇杷酒经处理后，双乙酰影响较小，色度的影响较大，分别下降了37%和55%，总酸分别下降1.43和1.35g·L~(-1)，柔和指数分别提高1.44和1.37。降酸处理使枇杷酒中的香气成分比例发生了改变，部分醇类物质比例提高，如苯乙醇、柏木醇等，部分酯类物质的比例提高，如丁二酸二乙酯等，部分酮类物质增加较多，如5-羟基-4-辛酮。
5、固定化重组酵母菌株CU-6降酸处理后的枇杷酒，其营养组成未发生明显变化。降酸前后的矿物质含量保留较好。降酸处理后游离氨基酸有不同程度的增加，其中必需氨基酸苏氨酸、缬氨酸、蛋氨酸、异亮氨酸、亮氨酸、苯丙氨酸等明显增加。早钟和解放钟枇杷酒中的抗氧化活性物质含总酚、总黄酮和维生素C。研究发现，两种枇杷酒降酸前后总抗氧化能力均比对照的国产解百纳干红低，但高于国产雷司令干白；羟自由基清除能力均高于国产解百纳干红，但低于国产雷司令干白；对DPPH自由基表现出较好的清除作用，且随着稀释倍数的增加能力逐渐减弱，当将其稀释20倍时DPPH自由基的清除能力均仍能维持在60%以上。
Malic acid cannot be degraded during loquat wine fermentation by Saccharomycescerevisiae and ultimately result in high total acid level in liquor. Malic acid taste sour andastringent, which impact flavor balance index and sensory quality of loquat wine. Malatepermease (mae1) gene of Schizosaccharomyces pombe was cloned, integration plasmid wasconstructed and expressed in Candida utilis in the study. Recombinant yeast strains wasemployed in loquat wine after primary fermentation to deacid, which can degrad malicacid,increase the aroma substrances and improve sensory quality of loquat wine.
Results of the study are as follows,
⑴Total RNA of Schizosaccharomyces pombe was extracted of by Trizol method,primers of mae1was designed based on reported sequence of Schizosaccharomyces pombe.Genome and cDNA as template was employed and Malate permease (mae1) wascloned.Recombinant expression plasmid pSH47-mae1was construct by double enzymedigestion, recovery, connection.Gal promoter pSH47in expression plasmid was replaced byPGK1promoter and recombinant expression plasmid pSH47-PG-mae1was construction.Recombinant plasmid pSH47-PG-mae1was linearized and homologous recombinated intogenome of Candida utilis to express. Recombinant yeast strains CU-6was screened bybacterium liquid PCR inspection and shake flask fermentation.It shows preferable passagestability through continuous subculture experiments.
⑵Effect on degradation of malic acid of recombinant yeast strains CU-6byconcentration of sulfur dioxide, brix, alcoholicity, inoculation, fermentation temperaturewere study.Concentration of sulfur dioxide, brix, alcoholicity were selected as main effectfactors. Response surface methodology (RSM) was applied to optimize deacidification inloquat wine by recombinant yeast strain. The degradation of malic acid was selected asresponse value and the mathematical model was established by Box-Benhnken centralcomposite design. The optimal fermentation conditions were: sulfur dioxide50mg·L~(-1),alcoholicity7.8%, brix4.3g·L~(-1). Under conditions of concentration of malic acid4.5g·L~(-1),inoculation1.5%, fermentation temperature24℃,1.82g·L~(-1)malic acid in loquat wine wasdegraded by Candida utilis CU-6within5days theoretically. With the optimal approch, the deacidification of loquat wine reaehed1.80±0.02g·L~(-1).
⑶Six kinds of immobilized materials were compared and deacidification ability ofrecombinant yeast strains CU-6applied in loquat wine was study. Group2%SOA+1.4%SO_2+0.6%PVA as immobilized material showed optimal deacidificationability.Tolerance capacity on sulfur dioxide and alcohol of recombinant yeast strains CU-6were increased reasonably by immobilization. The deacidification of loquat wine by CU-6immobilization reaehed1.60g·L~(-1)with concentration of sulfur dioxide80mg·L~(-1)andalcoholicity11%. Deacidification ability of recombinant yeast strains CU-6remains wellafter continuous employ for more than five times.
⑷Sensory quality of loquat wine were changed by immobilized recombinant yeaststrains CU-6after deacidification. After deacidification treatment, diacetyl and tanninchanged faintly, chroma were decreased37%and55%, total acid were decreased1.43g·L~(-1)and1.35g·L~(-1), flavor balance index were increased1.44and1.37in zaozhong andJiefangzhong loquat wine. Component of Aroma components in zaozhong andjiefangzhong loquat wine were varied. The proportion of partial ethanol such asphenethanol,cedrol were increased，partial esters such as diethyl succinate and partialketones such as5-hydroxy-4-octanone was increased via deacidification treatment.
⑸Nutrients in Zaozhong and Jiefangzhong loquat wine were changes faintly afterdeacidification treatmen by recombinant yeast strains CU-6. Content of minerals wereremained preferably and free amino acids were increased in different degrees afterdeacidification. Essential amino acid including methionine, threonine, valine, isoleucine,leucine, phenylalanine were increased significantly. Antioxidants in Zaozhong andJiefangzhong loquat wine contain total phenolics, total flavonoids and vitamin C. Totalantioxidant capacity of loquat wine before and after deacidification was lower thanindigenous Cabernet dry red, but higher than indigenous Riesling white; Hydroxyl radicalscavenging ability of loquat wine before and after deacidification was higher thanindigenous Cabernet dry red, but lower than indigenous Riesling white; loquat wine beforeand after deacidification showed commendable DPPH scavenging capacity,which wasweakened gradually with dilution increasing. It still maintained more than60%when itdiluted20times.
引文
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