Study On Changes In Microbial Communities And Quality During The Fermentation Process Of Bulang Pickled Tea

Bulang pickled tea is a tea product made from the leaves of Camellia sinensis(Linn.)var.assamica(Mast.)Kitamura under spontaneous fermentation,which is a traditional fermented food in Yunnan.The anaerobic fermentation is a key process in the processing of Bulang pickled tea and plays an important role in its quality formation.However,little is known with quality development principles during Bulang pickled tea fermentation process.In this study,based on high-throughput sequencing technology and metabonomics,the changes in microorganisms,non-volatile metabolites and volatile metabolites during threemonth fermentation of Bulang pickled tea were determined.The main physical and chemical indicators during fermentationwere also evaluated.Furthermore,the correlation between microbial communities,metabolites,and physicochemical indicators were analyzed during the fermentation process of Bulang pickled tea.The results would provide a theoretical basis for elucidating the mechanism underlying the quality development of Bulang pickled tea.The main findings are as follows:1.Dynamic changes in microbiome during fermentation of Bulang pickled tea were determined.Based on data with the culture-dependent method,during 0-2 month fermentation period,there was a significant increase in lactic acid bacteria,bacteria,and fungi.Afterwards,the numbers of lactic acid bacteria and fungi tended to stabilize,while bacteria significantly decreased.The results of 16 S rRNA and ITS high-throughput sequencing revealed that the dominant bacterial phyla during fermentation were Firmicutes and Proteobacteria,and the dominant bacterial genera were Lactobacillus and unclassified Enterobacteriaceae.At the species level,Bulang pickled tea were absolutely dominated by Lactobacillus plantarum and unclassified Enterobacteriaceae.The dominant fungal phyla were Ascomycota,Basidiomycota and an unclassified Fungi phyla.There were 30 fungal genera with a relative abundance of more than 1.00%.The top five dominant fungal genera were Cladosporium,Debaryomyces,Candida,Aspergillus and Wallemia,while 39 fungal species exhibited relative abundance with more than 1.00%.The top five dominant fungal species were unclassified Debaryomyces,Candida metapsilosis,Cladosporium sphaerospermum,unclassified Aspergillus and an unclassified Fungi.2.Quantitative analysis and dynamic changes of main physicochemical indicators during the fermentation process of Bulang pickled were studied.During fermentation,pH fluctuated slightly.Soluble protein and total titratable acidity reached the maximum value after 1 and 2 months of fermentation,respectively and then significantly decreased.The content of total soluble sugar changed in waves and reached the maximum value after 2months of fermentation.The level of total free amino acids continued to increase during fermentation,and reached the maximum value after 3 months of fermentation.Both total polyphenols and total flavonoids increased in early fermentation and then decreased.with fermentation,the content of butyric acid showed a trend of first increasing,then decreasing,and finally increasing,while acetic acid,propionic acid,isobutyric acid,isovaleric acid,valeric acid,and caproic acid showed a continuous increase trend.After 3months of fermentation,the content of all seven short chain fatty acids increased to their maximum value,with acetic acid content being the highest,followed by butyric acid,propionic acid,caproic acid,isobutyric acid,valeric acid,and isovaleric acid,respectively.4.Dynamic changes in non-volatile metabolites in Bulang pickled tea during fermentation were determined.Using ultra performance Liquid Chromatography Coupled with Quadrupole/Time-of-Flight Mass Spectrometry(UPLC-QTOF-MS)technology,a combination of fold change(FC),Student’s t-test,and OPLS-DA analysis was used to screen to obtain a total of 632 non-volatile differential metabolites(FC>2 or FC<0.5,P<0.05,and VIP>1).Four hundred and eighty five differential metabolites were screened between 0 and1 month of fermentation and 386 compounds were upregulated and 99 compounds were down regulated.When comparing 2 with 1 month fermentation,185 differential metabolites were identified,and 132 were upregulated and 53 were down regulated.Furthermore,132 differential metabolites between 2 and 3 months were identified,and 81 were upregulated and 51 were down regulated.Eight key differential metabolites were identified using analyzing the differential flavonoids,phenolic acids,tannins,alkaloids,fatty acids,amino acids and their metabolites,organic acids and their metabolites,carbohydrates and their metabolites,alcohols,aldehydes,ketones,esters,and terpenoids,and eight key differential metabolites were found,namely melibiose,Ser-Tyr-Asp-Glu,Phe4Cl-Asp-OH,Dglucopyranoside,naringenin-7-O-glucoside,D-glyceric acid,(S)-2-hydroxyglutaric acid and salicin-6-phosphate.5.Dynamic changes in volatile metabolites during fermentation of Bulang pickled tea were studied.Three hundred and ninety eight differential volatile compounds(FC>2 or FC<0.5,P<0.05,and VIP>1)were detected during fermentation of Bulang pickled tea using Headspace Solid-Phase Microextraction followed by Gas Chromatography-Mass Spectrometry(HS-SPME-GC-MS).The key aroma components of Bulang pickled tea were further explored through calculating odor activity values(OAV).A total of 82 aroma components had OAV>1 during fermentation primarily contributed to the flavor of Bulang pickled tea.Flavor compounds including 3-ethyl-phenol,guaiacol,1-p-menthene-8-thiol,(Z)-4-heptenal,1-hepten-3-one,2-methoxy-3-(1-methylpropyl)-pyrazine,1-(2-thienyl)-ethanone and 2,3-diethyl-5-methyl-pyrazine had the OAV values greater than 100,and the concentration increased with fermentation.With prolonged fermentation,the aroma components in Bulang pickled tea became more diverse,and OAV value showed a continuous upward trend.The unfermented tea mainly presents nutty,fatty,musty,green,woody and metallic flavor.The fruity,floral,roasted,coffee and earthy flavor of Bulang pickled tea increase after 1 month of fermentation.The sweet and phenolic flavor increased after 2 months of fermentation.The aroma is most abundant after 3 months of fermentation,and the spicy,vegetable,candy,smoky and wine flavor increased.6.Correlation analysis between microorganisms and physicochemical indicators,shortchain fatty acids,non-volatile and volatile metabolites during the fermentation process of Bulang pickled tea was performed.Spearman correlation analysis revealed that the changes in total titratable acidity,total free amino acids,total phenols and flavonoids content were positively correlated to Lactobacillus plantarum,Lactobacillus brevis,unclassified Wallemia,unclassified Xylariales,Cladosporium sphaerospermum and Apiotrichum montevideense.And,the changes in short-chain fatty acid content were related to the increasing relative abundance of Lactobacillus plantarum,Cladosporium sphaerospermum,Apiotrichum montevideense.Lactobacillus plantarum,Lactobacillus brevis,unclassified Xylariales,Cladosporium sphaerospermum,Apiotrichum montevideense and an unclassified Fungi were positively related with non-volatile metabolites such as Phe4Cl-Asp-OH,salicin-6-phosphate and naringenin-7-O-glucoside were positively,while negatively correlated with Ser-Tyr-Asp-Glu and melibiose,82 key aroma components are generally negatively correlated with dominant bacteria and positively correlated with dominant fungi.Taken together,microbes,physiochemical parameters and metabolites significantly altered during fermentation of Bulang pickled tea.Microorganisms including Lactobacillus plantarum,Lactobacillus brevis,Cladosporium sphaerospermum were correlated with changes in physicochemical indicators,short-chain fatty acid content,non-volatile metabolites,and key aroma compounds during fermentation of Bulang pickled tea.