| As an important part of the diet culture of many countries,fermented fish are deeply popular with consumers at home and abroad.With the continuous upgrading of consumers’ concept of healthy diet,the market value of Suanyu products that can be stored at room temperature and ready to eat with bones has increased significantly.Industrial production of Suanyu is an important developing direction for freshwater fish processing.Several dominant strains were isolated and purified from traditional Suanyu in our laboratory and the fermentation technology of mixed inoculation with Lactobacillus plantarum,Staphylococcus xylococcus and Saccharomyces cerevisiae was determined.The technical problems of long fermentation period,unstable flavor quality and safety of traditional Suanyu were solved,which laid a foundation for the industrial production of Suanyu.However,the mechanism of Suanyu flavor formation in fermentation process is still unclear,it is difficult to regulate Suanyu flavor in industrial production.Therefore,the typical flavor characteristics of Sunyu fermentation process were investigated in this study.The metabolism of characteristic flavor was explored,by analyzing the changes of flavor precursor and key enzymes related to the characteristic flavor.Species annotation of enzyme-coding genes in the flavor metabolic pathways was carried out,based on the fermented regulation.Meanwhile,the key microorganisms that were related to characteristic flavor were predicted.Furthermore,the succession of key microbial communities during Suanyu fermentation was revealed.This study is of great academic value to reveal the mechanism of fermentation flavor formation and is a theoretical basis for the directional regulation of characteristic flavor of Suanyu.To identify the characteristic flavor of Suanyu during fermentation,the Suanyu was inoculated and HS-SPME-GC/MS and GC-MS-O in combination with electronic nose techniques were used to identify the volatile compounds in different fermentation periods.The results showed that the increase of volatile compounds was most pronounced in the 1st week of fermentation,from 37 to 58 compounds,forming the fermentation flavor basis of Suanyu.Combining the odor activity and the results of GC-MS-O flavor intensity,a total of 21 key volatile flavors were found.The results of OPLS-DA model showed that the fermentation process of Suanyu could be divided into three stages: the first stage was raw material(fresh),the second stage was initial fermentation(0-2 weeks),and the third stage was the later fermentation(2-4 weeks),providing a key detection point for the whole fermentation process of Suanyu.Furthermore,nine differential flavor compounds at different fermentation stages were screened according to the OPLS-VIP value,including ethyl lactate,palmitic acid ethyl ester,4-hydroxy-3-methoxystyrene,2-octenal,(E,E)-2,4-decadienal,2-acetylfuran,ethyl octanoate,benzaldehyde and 3-methylbutanoic acid,which could be regarded as the key indicators for Suanyu quality monitoring during fermentation.To clarify the metabolic regulation of characteristic flavor,the precursors that probably formed characteristic flavor and key enzymes were further analyzed.The relationship between precursors,enzymes and characteristic flavor was also elucidated.According to the analysis of variable importance in projection of O2 PLS model,the amino acids with significant influence on the key flavor of Suanyu were aspartic acid,phenylalanine,methionine,glutamic acid,leucine,valine,isoleucine and cysteine;the fatty acids with significant influence on the key flavor of Suanyu were C16:0,C20:5,C20:4,C18:1,C22:6,C18:0 and C16:1.Moreover,the correlation between flavor and flavor precursors was analyzed.The results showed that ethyl lactate,acetaldehyde,benzaldehyde,phenethyl alcohol and 4-hydroxy-3-methoxystyrene were mainly correlated with amino acids.Hexanal,octanal,nonanal,(E,E)-2,6-nonadienal and(E,E)-2,4-decadienal were mainly correlated with fatty acids.The dynamic changes of other characteristic flavor were mainly correlated with amino acids and fatty acids.Furthermore,the changes of key enzymes activities in different metabolic pathways were analyzed.The results showed that the activities of phosphofructokinase,pyruvate kinase,lipoxygenase,hydroperoxidelyase and alcohol acyl transferase reached to the maximum after the 1st week of fermentation,which were 544 U/g,66 U/g,21.9 U/g,37.8 U/g and 26.4 U/g,respectively.However,the activities of hexokinase,branched amino acid transferase,aromatic amino acid transferase and esterase decreased continuously