| Red sour soup is a naturally fermented condiment with small tomatoes and peppers in Guizhou Province.Currently,domestic and foreign research focuses on the fermentation process,microbial diversity,nutrient components and so on.However,the characteristic flavour components of red sour soup and the metabolic mechanism of the dominant lactic acid bacteria on the formation of flavour quality need to be further studied.This study intends to identify the characteristic flavour substances of red sour soup and screen the dominant lactic acid bacteria.Through directed fermentation,the mechanism of the lactic acid bacteria affecting the formation of the red sour soup flavour was revealed by combining multi-group technology,providing a basis for the revelation of functional strains in red sour soup,directional regulation of fermentation technology and the promotion of standardised production of red sour soup.(1)In order to analyse the characteristic flavour substances of red sour soup,10 red sour soups from different regions were selected.The titratable acid,organic acid,amino acid and volatile flavour substances in the red sour soup were determined using high-performance liquid chromatography,an automatic amino acid analyser and gas chromatography-mass spectrometry.The results showed that lactic acid,pyruvic acid and citric acid were the main organic acids in the red sour soup,with an average content of 9.19 g/kg,3.62 g/kg and 3.61 g/kg.The total free amino acid content is 243.85-801.58 mg/kg.Through TAV comprehensive analysis,it is determined that glutamic acid and alanine are the characteristic fresh and sweet substances in red sour soup.A total of 158 volatile flavour compounds were detected.Among the flavour substances with OAV≥1,the volatile substances of eucalyptus leaf oleol and dimethyl disulfide are the main characteristics of the sour soup odour.Linalool,4-ethyl guaiacol,n-octanal,acetic acid,4-alcohol terpene α-Pinene,ethyl acetate,ethyl propionate,propyl propionate,isoamyl lactate,ethyl isovalerate,isobutyl acetate,heptadecane and isobutylthiazole are the key aroma substances of naturally fermented red sour soup.(2)In order to clarify the influence of the dominant bacteria and their succession on the flavour of red sour soup during the whole fermentation process,taking the post-fermentation stage of red sour soup which is not yet researched as the goal,the bacterial community and volatile flavour substances in post-fermentation stages of red sour soup were determined by high-throughput sequencing and HS-SPME-GC-MS.The results showed that lactic acid bacteria played a major role in the post-fermentation process with the extension of fermentation time,the abundance of lactic acid bacteria increased gradually.During post-fermentation,77 volatile flavour compounds were identified.Esters and aromatic hydrocarbons are the main volatile compounds in the post-fermentation of red acid soup,with the highest relative content of 39.8%and 25.79%,respectively.Through a correlational analysis of flora and volatile flavour,48 pairs of positive correlation and 19 pairs of negative correlation were obtained.Among these,Lactobacillus had the most significant correlation with volatile flavour(P<0.05)(20 pairs)and played a leading role in the formation of flavour during post-fermentation.(3)To provide a theoretical basis for realising the directional regulation of red sour soup.The lactic acid bacteria were screened from the naturally fermented red sour soup and six strains with high acid production,strong acid and bile salt resistance were obtained.They were identified as Lm.F1-2 Leuconostoc mesenterium,Lr.B2-7 Lactobacillus rhamnosus,Lp.F2-6 Lactobacillus plantarum,Lc.F2-1 Lactobacillus casei,Lpc.C2-1 Lactobacillus paracasei and Lz.C1-3 Lactobacillus zeae.The fermentation characteristics of the six lactic acid bacteria in red sour soup were evaluated using sensory and colour difference as indicators.The results showed that except for the Lz.C1-3 Lactobacillus cornis there was no significant difference in sensory scores of the other 5 strains of lactobacillus fermented red acid soup(P<0.05),while the Lz.C1-3 Lactobacillus cornis fermented red sour soup showed an overall brown color,leading to the lowest sensory score(5.8±0.32).(4)Although the five lactic acid bacteria screened had good fermentation characteristics,their role in the formation of the flavour quality of red sour soup was not clear.Lm.Fl-2,Lr.B2-7,Lp.F2-6,Lc.F2-1,Lpc.C2-1,as an enhanced starter of Red Sour Soup,combined with extensive targeted metabonomics and GC-IMS technology was used to detect the non-volatile metabolites and volatile flavour substances of Red Sour Soup,which were enhanced by the five lactic acid bacteria,respectively.The results showed that LrB2-7 and Lpc.C2-1 had a strong acid production capacity of 28.35±1.45 g/kg and 27.30±2.68 g/kg,respectively,while Lm.F1-2 had the weakest acid production capacity of 16.44±1.23 g/kg.A total of 536 nonvolatile metabolites were identified,of which lipids(28.9%),amino acids and their derivatives(25.75%)and organic acids(16.98%)were the three main nonvolatile metabolites.Through screening of different metabolites,it was concluded that L-lactic acid,the characteristic taste of Red sour soup produced by Lc.F2-1 metabolism,was the strongest(VIP=35544).The red acid soup fermented by Lr.B2-7and Lc.F2-1 is more abundant in sugars,nucleotides,fatty acids and their derivatives metabolites.Also,the characteristic flavour substances,such as acetic acid and ethyl lactate,produced by metabolism are significantly higher than those fermented by Lp.F2-6,Lpc.C2-1 and Lm.F1-2.Lr.B2-7 is more conducive to the formation of ethyl acetate-the characteristic flavour of red sour soup.