Effects Of Dominant Thermoduric Microorganisms On Pile-fermentation Processing Of Qingzhuan Brick Tea

Qingzhuan brick tea(QZBT)is a kind of pressed dark tea,which has significant hypoglycemic and weight-reducing functions and is an indispensable necessity for ethnic minorities in Chinese border areas.Pile fermentation is the key process for the formation of unique features of dark tea.Its essence is a solid-state fermentation process in which microorganisms and their extracellular enzymes are involved.At present,the fermentation procedure of QZBT is still be experience-controlled,leading to unstable product quality and large differences in different batches.In this study,combined with traditional microbial isolation methods and modern high-throughput sequencing technologies,the composition and dynamics of microbial community during the pile fermentation of QZBT were studied.Some microbial strains with important contribution to the formation of QZBT's characteristics were selected according to their population number on the tea leaves,and their ability to grow under high temperature and produce some crucial extracellular enzymes.The effect of specific stains on quality formation of QZBT was further studied by comparing the difference of the enzyme activity and quality component content of tea leaves fermented by single strain or mixed strains in artificial pile fermentation.The main results are as follows.1.Determining the temperature of tea leaves files during pile fermentation of QZBT indicated that the temperatures were always above 45? and fluctuated apparently throughout the process.The core temperature was higher by 3? than the average temperature.The operation of stirring the files could reduce the temperatures,but temperature would rise again quickly.The change of microbial population during the pile fermentation process determined by traditional microbial accounting method showed that bacterial strains were predominant in population quantity,followed by moulds,then by actinomycetes and yeasts.2.High-throughput sequencing of two tea samples which were evaluated as possessing the highest number of microbial population(day 9 and day 19 in file fermentation)were carried out.Results indicated that he procaroyotic community was diverse,there were 14 kinds of identified bacterial genera in the day 9 tea sample,and that was 13 kinds for the day 19 tea sample.Furthermore,there was obvious difference in species and proportion between different stage of file fermentation;Thermoduric Bacillus(8.00%)and Alicyclobacillus(1.58%)were detected in the day 19 tea sample.The sum proportion of them was just below that of Enterobacter(9.82%)which was in the third place in the rankings of prokaryotic population number.The fungal community was relatively simple and mainly contained three genera including Aspergillus and Saccharomyces,among which Aspergillus was definitely dominant.3.Using traditional microbial separation technology,thermoduric dominant microbes were selected under 45?.Then classical morphological,physiological and biochemical measurements and molecular biology methods were used to identify these stains.Finally,five bacterial strains(X1: Bacillus licheniformis,X2: Alicyclobacillus sp.,X3: Bacillus coagulans,X4: Bacillus subtilis subsp,X5: Pseudomonas),one actinomycetic strain(F1:Streptomyces thermoviolaceus),one yeast strain(J1: Cyberlindnera sp)and two mold strains(M1: Aspergillus fumigatus,M2: Aspergillus niger)were obtained.4.Three procaryotic strains(strain X1,X4 and F1)with high yields of cellulase,pectase and protease and one eucaryotic strain(strain M1)with high yield of polyphenol oxidase were further selected to be inoculated into sun-dried old green tea leaves in artificial file fermentation to explore their function on quality formation of QZBT.Enzymatic activity tests showed that the amount of cellulase,pectase and polyphenol oxidase in tea samples fermented by single strains were obviously lower than that fermented by mixed strains,but the latter fermentation pattern was unfavorable for prokaryotic strains to produce protease.Compared with control(non-inoculated tea sample),there was small change in the content of each quality component of tea samples inoculated with single prokaryotic strain,suggesting that single bacterial strain X1 and X4,and actinomycetic strain F1 has less influence on the biotransformation of sun-dried old green tea leaves.Nevertheless,strain M1 producing higher level of polyphenol oxidase could not only reduce the amount of tea polyphenols and catechins,increase the level of theabrownin,but have more influence on the amount of amino acids and soluable sugar than other stains(strain X1,X4 and F1),which implied that strain A.fumigatus M1 played an important role in the quality formation of QZBT.Two strain-mixed-fermentation was more beneficial than single-strain-fermentation in regard to the biotransformation of sun-dried old green tea leaves.Strain Bacillus subtilis X4 could enhance the ability of strain M1 to transform the sun-dried old green tea leaves.Whereas the efficacy of three strain-fermentation(strain X4,F1 and M1)was generally slightly lower than that of two strain-fermentation mode.5.After inoculating strain J1 into liquid medium containing extracts of sun-dried green tea leaves and cultivating at 45°C for 24 h,a total of 28 new substances were produced,including 10 alcohols,9 esters,4 ketones,2 terpenes,2 phenols and 1 aldehydes.Among them,according to previous studies,citral and cis-geranylacetone were the key aroma components of QZBT.6.Although coliform was present in the tested commercial products of QZBT,they were all below the threshold value of national standard(30 MPN/100 g);No Staphylococcus aureus and Salmonella were detected in the samples.The content of three common fungal toxins including aflatoxins,fumonisins and ochratoxins were lower than the detection limit(AFL :1?g/kg,Fumonisins B1 and B2:0.1 mg/kg,OTA:1 ?g/kg).These results provided data support for the safety of drinking QZBT.