with the progress of fermentation.Flavor derived from glycolysis metabolism was produced in the first week of fermentation.Flavor derived from lipoxygenase pathway was produced in the first two weeks of fermentation,and amino acid transamination occurred in the first two weeks of fermentation.According to the analysis of variable importance in projection of O2 PLS model,the key enzymes with significant influence on the early flavor of fermentation were branched amino acid transferase,hydroperoxidelyase and hexokinase.Aromatic amino acid transferase,esterase and alcohol acyl transferase were the key enzymes that affect the flavor in later fermentation.Metagenomic sequencing was used to analyze the possible pathways of characteristic flavor formation at the gene level and the key microorganisms involved in characteristic flavor of Suanyu were predicted according to the relationship between characteristic flavor and its precursors and key enzymes.It was found that the metabolic pathway accounted for the highest proportion of more than 61.9%,according to the annotation of KEGG metabolic pathway.Furthermore,in the carbohydrate metabolic pathway,the number of gene annotated to glycolysis/gluconeogenesis pathway was the most abundant,followed by citric acid cycle and oxidative phosphorylation.In the amino acid metabolic pathway,the relative abundance of aromatic amino acid and branched amino acid metabolic pathway increased gradually with the process of fermentation,and reached to 20.1% and 12% at the 4th week of fermentation,respectively.In the lipid metabolism pathway,glycerol-phospholipid metabolism and glycerolipid metabolism pathway accounted for more than 50%.According to the results of the annotation of the key catalytic enzymes in the characteristic flavor formation pathway,it was found that bacteria played a major role in the formation of Suanyu flavor.The microorganisms involved in the flavor derived from carbohydrate and lipid metabolism in the early stage of fermentation mainly included Staphylococcus,Pediococcus,Lactobacillus,Enterococcus,Macrococcus,Exiguobacterium,Vibrio,Kocuria,Saccharomyces and Candida.The microorganisms involved in the flavor derived from aromatic amino acid metabolism flavor and ester flavor in the later fermentation mainly included Wickerhamomyces,Rhodotorula,Debaryomyces,Staphylococcus,Leuconostoc,Enterococcus,Pediococcus,Lactobacillus and Vibrio.To regulate the characteristic flavor of Suanyu,the succession of microorganisms related to the characteristic flavor during fermentation was studied using high-throughput sequencing.The results showed that the relative abundance of Staphylococcus,which is related to flavor,increased sharply(P < 0.05)from an initial value of 1.95% to 12.52% after the 1st week of fermentation,then declined rapidly to 1.88% at 4th week.The flavor-related Lactobacillus and Macrococcus were predominant genera during the early stages of fermentation,accounting for 45.74% and 31.99% of sequences,respectively.Furthermore,the relative abundance of Lactobacillus reached to 71.57% at the end stage of fermentation.The relative abundance of Saccharomyces involved in carbohydrate metabolism reached to 88.83% in the 1st week of fermentation.Candida became the dominant genus(96.59%)after three weeks of fermentation,while Wickerhamomyces dominated the fungal community of the 4th week of fermentation(32.51%).However,the relative abundance of Lactococcus,Candida,Enterococcus and Vibrio associated with flavor was significantly inhibited during inoculation fermentation.Furthermore,the redundancy analysis showed that the major environmental factor contributing to the change of bacterial community was pH value,while the water content had greater influence on the change of fungal community.In addition,it was found that Lactobacillus was negatively correlated with several flavor-bacteria,such as Weissella and Vibrio,and Staphylococcus was positively correlated with Saccharomyces and Exiguobacterium,according to the analysis of microbial interaction.This study defined the relationships among characteristic flavor,flavor precursors and key enzymes,clarified the metabolism of characteristic flavor during Suanyu fermentation,predicted the key microorganisms in the flavor forming pathway,illustrated the succession of the key microorganisms which were related to flavor during Suanyu fermentation,and revealed the relationship between characteristic flavor formation and microbial community of fermented Suanyu.It could provide theoretical guidance for directional regulation of Suanyu flavor. |
PDF Full Text Request