【作者】 秦伟帅；

【导师】 翟衡；

【作者基本信息】 山东农业大学 ， 葡萄与葡萄酒学， 2013， 博士

【摘要】 高级醇是葡萄酒酿造过程中酵母酒精发酵的主要次生代谢物，如果葡萄酒中高级醇含量过高，则会使消费者饮用后“上头”、头疼，给人以辛辣腐臭感、刺激性和不愉快的苦涩味，其不仅严重破坏了酒的口感和风味，而且降低了葡萄酒的营养价值和保健效果。目前该问题在新生葡萄酒和家庭自酿葡萄酒中尤为多见。因此寻找有效降低葡萄酒中高级醇含量的方法，对提高葡萄酒质量，促进葡萄酒产业的发展具有重要的现实意义。论文基于对葡萄酒酵母高级醇代谢机制的综合分析，筛选出了高级醇生成量较低的葡萄酒酵母菌种，然后利用酵母遗传操作方法（基因组重排和同源重组技术）对其高级醇代谢途径进行了调控，并探讨了发酵工艺条件（温度、pH值和二氧化硫添加量）对改造菌株高级醇生成的影响，主要获得了以下研究结果：1.本试验综合比较了葡萄酒酿造业常用的6株葡萄酒酵母（EC1118、RC212、CY3079、D254、QA23、DV10）的凝聚性、耐二氧化硫性能、高级醇产生量及香气感官特征，结果表明菌株EC1118发酵性能优良，具有较低的高级醇生成量。通过描述性感官评定，菌株EC1118所酿酒的果香、花香浓郁，刺激性较小。以此菌株作为本试验的出发菌株。2.采用EMS对二倍体酵母菌株EC1118进行诱变，获得了具有充分遗传多样性菌株群，并通过有性重组来实现基因组重排的方法，构建了具有低产高级醇特性的葡萄酒酵母菌株CP1118。测定结果表明，菌株CP1118总高级醇生成量为237.43mg/L，较菌株EC1118总高级醇生成量（276.39mg/L）降低了近14%，其中以正丙醇和异戊醇降低幅度最为明显，较EC1118分别降低了34%和7%。异丁醇降低幅度不大。正己醇和β-苯乙醇生成量略有升高，较EC1118分别增加了近5%和12%。3.试验进一步利用同源重组技术成功敲除了二倍体菌株CP1118酯酶分解酶基因IAH1，并获得了低产高级醇菌株QC1118。测定结果表明IAH1基因敲除菌株QC1118总高级醇生成量较出发菌株CP1118降低了近10%。其中异戊醇和β-苯乙醇降低幅度较大，分别降低了22%和23%。异丁醇和正己醇降低幅度较小，分别降低了近18%和5%。正丙醇生成量增加了近25%。菌株QC1118总高级醇生成量与原始菌株EC1118相比降低了近23%。菌株QC1118和CP1118感官品评试验表明，两株酵母在相同的感官特性上所表达的每种特性的强度不同。菌株QC1118发酵的酒样花香和果香比较明显，具有较低的刺激性，整体评价较高。4.试验从发酵工艺角度研究了温度、发酵醪pH值和葡萄破碎时二氧化硫添加量对酵母QC1118高级醇生成的影响。测定结果表明17℃发酵时酵母高级醇生成量最大，为377.30mg/L。26℃发酵条件下高级醇生成量最低，为159.32mg/L。在17℃～26℃发酵温区内酵母高级醇生成量随着发酵温度的升高而降低。发酵醪pH值对酵母高级醇生成影响不大，在pH值3.4时酵母高级醇生成量为269.19mg/L，其它pH值条件下酵母高级醇生成量均在244mg/L左右。二氧化硫添加量在30mg/L时高级醇生成量最大，为326.88mg/L。在110mg/L的二氧化硫添加量时酵母高级醇生成量最低，为264.91mg/L，较高的二氧化硫添加量有利于降低葡萄酒中高级醇含量。更多还原

【Abstract】 Higher alcohols are the main secondary metabolites of yeast alcohol fermentation in thewinemaking process. If the wine has a high content of higher alcohols, it will give theconsumers a headache, a spicy sense of rancid, irritating and unpleasant bitter taste, which notonly seriously damages the taste and flavor of the wine, but also reduces the nutritional valueand health effects. The problem is particularly prevalent in the fresh and home-brewing wines.So finding an effective method to reduce the content of higher alcohols has an importantpractical significance to improve the wine quality and promote the development of brewingindustry. Based on the comprehensive analysis of the wine yeast alcohol metabolism, thestrains which produced less higher alcohols have been screened. Regulations to yeast higheralcohol metabolic pathways have been carried out by the means of genetic manipulation(genome shuffling and homologous recombination technology). In addition, the paper hasdiscussed the influence of fermentation conditions (temperature, pH and amount of sulfurdioxide) on the yeast higher alcohol production in the objective strain and obtained thefollowing results:1. The flocculation, resistance to sulfur dioxide, production of higher alcohols andsensory characteristic of six wine yeasts commonly used in winemaking (EC1118, RC212,CY3079, D254, QA23, DV10) were comprehensively compared in this paper. The resultsshowed that EC1118had an excellent performance of fermentation with low higher alcoholsproduction. Moreover, the wine made by EC1118had a strong fruity, flowery odour and lowpungency so that EC1118strain was selected as the original strain.2. The group strains with full genetic diversity were obtained by using EMS mutagenesisof the diploid yeast strain EC1118. And CP1118with low-yield higher alcohols wasconstructed by means of genome shuffling. The test showed that the total higher alcoholsproduction of CP1118was237.43mg/L, reduced by nearly14%compared with EC1118(276.39mg/l).1-propanol and isoamyl alcohol were significantly decreased by34%and7% while1-hexanol and2-phenethyl alcohol were slightly increased by5%and12%.3. Low-yield higher alcohols strains QC1118was obtained through the esterase-encodinggene IAH1of CP1118disruption. The results showed that the higher alcohols production ofQC1118was reduced by nearly10%compared with CP1118. Isoamyl alcohol and2-phenethyl alcohol were significantly reduced by22%and23%. Isobutyl alcohol and1-hexanol were decreased by18%and5%while1-propanol increased by25%. Theconcentration of total higher alcohols produced by QC1118was reduced by nearly23%compared with the original strain EC1118. The sensory analysis of QC1118and CP1118showed that the strength of each characteristic in the same organoleptic properties had somedifferences between the two strains. With a strong fruity, flowery odour and low pungency,the wine fermented with QC1118had a higher general evaluation.4. The impact of fermentation condition (temperature, pH, sulfur dioxide addition) on theQC1118higher alcohols production had been studied. The results showed that theconcentration of higher alcohols was the highest under17℃fermentation condition(377.30mg/L) while the lowest was26℃(159.32mg/L). The formation of higher alcoholsdecreased when the fermentation temperature increased from17℃to26℃. The pH offermented must had little influence on the production of higher alcohols and the highestconcentration was269.19mg/L when the pH value was3.4while others were approximately244mg/L. When the sulfur dioxide addition was30mg/L, the higher alcohols concentrationreached the highest (326.88mg/L). The lowest concentration of264.91mg/L appeared whenthe sulfur dioxide addition was110mg/L. Higher addition of sulfur dioxide was able to reducethe productions of higher alcohols.更多还原