Study On The Quality Formation Mechanism Of Naturally Fermented Peppe

Zao Chili(ZC)is a traditional fermented Chili that plays an important role in Chinese cuisine,especially famous in southwest China.Currently,there are various studies on fermented Chili peppers,especially the analysis of microbial flora and flavor in its fermentation system has become a hot topic.However,there are few reports on comprehensive analysis of the quality formation mechanism of ZC.Therefore,this study will provide a theoretical basis for improving the flavor quality of ZC by exploring the microbial community succession and changes in flavor substances during the fermentation process of Guizhou natural fermentation Zao Chili(NF-ZC),and deeply understanding the formation mechanism of flavor substances during the fermentation process of ZC based on metabolic networks.In this paper,the natural fermentation of ZC is conducted based on the 7% salt content in Zunyi,Guizhou,China.Analyze the changes in microbial community,organic acids,volatile flavor,and non-volatile metabolites of NF-ZC samples from different fermentation stages,and track the changes in their physicochemical factors to determine the fermentation speed of ZC.In addition,the relationship between dominant microorganism genus and physicochemical factors,volatile flavor compounds,organic acids,and nonvolatile metabolites was analyzed through correlation analysis,and a metabolic network was constructed to explore the mechanism of flavor quality formation in ZC.The main research results are as follows:(1)High throughput sequencing technology(HTS)based on the Illumina Miseq PE300 sequencing platform was used to detect and analyze the microbial community succession and related functions during the fermentation process of ZC.The original sequence readings of 1323887 and 781978 pairs of fungi and bacteria(average length:240 bp and 416 bp,respectively)were obtained from 18 samples of ZC.After quality control,1179846 and 712267 pairs of valid sequences were obtained,with an average ratio of 89% and 91%,respectively.At the gate level,a total of 2 fungal and 4bacterial phyla were identified based on relative abundance > 1%.They are Ascomycota,Basidiomycota,Firmicutes,Proteobacteria,Bacteroides,and Actinobacteria.Among the fungal genera,a total of 12 fungal genera have been identified(relative abundance > 1%),among which the dominant genera are Colletotrichum,Gibberella,Alternaria,Zygosaccharomyces,and Pichia.In terms of bacterial genera,a total of 14 bacterial genera(relative abundance > 1%)have been identified,mainly Lactobacillus,Weissella,Pectobacterium,Enterobacter,Kosakonia,Pseudomonas,etc.At the species level,based on the relative abundance > 1%,the species that have a significant impact on the fermentation process of fermented Chilli are Lactobacillus＿plantarum,Lactobacillus＿alimentarius,Gibberella＿intricans,Pectobacterium＿carotovorum,Zygosaccharomyces＿bisporus,Colletotrichum＿truncatum,Plectosphaerella＿niemeijerarum and Candida＿quercitrusa.The coexistence network relationship between microbial genera shows that there is a negative correlation between Enterobacter,Pseudomonas,Lactococcus,Pantoea,and Acinetobacter,and both Lactobacillus and Zygosaccharomyces(r <-0.8,P < 0.05),while there is no correlation between Lactobacillus and Zygosaccharomyces,indicating that the two can coexist under certain conditions.PICRUSt function prediction indicates that metabolism is the most important function of bacterial communities at the KEGG level＿1,which account for over 70%.At the KEGG level＿2,it mainly focuses on carbohydrate metabolism,amino acid metabolism,and cofactor and vitamin metabolism.In addition,the formation of metabolites by bacteria involves a total of 20 metabolic functions at the KEGG level＿3.(2)These physicochemical factors(pH,total acid,reducing sugar,nitrite,soluble solids,and salinity)of f ZC vary regularly during natural fermentation.A total of 90 volatile compounds were detected by headspace solid phase microextraction gas chromatography mass spectrometry(HS-SPME/GC-MS),mainly including 21 terpenes,15 alcohols,13 esters,13 aldehydes,8 acids,4 ketones,4 phenols,and 4ethers.The principal component analysis(PCA)study found that there were significant differences in the volatile flavor compounds of ZC at different stages of fermentation.Based on the variable importance in projection(VIP)in the supervised orthogonal partial least squares discriminant analysis(OPLS-DA)model and combined with analysis of variance(ANOVA),33 volatile flavor compounds with significant differences were selected based on VIP > 1 and P < 0.05,among which esters,alcohols,ketones,aldehydes,and phenols were more abundant.A total of 7organic acids were detected in ZC samples by HPLC,among which malic acid,lactic acid,acetic acid,and succinic acid gradually increased with fermentation time and tended to stabilize in the later stage.Citric acid gradually decreased with fermentation time.The content of oxalic acid and tartaric acid is low,so they have little impact on flavor.(3)The non-volatile flavor metabolites in the fermentation process of natural fermented ZC were analyzed using metabolomics using ultra high performance liquid chromatography quadrupole electrostatic mass spectrometry(UHPLC QE-MS)with non-targeted detection.A total of 1384 substances were detected during the entire fermentation process of ZC.After screening,a total of 112 effective non-volatile metabolites were obtained,mainly including amino acids,fatty acids,sugars,organic acids,aromatic compounds,alkaloids,flavonoids,organic heterocyclic compounds,phenylpropanoids,and phenols.Amino acids,fatty acids,sugars,organic acids,and aromatic compounds account for a large proportion and exhibit stable changes during fermentation.The PCA showed that the samples were within a 95% confidence interval,and the intra group samples were closely clustered,while the inter group samples were significantly separated.Based on the OPLS-DA model and combined with ANOVA analysis of variance,37 significantly different nonvolatile metabolites were selected based on VIP > 1 and P < 0.05.Through KEGG enrichment analysis and pathway analysis of 112 nonvolatile metabolites,it was found that vitamin B6 metabolism,alanine,aspartic acid,and glutamic acid metabolism,glyoxylic acid and dicarboxylic acid metabolism,glycine,serine,and threonine metabolism,and TCA cycle had a high enrichment rate(ER ≥ 5),and a significant pathway impact factor(PI > 0.1).(4)The redundancy analysis(RDA)relationship between dominant microbial genera and physicochemical factors indicates that both fungal and bacterial genera contribute significantly to pH.Based on the Spearman correlation coefficient analysis of the correlation between microorganisms and volatile flavor compounds and organic acids,it can be seen that Lactobacillus,Weissella,Enterobacter,Zygosaccharomyces,and Pichia may be potential volatile flavor contributing microorganisms in the fermentation process of ZC.The correlation between dominant microbial genera and non-volatile metabolites indicates that bacteria such as Lactobacillus,Weissella Leuconostoc,and Enterobacter have a significant positive correlation with a variety of non-volatile metabolites.Among fungal genera,only Candida and Pichia are related to a few metabolites,indicating that bacteria play a leading role in the formation of nonvolatile metabolites during the fermentation process of ZC.Through pathway enrichment analysis of 90 volatile flavor compounds and 7 nonvolatile organic acids using Metabo Analyst 5.0,a total of 14 main metabolic pathways were obtained.At the same time,combining the enrichment analysis results of non-volatile metabolites,microbial functional enzymes in high-throughput sequencing,the Kyoto Encyclopedia of Genes and Genomics(KEGG)database,and literature data,a related metabolic network was constructed to deeply analyze the formation mechanism of flavor quality during the fermentation process of ZC,providing a theoretical basis for improving the flavor quality of ZC.