Study On The Quality Formation Mechanism Of Pickled Pepper During Fermentatio

Pickled pepper is one of the most typical fermented vegetables in southwest China,a condiment and a unique appetizer.Most pickled pepper on the market are made by traditional fermentation technology,which makes use of the pepper’s own flora and microorganisms in the environment to ferment naturally,and its market demand is considerable.At present,there are numerous studies on the correlation between microorganisms and flavor quality in various fermented foods,however,there are few reports on the formation mechanism of flavor quality of pickled pepper.This study explored the dynamic changes and differences in microbial community structure,physicochemical properties and flavor substances during the traditional fermentation of pickled pepper,analyzed the potential effects of microbial community structure on physicochemical indicators and flavor substances,and analyzed the formation mechanism of pickled pepper flavor quality and its flavor-related core functional microorganisms.The main research results are as follows:(1)High Throughput Sequencing(HTS)technology was used to analyze the microbial community structure during the fermentation of pickled pepper.The results showed that 911339 and 1514169 pairs of raw sequence reads were obtained from the16 S r RNA and ITS1 genes of 18 pickled pepper samples,respectively,and generated into 720525 and 535513 pairs of valid sequence reads after quality control.A total of six dominant phyla(relative abundance > 1%)were identified from the phylum level during the fermentation of pickled pepper,including four dominant bacterial phyla and two dominant fungal phyla.They were Firmicutes,Proteobacteria,Bacteroidota,Actinobacteriota,Ascomycota and Basidiomycota,respectively.At the genus level,nine core bacterial genera and eleven core fungal genera(relative abundance > 0.01)were screened.PICRUSt functional prediction showed that metabolic function at KEGG level＿1 was the most dominant bacterial community function during pickled pepper fermentation,accounting for more than 70% at all stages.The metabolism of carbohydrates and amino acids on KEGG level＿2 was the main metabolism involved in microorganisms during pickled pepper fermentation,which may be closely related to the formation of pickled pepper flavor,and also indicated that the material basis of flavor formation was mainly protein and sugar substances.(2)Through physicochemical analysis,headspace solid phase microextraction gas chromatography mass spectrometry(HS-SPME-GC-MS),high performance liquid chromatography(HPLC)and ultra high performance liquid chromatography Q exactive orbitrap mass spectrometry(UHPLC-QE-MS)were used to determine the physicochemical parameters and metabolites of pickled pepper during fermentation.The results showed that the content of reducing sugar tended to be stable from the 9th day,and the p H value tended to be stable from the 24 th day.The total acid content continued to increase and began to level off on the 24 th day of fermentation.In the fermentation process of pickled pepper,the content of nitrite increased first and then decreased,while the content of soluble solid did not change significantly in the early stage of fermentation.The brightness value L*,redness value a* and yellowness value b* in the color difference were all higher at the end of fermentation than at the beginning.After fermentation,pickled pepper showed a brighter color.Based on SI >800,A total of 85 VFCs were identified during the fermentation of pickled pepper.Including 9 alcohols,9 acids,20 esters,8 alkanes,12 alkenes,11 aldehydes,7 ketones,6 phenols and 3 other groups,26 VFCs with significant differences were selected from the total VFCs(P < 0.05,VIP > 1).The main VFCs in the fermentation process of pickled pepper are esters.VIP > 1,P < 0.05 and MS2.score > 0.99 were used as the criterion for screening significant difference metabolites.A total of 38 significant difference metabolites were screened in pickled pepper.It includes 4 others,1 organic oxygen compound,1 plant hormone,3 organic heterocyclic compounds,6 fatty acids,3 amino acids,2 sugars,4 alkaloids,1 lipid and lipid molecule,1 nucleoside,1 other organic nitrogen compound,3 organic acids,3 aromatic compounds,2 coumarins,1fatty acyl,1 flavone,and 1 Lactone.The Metabo Anaylyst5.0 platform and KEGG database were used to analyze the enrichment of significantly different non-volatile metabolites in fermented pepper.It was found that the top 25 key metabolic pathways mapped by 38 different metabolites were: vitamin B6 metabolism;valine,leucine and isoleucine degradation;oxidation of branched chain fatty acids;phytanic acid peroxisomal oxidation;purine metabolism;propanoate metabolism;ketone body metabolism;arginine and proline metabolism;vitamin K metabolism;butyrate metabolism;mitochondrial electron transport chain;arachidonic acid metabolism;carnitine synthesis;phospholipid biosynthesis;citric acid cycle;aspartate metabolism;fatty acid biosynthesis;glutamate metabolism;warburg effect and tryptophan metabolism.These metabolic pathways are closely related to the flavor of pickled peppers.Tartaric acid,malic acid,lactic acid,acetic acid,citric acid and succinic acid were detected in the pickled peppers at different stages of fermentation,and the total organic acids content increased and then decreased during the pickled pepper fermentation.The highest level of total organic acids were reached on the 24 th day of fermentation.(3)The spearman coefficient was used to analyze the correlation between dominant bacteria and physicochemical indexes and metabolites.The results showd that: the redundancy analysis(RDA)method was used to study the potential correlation between bacteria and fungi and physical and chemical indexes in pickled pepper.At the level of bacterial species,it was found that Lactiplantibacillus level was negatively correlated with p H value,while other Lactic acid bacteria(LAB),such as Weissella,Leuconostoc,Lactococcus and Pediococcus were on the contrary,is positively correlated with p H value.Most dominant bacteria were positively correlated with reducing sugar.Compared with bacteria,the correlation between fungi and physicochemical indexes was generally weak.According to the Spearman correlation,a total of 9 kinds of bacteria and 8 kinds of fungi are highly correlated with VFCs and 5 kinds of organic acids(| r | > 0.8,P < 0.05).The results found that bacteria contributed more to the flavor of pickled pepper.The correlation between dominant fungi and dominant bacteria and 38 non-volatile metabolites with significant differences was analyzed.Lactiplantibacillus was found to be positively correlated with 6-Keto-prostaglandin F1 a,cytosine,D-proline,guanine,succinic acid,Prostaglandin J2 and malonic acid,and negatively correlated with guanosine,artesunate,dihydrocapsaicin,Hydroxytyrosol 1-O-glucoside and oleylglycine.The correlations between Weissella,Pectobacterium,Empedobacter,Pseudomonas,Pediococcus,and Lactococcus with these significantly different nonvolatile metabolites are the opposite of those between Lactiplantibacillus and these significantly different nonvolatile metabolites.The correlations with nonvolatile metabolites were roughly the same in the genera of Empedobacter,Pediococcus,and Pseudomonas.There was no significant correlation between fungi and 38 different nonvolatile metabolites,again confirming that fungi were not the major contributors of flavor in pickled pepper.To determine the relationship between microorganisms and metabolites during the fermentation of pickled pepper,metabolic pathway and enrichment analysis(Metabo Analyst 5.0)were used to evaluate all metabolites.The metabolic network related to metabolite formation in pickled pepper fermentation was predicted based on microbial annotation related enzymes combined with the KEGG database and related literature.Pyruvate and acetyl-co A were central compounds in the whole metabolic pathway.The enzyme abundance related to flavor synthesis in pickled pepper was at a high level in the early fermentation stage,which was conducive to the rapid formation of flavor substances in the early fermentation stage.In conclusion,the results showed that the expression of microbial community and related enzymes had a certain effect on the flavor of pickled pepper.Carbohydrate metabolism,amino acid metabolism and fatty acid metabolism seem to play an important role in the formation of flavor compounds during the fermentation of pickled pepper.