| As a natural fermented agent for traditional fermented milk kefir in Nyingchi Prefettura,Tibet,Tibetan kefir mainly includes lactic acid bacteria and yeasts as the main strains and also contains acetic acid bacteria,which form lactic acid,low abundance of ethanol,carbon dioxide and acetic acid that offer the fermented milk a mild aroma and foaming properties.Due to the complex microbial structure and abundance,Tibetan kefir used as a commercial starter directly results in unstable fermentation quality,which prevents the large-scale popularization of Tibetan kefir yogurt.As a natural fermentation agent with Chinese characteristics,Tibetan kefir is rich in precious strain resources,however,it hasn't been clarified that the effect of different strains on fermentation quality of Tibetan kefir yogurt.Based on modern molecular biology technology to analyze its diversity and fermentation performance,reconstruction of microorganisms composition in Tibetan kefir to analyze the relationship between strains and flavor and quality of Tibetan kefir is of great significance for the development of new starter culture to fermented milk with Chinese independent intellectual property rights.In this study,8 Tibetan kefir grains collected in Tibet were used as the research object to connected the microbial diversity with fermented quantity indices(pH value,titratable acidity,total number of colonies,syneresis,water-holding capacity and volatile flavor compounds)in fermented milk.In addition,the type of freeze-dried starter culture and combination of predominant strains were simulated to explore the correlation and contribution of fermented starter culture and dominant fermented strains to fermented milk quantity of Tibetan kefir.The main conclusions of this study are as follows:(1)The results of microbial diversity showed that Lactococcus sp.,Lactobacillus sp.,Acetobacter sp.,and Leuconostoc sp.are the high abundant bacterial genus in Tibetan kefir fermented milk.Kazachstania unispora was the only species of fungus.The less abundant Acinetobacter sp.,Rhodococcus sp.,and Acidovorax sp.were detected in TK3.The abundance of microbial communities was significantly different among the samples,among which TK6 had the highest bacterial abundance and TK3 had the most species.Through correlation analysis and cluster analysis of physicochemical indices of 8 cups of Tibetan kefir fermented milk with main bacterial communities at the genus level,we found Leuconostoc sp.has a positive effect on pH value,and both can promote the increase of the total number of bacterial colonies with Lactobacillus sp.,however,Lactobacillus sp.have a negative effect on bacterial colonies number.It's predicted that and Acetobacter sp.has a significant negative correlation with the total number of yeast colonies number and diacetyl content in yogurt,while Lactobacillus sp.is the main contributor to diacetyl.In addition,Acetobacter sp.is main contributor to the content of 2-heptanone and acetic acid in fermented milk,on which Leuconostoc sp.shows an significantly opposite effect.(2)There is a significant difference in fermented milk quality between freeze-dried starter(FD)and Tibetan kefir(TK).FD is more stable and stable than TK in total number of colonies,and the yogurt fermented by FD can control the number of microorganisms in each batch to a certain extent.The overall acidity of FD fermentation is higher than that of TK,and the variation in acid production between batches is small,but the water-holding capacity of FD is poor.Analysis of the key aroma components of yogurt,ethanol and isopentanoll contents produced by FD decreased significantly compared with TK fermentation,while diacetyl and acetic acid content increased significantly.It was found that most of TK and FD had significant differences in fermented flavors through sensory evaluation of Triangle Test.Yogurt directed vat set of FD that made from Tibetan kefir fermented milk has significantly different on flavor and quality from TK fermentation.Through the Preference Ranking Test and the Paired Comparison Test,there was no significant difference in flavor quality among 8 samples of yogurt fermented by Tibetan kefir that cannot be directly judged by sensory evaluation.Based on the highest preference score,the sample TK3 was selected to further explore the relationship between flavor quality and the dominant fermented strain of Tibetan kefir fermented milk.(3)266 isolated strains were identified from 8 Tibetan kefir fermented milks,including 72 strains of Lactobacillus kefir,70 strains of Kazachstania unisporus,41 strains of Enterococcus durans,39 strains of Leuconostoc pseudomesenteroides,25 strains Acetobacter cibinongensis,5 strains of Acetobacter fabarum,3 strains of Lactococcus lactis subsp.lactis,3 strains of Leuconostoc mesenteroides,3 strains of Enterococcus hirae,2 strains of Lactobacillus delbrueckii subsp.bulgaricus,2 strains of Acetobacter orientalis and 1 strain of Leuconostoc citreum.There are 23 strains isolated from TK3 and used in food fermentation,from which 5 strains of Lactobacillus kefir with excellent acid production capacity,1 strain of Acetobacter cibinongensis,2 strains of Acetobacter fabarum,1 strain of Leuconostoc mesenteroides,1 strain of Lactococcus lactis subsp.lactis and 4 strains of Kazachstania unisporus were screened for recombination test.The contribution of different strains to the quality of fermented milk was evaluated by the absence of dominant strains and recombination.As a result,Acetobacter fabarum and Acetobacter cibinongensis significantly contribute to titratable acidity,total microbial colonies,and have a dominated effect on 2-heptanone and acetic acid content.The water-holding capacity of yogurt is significantly improved due to Acetobacter cibinongensis,and thus the syneresis can be inhibited.Leuconostoc mesenteroides and Lactococcus lactis can affect the increase in pH value,which is not conducive to the formation of an acidic environment.Both can contribute to the improvement of total microbial colonies,diacetyl and 2-heptanone contents of yogurt.However,Lactococcus lactis is not conducive to the water-holding capacity of yogurt,increases the degree of syneresis of yogurt,and reduces the content of acetic acid.As one of the dominant species in the Tibetan kefir,Lactobacillus kefir plays a decisive role in the colony forming unit of bacteria and the content of diacetyl and 2-heptanone,and it also affect the colony forming unit of yeasts partly.A low-acid environment during fermentation is also not conducive to form due to Kazachstania unisporus,which reduce the water-holding capacity of yogurt,affect the number of bacterial colonies,and determine the abundance of ethanol,isopentanol,diacetyl and acetic acid. |
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