| Background and ObjectiveThe traditional sun-dried vinegar is produced in Fushun,Zigong.It uses bran as the main fermentation material,adds the natural fermentation liquid after cofermented with rice porridge and homologous Chinese herbal medicine of hundred herbs in the same pool for 30 days,and then has the process characteristics of open aging for 3 years as its technological characteristics,forming sun-dried vinegar with unique flavor and taste.It is one of the representatives of intangible cultural heritage in Sichuan Province.The preliminary experiments showed that the sun-dried vinegar was rich in nutrients,and the content of y-aminobutyric acid(GABA)was particularly high.However,there were no reports on its nutritional composition and fermentation characteristics.Therefore,this study will determine the safety indicators and flavor components of traditional sun-dried vinegar,evaluate its food safety and ingredient advantages,explore the correlation of flavor and nutrients substances with microbial succession in the fermentation process,and screen functional strains that can produce high GABA.This study lays a foundation for further exploring the health effects of traditional sun-dried vinegar and developing healthy vinegar and related health products,so as to promote the development of local industry and rural revitalization.MethodsPart 1 Safety of traditional sun-dried vinegar and comparison of main flavor and nutrients substances with similar vinegar productsAccording to the method of national food safety standards,the contents of harmful inorganic elements(Ni,Sb,Cd,As,Pb,Sn,Cr and Hg),aflatoxin B1(AFB1),nitrate and nitrite,and 3-chloro-1,2-propylene glycol(3-MCPD)in traditional sun-dried vinegar in different years were determined by inductively coupled plasma mass spectrometry(ICP-MS),ELISA,ion chromatography and gas chromatography-mass spectrometry,respectively.The total number of bacterial colonies and coliforms were determined by traditional culture method,and the safety of traditional sun-dried vinegar was comprehensively evaluated.Then,the contents of 94 amino acids and their derivatives,65 organic acids and their derivatives in different brands of vinegar(Qian Tian Baiwei,Bao Ning,Qian He,Tai Yuanjing)were analyzed by ultra-performance liquid chromatography tandem high resolution mass spectrometry.The contents of,amino acid nitrogen,pH,total acids,reducing sugars,total phenols and total flavonoids in vinegars were determined by chemical methods.In addition,principal component analysis(PCA)was used to compare the content and composition characteristics of amino acids and organic acids in different brands of vinegar,and to find the dominant flavor substances and potential characteristic components in traditional sun-dried vinegar.Part 2 Study on the composition and variation characteristics of flavor and nutrients substances during fermentation of traditional sun-dried vinegarThirteen samples of Cupei at different fermentation stages(SS,AF,AAF stages)were collected.The contents of water,pH,total acid,and amino acid nitrogen in the samples were measured according to the national standard method for food safety.The contents of total phenols and flavonoids in the samples were determined by spectrophotometry.Eight organic acids and seventeen free amino acids in the samples were detected by high performance liquid chromatography(HPLC).Principal component analysis(PCA)was used to explore the potential characteristic organic acids and free amino acids in different fermentation stages(starch saccharification stage,alcohol fermentation stage and acetic acid fermentation stage),and to explore the composition and variation rule of main flavor substances in the fermentation process of traditional sun-dried vinegar.Part 3 Microbial community succession and its correlation with flavor and nutrients substances during traditional sun-dried vinegar fermentationTo investigate the changes in the number of culturable microorganisms in Cupei samples at different fermentation stages,the total number of bacteria,mold,and yeast colonies and lactic acid bacteria in different Cupei samples were counted by traditional cultivation method.Illumina high-throughput sequencing technology was used to study the structure and dynamic evolution of microbial communities(bacteria and fungi)in different fermentation stages of traditional sun-dried vinegar samples.The α diversity,β diversity and UPGMA cluster analysis of sequencing data were performed by R