Study On Constructing Of Deacidification Yeast Strain And Its Application In Loquat Wine Brewage

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^-1malic 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%SO₂+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^-1with concentration of sulfur dioxide80mg·L^-1andalcoholicity11%. 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^-1and1.35g·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.