Optimization Research Of Screening And Proliferation Culture Of Dairy Yeast (Kluyveromyces Marxianus) And Preparation Of Freeze-dried Powder

Milk beer is a new milk beverage beneficial to human health,which is fermented by lactobacilli and yeasts.It has the characteristics of“rich nutrition,palatedfullness and pleasant feeling”and was pioneered by Chinese.At now,it is increasingly popular with Chinese consumers.Yeast is the key strain in the fermentation production of milk beer,which can provide milk beer with major flavors such as alcohols,acids and esters and determine the flavor and quality of the milk beer products.At present,most domestic enterprises use commercially available beer yeast and Saccharomyces cerevisiae strains to produce milk beer products.As the method of producing milk beer results in products with a single variety and taste,which seriously affects the promotion and market share of milk beer.On the other hand,excessive hops and malt added during the production process result in complex production processes and high costs.Although some enterprises used screened high-quality dairy yeast to improve the quality of milk beer,the lack of active dry yeast powder is not conducive to the long-term stability development of the milk beer industry.Therefore,in recent years,research on milk beer beverages has gradually transferred to the screening of high-quality dairy yeast strains and the preparation of active dry yeast powders with the intention of directly producing milk beer with milk from lactic acid bacteria.In this study,the screening and identification of yeast strains were carried out at first,which provided high-quality strains for the preparation of active bacteria powder.The high activity freeze-dried yeast powder was prepared,which provided the basis for industrial application of high-quality milk beer milk yeast active dry powder.The main scientific research achievements of this research are as follows:?1?High-quality milk beer fermentation strains were screened from wild Kefir grains.After a lot of screening and identification,a strain of high-quality strain?Kluyveromyces marxianus BJ1?suitable for milk beer fermentation was obtained,which was initially put into pilot-scale production of milk beer.In the experiment,a total of 79 strains were isolated from wild kefir grains.from different regions of China.According to morphological analysis,microscopic observation and molecular biological identification,15 strains were identified as yeast strains.There were 4 strains of Kluyveromyces marxianus,5 strains of Saccharomyces cerevisiae and 6 strains of Pichia.Using the commercial strain,Kluyveromyces marxianus TRJ1 as control,15 yeast strains were analyzed for milk beer fermentation samples,and the quality of milk beer fermented by strain BJ1 was found to be the best by physical,chemical test,genetic stability test,sensory evaluation and volatile components test.The biological phylogenetic tree of strain BJ1 was constructed and the strain BJ1 was further determined to be Kluyveromyces marxianus.?2?A high concentration of proliferation culture of Kluyveromyces marxianus strain BJ1 was obtained for the preparation of the freeze-dried powder by the optimization of the proliferation medium formula and the culture conditions.The medium of strain BJ1 was optimized by single factor and response surface analysis.The best proliferation medium was found to be:wort medium as the base mediumwith 2.94%molasses,1.56%bran,0.21%CaCO₃.The maximum concentration of the viable strain BJ1 was 6.8×10⁸ CFU/mL.Based on this,the culture conditions were optimized by orthogonal test.The optimal culture conditions of strain BJ1 were 28°C,220 rpm,and the inoculum concentration was 1.5×10⁶CFU/mL.The maximum concentration of viable strain BJ1 could reach 7.8×10⁸CFU/mL,which was increased 4.11 folds as compared with in the YPD medium.?3?The formulation of freeze-drying protectant and the storage condition of high-activity freeze-dried powder for Kluyveromyces marxianus strain BJ1 were optimized to provide a reliable basis for the preparation of lyophilized powder.The optimal preparation conditions of the highly active strain BJ1 yeast lyophilized powder were:?1?6000 rpm,15 min,the collection rate of yeast slime can reach 86.09±1.28%.?2?Through the single factor and response surface analysis,the optimal lyoprotectant formulation was determined to be 11.27%sucrose,4.88%mannitol,15.43%maltodextrin,7.82%skim milk powder,and the optimal mixing ratio of slime and protectant was 1:1.5,and the optimum equilibrium time was 30min,the freeze-drying survival rate can reach 90.21±1.04%,reaching the domestic advanced level;?3?After storing under the condition of vacuum at-20°C for 3months,the survival rate of strain BJ1 lyophilized powder was 72.08±1.31%.?4?The conditions for fermentation of Kluyveromyces marxianus strain BJ1 were optimized while freeze-dried bacteria powder is used as a starter to produce milk beer.Through the single factor and orthogonal test design,the optimum process conditions for milk beer fermentation were as follows:time of 18 h,temperature of28°C,inoculum concentration of strain BJ1 1.5×10⁶CFU/mL.Under this condition,the sensory score ofthe strain BJ1 freeze-dried powder fermented milk beer sample is up to 92±1.9 points;the alcohol content is about 0.45%,which meets the requirements of the state for non-alcoholic beverages;the volatile component analysis of the sample shows that the relative content of alcohol,ester and other flavor substances in aroma components of milk beer are significantly increased,and the flavor and quality of milk beer are significantly improved compared with previous.Through this study,a high-quality milk beer fermented milk source Kluyveromyces marxianus BJ1 strain was obtained,and the high-concentration culture of the strain and the preparation of the freeze-dried powder were completed.The freeze-drying survival rate was as high as 90.21±1.04%,reaching the domestic advanced level.The strain BJ1 freeze-dried powder has stable performance in the production of milk beer at laboratory.The milk beer product has good taste and flavor with stable quality,which meets the requirements of the next industrialized pilot production.