software to explore the succession characteristics of microbial communities(bacteria and fungi)and the similarities and differences of microbial communities at different fermentation stages of traditional sun-dried vinegar.Spearman correlation analysis was used to analyze the correlation between microbial communities and main flavor substances,and redundancy analysis(RDA)was used to explore the highly related microorganisms that played an important role in the changes of flavor substances.This study provided data support for finding the core flora and functional microorganisms in the traditional sun-dried vinegar fermentation process.Part 4 Identification of facultative anaerobic lactic acid bacteria in Cupei and screening of GABA-producing lactic acid bacteriaTo screen out facultative anaerobic lactic acid bacteria,we spread dilutions of Cupei samples on MRS plates and cultured them in anaerobic and aerobic environments,respectively.The single colonies with lactic acid bacteria characteristics cultivated in anaerobic environment were transferred to MRS plates and then cultured in aerobic environment.The single colonies with the characteristics of lactic acid bacteria cultured in aerobic environment were transferred to MRS plate,and then cultured in anaerobic environment.The bacteria that could grow in both aerobic and anaerobic environments were preserved for subsequent Gram staining,MALDI-TOF-MS and 16s rDNA sequencing.Berthelot colorimetric method was used to screen out the bacteria with high GABA production.The acid-resistant bacteria were further screened out by the tolerance test of artificial gastric fluid,which were used in the subsequent tolerance experiments of artificial intestinal fluid tolerance test,bile salt environment tolerance test,high salt environment tolerance test,safety test(hemolysis test and plasma coagulase test),antibiotic sensitivity test(14 antibiotics including cephalexin,rifampicin,chloramphenicol and so on),growth curve determination and GABA production stability test were used to further test the high GABA producing strains to obtain lactic acid bacteria with strong GABA production ability,excellent performance and high safety.The whole genome sequencing of the strain was performed to comprehensively analyze the genomic function of the strain,so as to predict and discover its potential probiotic effects,and to accumulate data for the development of healthy vinegar and related health products.ResultPart 1 Safety of traditional sun-dried vinegar and comparison of main flavor and nutrients substances with similar vinegar products1.The four harmful heavy metal elements of Pb,Cr,Sb and Hg were not detected in all types of traditional sun-dried vinegar samples.The content of four harmful inorganic elements of Ni,Cd,As and Sn,AFB1,the total number of bacterial colonies,coliform bacteria,nitrate and nitrite,and 3-MCPD were all in line with the limit requirements of the national food safety standard of China.2.A total of 77 amino acids and their derivatives and 52 organic acids and their derivatives were detected in the 4 brands of vinegar.Compared with other brands of vinegar,traditional sun-dried vinegar had significant advantages in the content of 39 amino acids and 23 organic acids,respectively.The contents of γaminobutyric acid(117.65 mg/mL)and 3-aminoisobutyric acid(32.79 mg/mL)were particularly high,which were more than 10 times of other brands of vinegar,followed by benzoic acid,which were about 3 times,6 times and 4 times of Taiyuanjing vinegar,Qianhe vinegar and Baoning vinegar samples,respectively.In addition,the contents of total acid(83.52±0.78 g/kg)and amino acid nitrogen(9.41±0.16 g/kg)were also the highest in this sun-dried vinegar.The contents of total phenol(11.40±0.26 g CAE/kg)and total flavonoids(8.53±0.15 g RTE/kg)were also higher than those of Qianhe vinegar and Baoning vinegar samples.3.PCA analysis showed that the cumulative contribution rates of amino acids and their derivatives,organic acids and their derivatives in the first two principal components(PC1 and PC2)of the four different brands of vinegar were 81.40%and 83.50%,respectively,indicating that PCA analysis was effective.Among them,11 amino acids,such as γ-aminobutyric acid,3-aminoisobutyric acid,alanine,glycine and threonine,and 5 organic acids,such as DL-3-phenyllactic acid,benzoic acid,3-hydroxyisovaleric acid,subaric acid and homovanillic acid,are the characteristic components of traditional sun-dried vinegar.Part 2 Study on the