Construction Of Wine Yeast Strain With Low-Yield Higher Alcohols By Genetic Manipulation

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.