Biodiversity Of Traditional Koumiss Microbiota And Its Characteristic Index Of Fermentation

Koumiss,a traditional dairy product with special regional character,has a long history and rich nutritional and health value.Koumiss is mainly popular with nomadic ethnic groups such as Mongolian and Kazakh in Xinjiang and Inner Mongolia,which is produced in different ways due to the difference of environmental climate,regional customs and taste preference.In addition,Koumiss is produced by natural fermentation at present.As a result,it’s composition of microorganism is very complicated.However,there is a lack of systematic research of the microbial composition of and it’s phase structure of Koumiss,the effect of temperature on the stage of fermentation and after-ripening,and the correlation between microbial and physicochemical index and flavor substances in the fermentation process.The Koumiss samples in different regions were collected and analyzed the composition of the microorganisms and flavor substances;analyzed the changes in the composition of microbes and flavor substance during the different stages of fermentation of Koumiss;analyzed the effects of temperature on dominant flora and physicochemical index of quality during the fermentation and after-ripening stage of Koumiss;analyzed the correlation between microbial and physicochemical index of quality and flavor substances in the fermentation process.The aim was to provide theoretical basis and data support for research of microbial resources development,the mechanism of microbial metabolism and quality control of Koumiss.The main results of the study are as follows:1.The strains of lactic acid bacteria,yeast and acetic acid bacteria were isolated and purified with the traditional isolation culture method and were identified by basic morphology,microscopic observation,physiological and biochemical tests and nucleotide sequence analysis.The lactic acid bacteria were identified as L.paracasei,L.casei,L.plantarum,L.fermentum,L.helveticus,one strain of acetic acid bacteria was identified as Acetobacter pasteurianu,The yeast were identified as Kazachstania unispora,Pichia fermentans,Kazachstania servazzii,Candida glabrata.The high-throughput sequencing(HTS)analysis of composition of the microorganisms of Koumiss in different regions found that the correlation between the phase structure of microorganism and the geographical distribution was relatively close,and each region has a unique micro-ecological environment.In terms of bacteria,there were 6 bacterial genus with an average relative content of more than 1%.Lactic acid bacteria,especially the genera Lactobacillus,was widely distributed in Koumiss,which was one of the dominant bacteria of Koumiss;The genera Acetobacter was the most advantageous non-lactic bacteria in Koumiss.In terms of fungi,there were 5 yeast genus with an average relative content of more than 1%.The genera Kazachstania was one of the dominant flora in the fungi phase of Koumiss,and the genera Kluyveromyces and Clavispora were the main flora of the Koumiss.In addition,a large number of unculturable or unclassified bacteria and yeasts were found in Koumiss,which also played an important role during fermentation process.2.Four growth models were used to evaluate the growth status of microorganisms of Koumiss in different regions,the sample MC2 was determined as the starter for the subsequent fermentation and Gompertz model,Logistic model and Baranyi model were determined as the primary models.The total number of colonies and the number of lactic acid bacteria,yeast and acetic acid bacteria under the 4,22,28 and 37℃were measured,then the three primary models were used to construct the growth model and the Belehradek model to determine kinetic parameters of the model.The results showed that the Baranyi model was selected as the primary model,and statistical indices of R2 and RMSE of Belehradek model were 0.9941,0.9910,0.9949,0.9999 and 0.0538,0.0254,0.0099,0.0009,respectively.Bf and Af of internal and external verification were both less than 1.15,which illustrated the applicability and reliability of model established.Study on effect of after-ripening stage at different temperatures on the Physicochemical Indexes of Koumiss,the results indicated that the effect of after-ripening temperature on the microbial abundance,acidity,butanedione,lactose,vitamin C,lactic acid,acetic acid and ethanol,and the quantity of acids,alcohols and esters were significant(P<0.05),however,the effect on protein content was not significant(P>0.05).The correlation between microbial and physicochemical index was analyzed by partial least squares regression model(PLSR).The result indicated that the correlation between lactic acid bacteria and acidity,butanedione,lactose,lactic acid,acid flavor substances was higher than that of yeast and acetic acid bacteria,and The changes of protein,ethanol,acetic acid,alcohols and esters were more closely related to acetic acid bacteria and yeasts.However,the relationship between vitamin C and lactic acid bacteria,yeast and acetic acid bacteria was not close.3.The high-throughput sequencing analysis of bacteria and fungi in the fermentation process of Koumiss found that the abundance of bacteria in the early fermentation stage was higher than in the late fermentation stage,and reached the maximum value at 96 h.The main flora was the genera Lactobacillus,Lactococcus,Acetobacter,Streptococcus during fermentation process.In the terms of fungi,the genera Kazachstania was the dominant flora in the fungi phase of Koumiss,which relative abundance was up to 61.08%;the genera Kluyveromyces,Pichia and Trichosporonaceae were the main flora.In addition,a large number of unculturable or unclassified bacteria and yeasts were found in Koumiss,which also played an important role during fermentation process.The detection of flavoring substances by SPME-GC-MS in Koumiss in different regions illustrated that ethanol,3-methyl-l-butanol,phenylethyl alcohol,2-nonanone,ethyl acetate,octanoic acid ethyl ester,decanoic acid ethyl ester,hexanoic acid,acetic acid,octanoic acid,n-decanoic acid,9-decenoic acid,dodecanoic acid were widely distributed compounds.The proportion of acids and esters was higher than other flavoring substances.The acids,alcohols,esters,aldehydes,ketones and other compounds were produced in large amounts while alkanes decreased sharply or even disappeared during the fermentation process,and up to 71 compounds were detected at 96 h of fermentation.It was found that only 27 compounds at 168 h,and alkanes,olefins and ketones disappeared in large amounts.To explore the correlation between microbial and flavor substance,the partial least squares regression model(PLSR)was used to evaluate the association between microbial and volatile compunds in different fermentation stages.According to the results,the alcohols,an important flavor substance of Koumiss,were related to the genera Kazachstania,Pichia and Kluyveromyces;ethyl acetate was related to the genera Lactococcus,Acetobacter and Saccharomyces;octanoic acid was related to the genera Lactobacillus and Streptococcus.In addition,the relationship between some microbes and compounds was not close.