composition and variation characteristics of flavor and nutrients substances during fermentation of traditional sun-dried vinegar1.The water content of Cupei samples was maintained between 60%-65%during the traditional fermentation process.The contents of total acid and amino acid nitrogen in Cupei samples increased with fermentation time,from 3.48±0.18 g/kg and 0.06±0.02 g/kg before fermentation to 10.50±0.18 g/kg and 1.31±0.03 g/kg on the 30th day,respectively.There were significant differences among different fermentation stages(P<0.05).The pH value of Cupei samples was maintained at about 4.5 during fermentation process,with no significant difference among different fermentation stages(P>0.05).In addition,the contents of total phenols and flavonoids in Cupei samples showed an increasing trend in SS and AF stages,with statistically significant differences(P<0.05),but did not increase significantly in AAF stage.The total phenolic content increased from 0.47±0.06 g CAE/kg before fermentation to 1.41±0.04 g CAE/kg on the 30th day of fermentation,and the total flavonoid content increased from 0.45±0.04 g RTE/kg before fermentation to 0.88±0.02 g RTE/kg on the 30th day of fermentation.2.The total organic acid content of traditional sun-dried vinegar showed a rising trend during fermentation,which increased from 1.54±0.30 g/kg on the 0th day of fermentation(SS0)to 24.29±1.21 g/kg on the 30th day of fermentation(AAF 10),and the total amount of organic acid increased about 15 times.Acetic acid,lactic acid and succinic acid were the most abundant organic acids,accounting for more than 80%of the total organic acids.At the same time,the content of total free amino acids also showed a rising trend,which increased from 29.64±4.39 mg/100g on the 0th day of fermentation(SS0)to 705.35±56.03 mg/100g on the 30th day of fermentation(AAF 10),and the total amount of free amino acids increased about 24 times.The content of threonine,arginine and alanine increased significantly in the middle and late stages of fermentation,and their content together accounted for more than 50%of the total free amino acids.There were significant differences in total organic acids and free amino acids among the three stages of SS,AF and AAF(P<0.05).Part 3 Microbial community succession and its correlation with flavor and nutrients substances during traditional sun-dried vinegar fermentation1.The results of traditional culture showed that the number of bacteria,lactic acid bacteria,yeast and mold all showed an increasing trend with the increase of fermentation time,and reached the maximum on day 7(SS7).The total counts of bacteria,yeast,mold colonies and lactic acid bacteria in 13 Cupei samples were 8.05±0.64 logCFU/g,5.51±1.03 logCFU/g,3.72±0.49 logCFU/g and 8.11±0.84 logCFU/g,respectively.There was no significant difference in the total number of colonies between bacteria and lactic acid bacteria.The differences of the total number of colonies among other microorganisms were statistically significant(P<0.05),indicating that bacteria were the dominant bacteria in Cupei samples during fermentation.Notably,although the lactic acid bacteria counts were lower than the total bacterial colonies at the beginning of fermentation,from day 4(SS4)onwards,the lactic acid bacteria counts(9.11 logCFU/g)exceeded the total bacterial colonies(8.70 logCFU/g)and became the dominant bacteria.2.The results of high-throughput sequencing showed that the composition of microbial communities in Cupei samples at different fermentation stages was different.At the phylum level,the bacterial communities of all Cupei samples were mainly composed of Firmicutes and Proteobacteria.The relative abundance of Firmicutes increased from 66.48%at SS stage to 97.58%at AAF stage,while the relative abundance of Proteobacteria decreased.It decreased from 33.31%in SS stage to 1.67%in AAF stage.In the fungal community,Ascomycota dominated the whole fermentation stage,with a relative abundance of about 90%,followed by Basidiomycota,whose relative abundance varied from 1%to 10%.At the genus level,Lactobacillus and Acetobacter were the dominant bacteria,and the abundance of Lactobacillus increased from 37.55%in SS stage to 92.50%in AAF stage,becoming the absolute dominant genus.For yeasts,the dominant species at the SS and AF stages were Kazachstania and Issatchenkia,but at the AAF stage Zygosaccharomyces(69.24%)and Schwanniomyces(15.50%)were the main dominant yeasts.Notably,the abundance of the Zygosaccharomyces rose