Biodiversity Of Chinese Traditional Stinky Tofu Microbiota And Its Biogenic Amines Metabolism

Stinky tofu,a traditional Chinese snack,has a long history and rich nutritional value.Stinky tofu has its origins in all parts of China.Due to the differences in climate,customs and tastes,stinky tofu is produced in different ways.In addition,stinky tofu has been naturally fermented.As a result,its composition is very complicated.Besides,the accumulation of harmful substances such as biogenic amines is not well controlled.However,there is a lack of systematic research of the microbial composition of the stinky tofu and its structure of the colony,flavor substances and metabolism of biogenic amines in the fermentation process.The stinky tofu in different regions was collected and analyzed the composition of the microorganisms,the composition of the flavor substances,the type and content of biogenic amines;analyzed the changes in the composition of microbes,of flavoring substances and of biogenic amines during the different stages of fermentation of stinky brine.In addition,for the isolated strains,the metabolism of biogenic amines,especially putrescine,was analyzed under different conditions.In combination with RNA sequencing,the gene expression of strains under different conditions was analyzed as also.The microbial composition of stinky tofu was both uniformed and unique and has no obvious correlation with the geographical distribution.Each stinky tofu was a unique micro-ecological environment.Different lactic acid bacteria were widely and abundantly present in stinky tofu.Lactobacillus,Lactococcus and Streptococcus may be the dominant lactic acid bacteria in stinky tofu,and Lactococcus lactis was the most widely distributed dominant bacteria in stinky tofu.Prevotella was the most important non-lactic bacteria in stinky tofu.In terms of fungi,26 species fungi were isolated by culture-dependt method and 17 of them were yeast.Among them,Yarrowia lipolytica was the main fungal species and also was the dominant yeast in stinky tofu.The Irpex lacteus and Phlebia subserialis were isolated from stinky tofu and brine samples for the first time.The high-throughput sequencing(HTS)analysis of the bacteria in the process of stinky tofu fermentation found that the bacterial content was highest in the initial stage of fermentation,and the population and diversity of the microorganisms in the early fermentation stage was higher than that in the late fermentation stage.The main flora was the genera Prevotella,Lactobacillus,Halomonas and Christensenella during the fermentation process.Traditional culture depended methods were used to complement the changes in microorganisms at the species level.As a result,it was found that lactic acid bacteria were distributed in various stages of fermentation,and among which Lactobacillusparacasei was predominates.Lactobacillus mixtipabuli and Leuconostoc mesenteroides appeared in the early stage of fermentation,while Lactobacillus plantarum and Lactobacillus coryniformis appeared in the later stage of fermentation.The Cutaneotrichosporon curvatus and Mucor circinelloides appeared in the early stage during fermentation,but Geotrichum candidum and Pichia fermentans were more easily isolated in the middle and late stages of fermentation.The detection of flavoring substances in stinky tofu and brine in different regions revealed that 1-hexanol,1-heptyn-3-ol,2-octenal,hexanal,furfural,valeric acid,butyric acid,phenol,guanidine and D-limonene butyl were widely distributed compounds.In addition,the main compounds in the brine included phenethyl alcohol,4-nonenol,3 hydroxy dodecanoic acid,ethyl butyrate,cyclohexene,furanone and acetophenone.It was found that the flavoring substances in the stinky tofu mainly come from the brine fermentation.The formation of acids and esters of the flavor substances in the brine was mainly concentrated in the later stage.The soaking process was an important part of the formation of stinky tofu flavor.The stinky tofu was increased flavor substance including the butyric acid,valeric acid,phenol,hydrazine,thiol,isopropyl ester,sulfide and other flavor compounds by soaking.It was found that biogenic amines were widely exitance in stinky tofu,but the distribution of biogenic amines has no obvious regional characteristics.As a whole,the main biogenic amines in the stinky tofu(distribution and content)are tyramine,putrescine,and cadaverine.Different biogenic amines have different trends during the fermentation process,but generally reach the first peak on the 15th day.The would fluctuate and rise.When the brine fermentation was over,the content of putrescine reached the maximum while spermine disappeared.After soaking,the biogenic amines in the br:ine would rise to varying degrees,while the tofu does not change much.To explore the correlation between bacteria and flavor substances/biogenic amines,a cross-validated PLS regression model was used to assess the association between bacterial taxa(explaining variables)and volatile compounds or biogenic amines(response variables).As a result,it was found that 7 methylene-9-oxabicyclo[6.1.0]non-2-ene,tans-2-dodecenal,trans-2-octenal,hexanal,furfural,2,6,11-trimethyl-dodecane were not relevant to bacteria,and these volatile substances may be due to chemical reactions or from the packaging and the raw material itself.The more important flavor substances in stinky tofu were related to Porabacteroides,RC9 gut group,and Mogibacterium,and 3-methylphenol was mainly related to the Kurthia.Phenethyl alcohol may be related to Rhodococcus and Bavariicoccus.The relationship between some compounds and bacteria was not relatively tight,and these substances may be generated by chemical reactions or come from the packaging and raw materials themselves.Analysis of biogenic amines revealed that the formation of amines was highly dependent on the bacterial strain and not the genera or species of microorganism.The production of biogenic amines of Lactobacillus oryzae and Meyerozyma guilliermondi,were test at different condition.It was found that Lactose and glucose inhibited the putrescine activity of Lactobacillus oryzae,but the inhibition of Meyerozyma guilliermondii was not significant.For Meyerozyma guilliermondii,different types of carbon sources had little effect on the synthesis of cadaverine and histamine,while different proportions of carbon sources affected the synthesis of cadaverine and histamine,and there was also a positive correlation between cadaverine and histamine.Metabolic pathways of different biogenic amines may interact with each other.The intermediates and key genes involved in the synthesis of putrescine of Lactobacillus oryzae and Meyerozyma guilliermondii were researched and it showed that neither of the two strains did not synthesize putrescine via the ADI pathway and the ODC pathway.The putrescine synthetic pathway of Lactobacillus oryzae was mainly the AGM pathway,and the main pathway for the production of putrescine in Meyerozyma guilliermondii was the AgDI pathway.