Study On The Spatiotemporal Characteristics Of The Microbial Community And Metabolites In Pixian Doubanjiang

Pixian Doubanjiang(PXDBJ)is an essential condiment of Sichuan cuisine,which is one of the eight famous Chinese cuisines.The unique brewing technique of PXDBJ is that it refers to uncooked materials in douban-koji manufacturing and fermenting,compared with other types of traditional fermented sauces;thus,its technique has been registered in the national intangible cultural heritage(ICH)list.Meanwhile,the excellent coloring and aroma & flavoring producing function is not only closely related to the unique technique but also caused by the defined raw materials origin and manufacture area.Therefore,PXDBJ has been listed as one of a few protected geographical indication(PGI)products,and recognized by both China and European Union.In the last decade,it has been widely paid attention to and become one of the focuses in the traditional fermentation food industry that exploring the succession of microbial community and their metabolic regulation hidden behind the unique technique.However,it is being faced with an enormous challenge since the inheritance of the ICH technique is inconsistent with its promotion and development.There are indeed some difficulties in this work,especially the unique flavor components that are characterized quantitatively,and the typical markers in connection with the defined raw materials and geographical environment.In fact,the unique flavor forming is responsible for the interaction relationships between microecosystems and environmental stress,which included side-and isolation-effect of micro-ecosystems,as well as the tendency of directed evolution in the extreme environment.Meanwhile,the formation of flavor features is also affected by its spatiotemporal characteristics;however,those are unclear so far.In the present research,the integration platform technology was established by coupling omics technology,macroecology technology and multivariate statistics,to know the characteristics prescribed by PGI and ICH technique.Firstly,the characteristic flavor components of representative traditional PXDBJ were identified with the help of polyphase detection technology,and the correlations of dominant constituents with keystone species were explored during the ripening process.And then,the effects of the main factors on the unique flavor profile and microbial diversity were investigated,including varieties of raw materials,ripening periods and niches.Subsequently,the spatiotemporal characteristics of Banpei and its correlations with the dominant microbes were explored,as well as the micro-ecological environment.The PGI characteristics were revealed using the integration platform technology on multiple scales;in particular,the indicators were identified and a discriminating model was developed.Finally,the scientific attribute of the PGI and ICH technique for PXDBJ was analyzed by simulated fermentation,Doubanjiang sample was bioturbated and ripened in the different geographical environments.The revealed brewing mechanism of PXDBJ lays a theoretical foundation for the scientifically inheriting ICH technology,not just formal inheriting.Furthermore,it provides the necessary technical means and methodology for distinguishing the PGI product of PXDBJ.The main results are as follows:(1)Unique flavor components of the traditional PXDBJ were characterized,the correlations between key components and core microbes were revealed.The metabolites of representative PXDBJ were examined qualitatively and quantitatively by polyphase detection technologies,which included Amino acid analyser,HPLC,GC-MS/MS,GC×GC-TOF-MS,TDU-GC-MS,Illumina Mi Seq sequencing and conventional analysis methods.There were seventeen kinds of dominant metabolites among these representative samples,which characterized the flavor feature of PXDBJ.Among them,citric acid,malic acid,Asp and Glu were the key taste constituents.In addition,the other thirteen kinds of volatiles represent aroma features,which was 4-ethylguaiacol,4-ethylphenol,phenyl ethanol,phenylacetaldehyde,(EZ)-β-ionone,linalool,undecane-2-one,ethyl phenylacetate,dimethyl trisulfide,3-octanone,2-pentylfuran,2-acetylpyrrole and 1-octen-3-ol.In particular,most of these aroma components could be detected by HS-SPME-GCMS/MS.The differences of the flavor feature among enterprises and raw materials can be distinguished by partial least squares-discriminant analysis(PLS-DA,Q2(cum)= 0.988).Cupriavidus,Burkholderia-Caballelonia-Paraburkholderia and Saccharomycetales were the dominant microbes during the ripening process.The abundance of the five genera was closely correlated with the major unique flavor components,which were Bacillus,Lactobacillus,Staphylococcus,Pichia and Zygosaccharomyces.The result predicted by PICRUSt2 showed that the contents of various flavor components were closely correlated with the intensity of corresponding enzymes in the metabolic pathway of the community,and that 4-ethylguaiacol and 4-ethylphenol were the results of the synergistic action of biological metabolism and non-biological synthesis.(2)The main factors affecting the flavor characteristics and microbial community diversity of PXDBJ were analyzed.It was examined that the effects of various factors on the PXDBJ feature,which originated from the major enterprises located in Pixian county.These factors involved in raw materials varieties,ripening periods and niches.The results showed that the varieties of raw materials significantly affected the contents of organic acids,especially the proportion of citric acid and malic acid.Niches and ripening periods affected the amino acids contents,while the proportion of dominant components(Asp and Glu)was relatively stable.The mixing ratio of different raw materials varieties slightly affected the total contents of volatiles.In general,the differences in aroma volatiles caused by the raw materials varieties were revealed based on the PLS-DA model.In addition,the differential metabolites(also called variable importance in the projection,VIP > 1)could be used to analysis the influences of niches and ripening periods among identical variety Doubanjiang.Moreover,the discrepancies of taste and flavor profiles among different varieties Doubanjiang were explained by Orthogonal PLS-DA(OPLS-DA),the fitting degree and predictability of the matrix were 95.3% and 91.8%,respectively.The results obtained by the Spearman correlation coefficient demonstrated that the ripening periods significantly affected the total acid,the ratio of various raw materials was closely correlated with citric acid and malic acid.However,the diversity of volatiles was influenced by various factors and microbial metabolism simultaneously.Further co-occurrence network analysis indicated that the contents of flavor components slightly varied with the abundance of dominant microbes.