(5)In order to explore the interaction of the screened dominant bacteria in the fermentation process of red sour soup.By 16S sequencing,metabolomics and GC-MS,7 different stages of multi-strain cooperative fermentation of red acid soup were used to detect the bacterial diversity and flavor.The synergetic fermentation of lactic acid bacteria significantly affected the microbial diversity and metabolite content during the fermentation of red sour soup.A total of 393 non-volatile metabolites were identified and the content of 10 metabolites was significantly increased through screening and analysis of different metabolites.According to the correlation analysis between dominant bacteria and non-volatile metabolites,lactic acid bacteria were significantly positively correlated with L-isoleucyl-L-aspartate,2-hydroxy-4-methylvaleric acid,thymine,3-aminosalicylic acid,fructose-6-phosphate.Lactobacillus rhamnosus was positively correlated with lactic acid,ascorbic acid and chlorogenic acid.A total of 80 volatile substances were detected.The total ester content reached 239.91±4.17 μg/kg,after 20 days of fermentation,which acetate,ethyl acetate,isoamyl lactate,ethyl lactate and ethyl propionate combine to form the characteristic ester flavour of red sour soup.The alcohol content reached its highest,103.12±5.86 μg/kg,after 15 days of fermentation,Among them,linalool,isoamyl alcohol,benzyl alcohol,α-Terpineol,3-methyl-2-hexanol,4-ol terpene and 4-ethyl guaiacol,which constitutes the characteristic mellow aroma of red sour soup.The correlational analysis between dominant bacteria and volatile flavour substances found 18 key flavour substances were positively and negatively correlated with the dominant lactic acid bacteria;eucalyptus oleol α-Pinene and isobutylthiazole were significantly negatively correlated with Lactobacillus rhamnosus,Lactobacillus casei,Lactobacillus paracasei and Leuconostoc mesenterium.Linalool and 4-ethyl guaiacol only showed significant positive correlation with Leuconostoc mesenterium and Lactobacillus plantarum only showed significant positive correlation with ethyl butyrate.(6)To explore the interaction of dominant bacteria in the fermentation process of red sour soup,macro gene technology was used to predict the pathway of flavour formation,involving dominant lactic acid bacteria at the gene level,and a metabolic network diagram was constructed.The results showed that lactic acid bacteria converted glucose into pyruvate through glycolysis and then converted pyruvate into various flavour compounds through different metabolic pathways.First and foremost,acetic acid and lactic acid are formed through pyruvic acid metabolism.Ethyl acetate and ethyl lactate can be formed by the esterification of acetic acid and lactic acid with ethanol,respectively.At the same time,pyruvate can also metabolize to produce acetyl CoA,which is the main precursor of the TCA cycle,and then metabolize to generate organic acids,such as malic acid.Glutamic acid and alanine,the key taste substances,are mainly produced by lactic acid bacteria through amino acid metabolism.Lm.Fl-2 Leuconostoc mesenterium,Lr.B2-7 Lactobacillus rhamnosus,Lp.F2-6 Lactobacillus plantarum,Lc.F2-1 Lactobacillus casei and Lpc.C-2 Lactobacillus paracasei all participate in the decarboxylation of aspartic acid to pyruvate by aspartic acid decarboxylase(EC:4.1.1.12)and the pathway of alanine transaminase catalysing pyruvate(EC:2.6.1.2)to produce alanine.At the same time,Lc.F2-1 Lactobacillus casei and Lpc.C-2 Lactobacillus paracasei can also produce alanine through pyruvate under alanine dehydrogenase(EC:1.4.1.1).Lm.F1-2 Leuconostoc mesenterium,Lr.B2-7 Lactobacillus rhamnosus,Lp.F2-6 Lactobacillus plantarum,Lc.F2-1 Lactobacillus casei and Lpc.C-2 Lactobacillus paracasei are involved in the hydrolysis of L-glutamine to L-glutamic acid,catalysed by glutaminase and the production of glutamic acid by ketoglutarate and glutamine under the action of glutamic acid synthase.Lactobacillus rhamnosus Lr.B2-7 and Lactobacillus casei Lc.F2-1 are also involved in the α-Ketoglutarate generated glutamate(EC 1.4.1.2)pathway under the action of glutamate dehydrogenase.There were seven enzymes related to the formation of esters and five lactic acid bacteria contributed. |
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