Breeding And Application Of Excellent Yeast Strains For Wolfberry Wine Brewing

Lycium barbarum is a member of the Solanaceae family and is cultivated in China’s Xinjiang,Ningxia,Gansu,and Qinghai provinces.It is a good source of amino acids,organic acids,crude fiber,crude protein,and functional components such as the L.barbarum polysaccharide.Brewing of wolfberry wine has the potential to resolve the issue of L.barbarum deep processing and to promote the growth of the L.barbarum industry.At the moment,the Saccharomyces cerevisiae used to ferment wolfberry wine is generally the wine yeast strain or a universal fruit wine yeast strain.Due to a lack of unique S.cerevisiae,issues such as insufficient aroma and unremarkable typicality of wolfberry wine limit the industry’s growth.Thus,the selection and breeding of specialized brewing strains for wolfberry wine is critical to enhance the wine’s quality.In this study,the brewing microorganisms were screened and isolated from the surface of L.barbarum and the orchard soil,and were chosen using a combination of atmospheric and room temperature plasma（ARTP）mutagenesis technology and high throughput screening（HTS）.S.cerevisiae with excellent aroma production and suitable for the fermentation of wolfberry wine was selected;at the same time,non-S.cerevisiae withβ-glucosidase was screened to improve the flavor substances in wolfberry wine;the fermentation process conditions of wolfberry wine were further optimized,and the wolfberry wine was clarified.The research content and results of the thesis are as follows.（1）The flavor components of two commercially available fermented wolfberry wines（"Health Express"and"Golden Chuanqi"）were qualitatively determined using gas chromatography-mass spectrometry（GC-MS）,and the major flavor components of the wolfberry wine were identified and quantified,as were seven key aroma components in two commercially available fermented wolfberry wines based on their aroma activity value（OAV）.The seven substances studied were isoamyl acetate（3.4-4.4）,ethyl butyrate ester（2.9-3.6）,ethyl acetate（3.2-5.6）,ethyl caproate（2.1-4.8）,isoamyl alcohol（4.0-5.5）,propanol（5.5-5.7）,and phenethyl alcohol（1.8-2.6）.（2）A strain of S.cerevisiae M-23 with good performance was screened from the fruit of Ningqi No.5 and the orchard soil,and further directional screening was found to be suitable for L.barbarum using a combination of ARTP mutagenesis technology and the HTS method.In comparison to the starting strain M-23,the fermented Saccharomyces cerevisiae M-23-7-14showed no significant difference in the basic physical and chemical indexes of the wolfberry wine fermented by this strain,but the content of key flavor compounds increased 37.0%.Quantitative analysis of the flavor components found in various wolfberry wines.Among them,the total amount of seven different aroma substances in M-23-7-14 fermented wolfberry wine is 41.7%greater than in"Health Express"and 29.9%,greater than in"Golden Chuanqi",which is 36.8%more than wolfberry wine fermented with Angel yeast SY.For seven consecutive generations,the strain M-23-7-14 was cultivated.Under the premise of maintaining consistency in terms of alcohol content,total sugar,and total acid,the alcohol content and total flavor substance content of the obtained wolfberry wine remained relatively stable,which showed a high level of genetic stability.（3）Six non-S.cerevisiae strains producingβ-glucosidase were isolated from L.barbarum Ningqi No.5 fruit and orchard soil and their physiological characteristics were determined using growth curve analysis,alcohol and osmotic pressure tolerance tests.Wickerhamomyces anomalus Y11 and S.cerevisiae M-23-7-14 were used to ferment wolfberry wine.The results indicated that when the yeast M-23-7-14 and yeast Y11 were mixed in a 10:1 ratio and yeast Y11 was inoculated first,followed by yeast M-23-7-14 at a 12-hour interval,the system performed optimally during fermentation.In comparison to pure-bred fermentation,the total content of flavor substances increased by 21.7%,while the contents of esters and acids increased by 85.8%and 111.3%,respectively.（4）The fermentation strains for wolfberry wine were yeast M-23-7-14 and yeast Y11,and Y11(1×10⁶ CFU·m L^-1)was initially inoculated with M-23-7-14(1×10⁷ CFU·m L^-1)at 12-hour intervals.The single-factor experiment was used to optimize the fermentation process of wolfberry wine,and the best fermentation process was obtained:the initial sugar content was200 g·L^-1,the fermentation temperature was 16°C,and the solid-liquid ratio was 1:10.The quantitative analysis of key flavor compounds revealed that the total amount of key flavor compounds in mixed-bacteria fermentation wolfberry wine after process optimization was436.66 mg·L^-1.It increased by 17.6%when compared to the mixed bacterial fermentation prior to optimization.Here,the results of 20 L pilot-scale fermentation showed that the physicalchemical characterization and key flavor compounds of fermented wolfberry wine with M-23-7-14 and Y11 was basically consistent with that of flask fermentation.The results indacted that the yeast strains bred in current study could be used for industrial manufacture of wolfberry wine.