(3)Revealing the spatiotemporal characteristics of Banpei and their correlations with flavors and microbial profilesIt was investigated that the effects of various factors on microbiota and metabolites of Banpei,which is a mixture of broad bean-koji and brine.These factors include broad bean origin,fermentation methods and periods.The result showed that the fermentation methods of Banpei mainly affected the contents of the characteristic flavor components,including citric acid,malic acid and succinic acid,bitter and umami amino acids,and volatiles such as palmitate,methyl linoleate and phenyl ethanol.These components were also affected by the other factors.For example,both Banpei fermented by temperature controlling and by ambient temperature were influenced by the broad bean origin and fermentation periods.However,the flavor feature of Banpei fermented by weather-exposed pattern depended significantly on the enterprises,including its geographical environment and process regulation parameters.The relative abundance of Pelomonas and Ralstonia ranged from 71.74%to 90.74%,and were relatively stable.The abundance of Saccharomycetales in Yunnan Banpei was higher than that in Sichuan Banpei.In addition,Saccharomycetales in Banpei fermented by weather exposed pattern was also higher than that in Banpei fermented by ambient temperature.The abundance of Zygosaccharomyces was sharply decreased from 86.72% to 9.62% during the fermentation periods.Redundancy analysis showed that most amino acids were positively correlated with Bacillus.Six organic acids,except lactic acid,were positively correlated with Pichia and Saccharomycetales.Methyl linoleate and methyl palmitate were positively related to Wickerhamomyces and Staphylococcus,while ethyl palmitate and phenethyl alcohol were associated with Zygosaccharomyces.PICRUSt2 predicting indicated that the most active pathways were the same in both the bacterial and fungal communities,which were biosynthesis,degradation/utilization/assimilation,precursor metabolites and energy.In addition,the influences of chili varieties and the fermentation methods of Banpei on Doubanjiang were revealed,the results demonstrated that both were significantly affected the taste characteristics and the aroma components contents.(4)PGI feature of PXDBJ was characterized by multiple scales,and proved the discriminant model based on distinguishing indicators and statistical analysis coupling is effective and utility.Polyphasic detection approaches were used to identify the PGI feature of PXDBJ,and further compared with the flavor metabolites of various sauces.These sauces include miso,Doenjang,rice sauce,soybean sauces,chili-bean pastes,etc.,originated from Japan,Korean and outside Pixian origin,even contiguous county.Therefore,the taste markers of PXDBJ were that the proportion of citric acid and malic acid(44.89%～ 67.48%),Asp and Glu proportion(22.34% ～ 34.30%).The aroma marker was that the proportion of ethyl palmitate and ethyl laurate in ester components(17.09% ～38.03%).In general,the differences in flavor profiles between PXDBJ and other sauces were revealed by PCA and(O)PLS-DA matrix.Furthermore,γ-elemene,(Z)-anethole and ethyl decanoate were the specific volatiles in the analogous Doubanjiang,which were manufactured in the contiguous county according to the same technique.In addition,nineteen potential markers could be used to distinguish PXDBJ and the other fermented sauces based on the aroma volatiles,such as(EZ)-β-ionone and 4-ethylguaiacol,etc.Therefore,the discriminant model was established by coupling indicators and multivariate mathematical statistics methods,in which these indicators were determined by polyphase detection technology.Taken as a whole,the model is suitable for protecting the place of origin of PXDBJ.(5)Studying the attribute of peppers variety and the attribute of ripening region of PXDBJ,and interpreting the scientific nature of its PGI productsDefinitely,the chili variety used for PXDBJ manufacturing was restricted according to the PGI standard(GB/T 20560-2006).Therefore,two kinds of Doubanjiang were used to reveal the attribute of chili variety,which was produced by Capsicum annuum L.Erjingtiao and Capsicum annuum L.Meiguohong,respectively.Results showed that the CIELAB color space was higher in the latter.Similarly,the content of phenols components in Meiguohong was 2.69 times higher than that in Erjingtiao.There were 10 differentiated volatiles only detected in Meiguohong,such as 3-methylthiopropanol and phenethyl acetate,etc.;in addition,12 volatiles including(R,R)-(-)-2,3-butanediol and(Z)-2-heptenal,etc.were detected in Erjingtiao merely.According to the ICH technique,Doubanjiang was ripened in two ways(natural and fortified)by the same raw materials at both the inside and outside the Pixian region.The results demonstrated that the geographical attribute of PXDBJ was the main factor limiting the synthesis of 4-ethylguaiacol and 4-ethylphenol.In addition,the discrepancies in the microbial structure were significantly affected by the regional differences.The dominant microbes were Lactobacillus and Hortaea in the Doubanjiang of the inside region,while Aquabacterium and Zygosaccharomyces were dominant in the outside region.In addition,microflora was changeable as a result of the biological disturbance,especially,the interaction of Candida and Pseudomonas was altered,Kazachstania and Caulobacter as well.Furthermore,biological disturbance could accelerate the formation rate of the main flavor components;however,(Z)-2-heptenal,4-ethylguaiacol,trans-2-octenal,2,3-butanediol and guaiacol were only detected in PXDBJ fermented in the domain.Therefore,the limited raw materials and regions are the scientific connotation of PGI of PXDBJ.