Analyses Of Differences In Microbial Community Structure Between Tibetan Kefir Grains And Their Fermented Milks With Analyses Of Key Flavor Compounds In Tibetan Kefir Fermented Milks

The exploration and utilization of natural microbial resources in nature have produced important social and economic benefits.It is a research hotspot in the field of modern biology.Original Tibetan kefir grains are precious natural dairy microbial resources originated from Tibet region in China.Traditional Tibetan kefir is functional fermented milk with feature flavor prepared by inoculated with original Tibetan kefir grains.Not all varieties of original Tibetan kefir grain have good industrial application potential to prepare high-quality kefir.Therefore,the screening of original Tibetan kefir grain is a necessary basic work for research and development of Tibetan kefir mix milk dairy product.This work is of great significance for excavation,protection and utilization of precious microbial resource of original Tibetan kefir grain superior varieties originated from Tibet region in China.In this thesis,five varieties of original Tibetan kefir grain(TKG1-5)collected from different orientation regions of Nagqu prefecture in northern Tibet and their fermented traditional Tibetan kefir milks(TK1-5)were taken as the research objects.On the one hand,high-throughput sequencing technology was used to analyze and compare the microbial community structures of each TKG and its fermented TK systematically,aiming at to clarify the difference in microbial community structure between Nagqu prefecture original Tibetan kefir grains(TKG1-5)and their fermented traditional Tibetan kefir milks(TK1-5);On the other hand,electronic nose technology and HS-SPME-GC-MS combined with principal component analysis(PCA)and odor activity values(OAVs)were used to compare the differences in flavor profile and volatile flavor compound composition among each TK,to investigate the relationship in differences of flavor compounds and genera among traditional Tibetan kefir milks(TK1-5),to characterize the key flavor compounds in each TK,and to discuss the flavor differences between traditional Tibetan kefir milks(TK1-5)and yogurt,aiming at to clarify the flavor characteristics and feature location of Nagqu traditional Tibetan kefir milks(TK1-5).Finally,based on the results of above two aspects,the research on both screening of original Tibetan kefir grain superior variety and preparation of Tibetan kefir mix milk were carried out.High-throughput sequencing technology combined with multifarious bioinformatics analysis methods were applied for systematic analyses and comparisons in the bacterial and fungal community structure of each original Tibetan kefir grain and its fermented traditional Tibetan kefir milk at three high taxonomic levels(family,genus and species).The foundings were as follows:(1)bacteria in original Tibetan kefir grains(TKG1-5)and their fermented traditional Tibetan kefir milks(TK1-5)were classified and annotated as five major families(Lactobacillaceae,Streptococcaceae,Leuconostocaceae,Acetobacteraceae and Enterobacteriaceae),five major genera(Lactobacillus,Lactococcus,Leuconostoc,Acetobacter and Enterobacter),eight major species(Lactobacillus kefiranofaciens,Lactococcus lactis,Leuconostoc mesenteroides,Lactobacillus kefiri,Lactobacillus helveticus,Acetobacter okinawensis,Acetobacter orientalis and Enterobacter aerogenes);(2)fungi in original Tibetan kefir grains(TKG1-5)and their fermented traditional Tibetan kefir milks(TK1-5)were classified and annotated as three major families(Saccharomycetaceae,Pichiaceae and Trichocomaceae),five major genera(Kazachstania,Kluyveromyces,Saccharomyces,Dekkera and Aspergillus),six major species(Kazachstania unispora,Kazachstania turicensis,Kluyveromyces marxianus,Saccharomyces cerevisiae,Dekkera anomala and Aspergillus amstelodami);(3)difference in bacterial community structure was the main difference in microbial community structure between the original Tibetan kefir grain group(TKG1-5)and traditional Tibetan kefir milk group(TK1-5).Lactobacillus kefiranofaciens(mean relative abundance of samples within grain group was 80.85%)was dominant bacteria in the original Tibetan kefir grain group(TKG1-5),whereas Lactococcus lactis(mean relative abundance of samples within milk group was 66.64%)and Leuconostoc mesenteroides(mean relative abundance of samples within milk group was 30.96%)were dominant bacteria in the traditional Tibetan kefir milk group(TK1-5);(4)there was no significant difference in fungal community structure between the original Tibetan kefir grain group(TKG1-5)and traditional Tibetan kefir milk group(TK1-5).The fungal community structures in each TKG and its corresponding TK were similar,respectively;(5)the microbial community structures of traditional Tibetan kefir milks(TK1-5)were different from each other: bacterial and fungal species richness in TK2 was highest,with the bacterial ace and chao indices were 18.31 and 18.00 respectively,and the fungal ace and chao indices were 30.44 and 30.00 respectively.Comparison in flavor profiles based on electronic nose technology indicated that the flavor profiles of traditional Tibetan kefir milks(TK1-5)were different from each other.Analysis in flavor compound composition based on HS-SPME-GC-MS showed that a total of 32 volatile flavor compounds were identified in the traditional Tibetan kefir milks(TK1-5),among which alcohols(relative content range from 45.27% to 60.39%)and acids(relative content range from 27.98% to 47.67%)were dominant in each TK.Analysis of flavor compound composition in each TK,investigation of relationship in differences of flavor compounds and genera among the traditional Tibetan kefir milks(TK1-5)based on flavor compound or genera composition combined with PCA indicated that flavor compound composition of each TK was different from each other.Higher dominant position of esters made the flavor compound composition of TK2 significantly different from the other four traditional Tibetan kefir milks,which was speculated to be related with the exclusive genus Saccharomyces in TK2.Characterization of key flavor compounds in each TK based on combination of flavor compound compositions and odor activity values(OAVs)indicated that ethyl octanoate,phenylethyl alcohol and hexanoic acid were the common key flavor compounds in traditional Tibetan kefir milks(TK1-5).Esters were the most important flavor contributors with highest aroma contribution in each TK,which made the highest contribution to overall aroma of TK2.Discussion on the flavor difference indicated that “bouquet/estery” was prominent differential flavor characteristic and feature of Nagqu traditional Tibetan kefir milks(TK1-5)different from yogurt.Original Tibetan kefir grain TKG2 was screened as the best original Tibetan kefir grain variety based on comprehensive comparisons of total fermentative activity of original Tibetan kefir grains(TKG1-5),differences in microbial community,flavor and sensory quality of traditional Tibetan kefir milks(TK1-5).Afterwards,by genotyping and flavor production performance screening,10 lactic acid bacteria strains and 8 yeast strains isolated from fermented kefir milk prepared by the screened best original Tibetan kefir grain variety TKG2 were determined for the preparation of Tibetan kefir mix milk.Finally,based on D-optimal mixture design,proportion of inoculated microbial strains was optimized.Tibetan kefir mix milk-TKM prepared by inoculating the mix microbial strains of optimal proportion(instead of original Tibetan kefir grain TKG2)could not only possess the prominent feature “bouquet/estery” flavor characteristics compared with yogurt,but also better than TK2 in terms of flavor quality and texture,realizing the aim that both free from inoculation dependence on original Tibetan kefir grains and maintaining the feature flavor of traditional Tibetan kefir milk-TK2.