rapidly from 0.08%at the SS stage to 69.24%at the AAF stage.3.Alpha diversity analysis showed that the Observed abundance indices of bacterial and fungal communities decreased from 195.6 and 83.8 in SS stage to 109.8 and 53.0 in AAF stage,respectively,suggesting that the abundance of microbial communities in Cupei samples decreased with the increasing fermentation days.However,there were no significant differences in the abundance of bacteria and fungi at different fermentation stages(P>0.05).The Shannon diversity index of bacterial community decreased from 3.16 to 0.92,and the Simpson diversity index increased from 0.13 to 0.70.The Shannon diversity index of fungal community increased from 1.74 in SS to 2.28 in AF and then decreased to 1.05 in AAF.The Simpson diversity index decreased from 0.36 in SS to 0.17 in AF and then increased to 0.58 in AAF.There were significant differences in the diversity of microbial communities at different fermentation stages(P<0.05),suggesting that the diversity of bacterial communities in Cupei samples decreased with the increase of fermentation days,while the diversity of fungal communities increased first and then decreased.The results of β diversity and UPGMA cluster analysis showed that the microbial communities in Cupei samples were well separated at SS,AF,and AAF stages,indicating that the composition of bacterial and fungal communities in fermented grains samples at different fermentation stages was significantly different(P<0.01).Compared with the samples in SS stage,the distances between samples in AF and AAF fermentation stages were close to each other.Especially,the three samples in the late fermentation stage(AAF4,AAF7,and AAF 10)were all on the same branch,indicating that the microbial community composition in the samples was similar.These results suggested that the differences in microbial community composition in Cupei samples supported the experience-based fermentation stage classification.4.Spearman correlation analysis showed that Lactobacillus,Zygosaccharomyces,Schwanniomyces,Corynebacterium,Millerozyma were positively correlated with total acid,amino acid nitrogen,total phenols,total flavonoids,7 organic acids and 9 amino acids.Oceanobacillus were positively correlated with succinic acid,suggesting that these genera may increase the content of these flavors and nutrients,while Weissella and Alternaria may cause a decrease in total acid,total flavonoids and methionine.Acetobacter,Kazachstania,and Pichia were negatively correlated with pH,which might be more meaningful in maintaining the acidic pH value of Cupei samples.These results suggest that these bacteria may be the core flora affecting the flavor and nutrient formation during the fermentation of traditional sun-dried vinegar.In addition,RDA analysis showed that Lactobacillus,Zygosaccharomyces,and Schwanniomyces were highly related to the changes of flavor and nutrients during traditional sun-dried vinegar fermentation,which were important microbial communities.Part 4 Identification of facultative anaerobic lactic acid bacteria in Cupei and screening of GABA-producing lactic acid bacteria1.A total of 201 strains of bacteria were isolated from 13 Cupei samples.170 strains could grow in both aerobic and anaerobic environments,of which 168 were Gram-positive bacteria.By 16s rDNA sequencing,104(61.2%)of the 170 strains were identified as Lactobacillus and 47(27.6%)were Bacillus.Among the 170 strains identified by MALDI-TOF-MS,102 strains(60.0%)were Lactobacillus and 39 strains(22.9%)were Bacillus.A total of 140 strains were identified consistent by the two methods,with an agreement rate of 82.4%.The Kappa value was 0.644,P<0.001.The results of the two methods were highly consistent for the identification of bacteria isolated from Cupei samples.2.Six strains(SC25,SC26,SC36,SC68,SC70 and SC114)producing GABA more than 2 g/L were screened by Berthelot colorimetric method.Three strains(SC25,SC26 and SC114)with survival rates higher than 80%were further screened by simulated artificial gastric juice tolerance test(pH=5.5).These three strains did not show hemolytic rings on blood plate and did not have plasma coagulase activity,suggesting that they were safe.In the simulated intestinal fluid tolerance test,the viable counts of the three strains were all maintained at about 8.2 log CFU/mL,indicating that they had strong tolerance to artificial intestinal fluid.However,the viable counts of the three strains after 24 h culture in different concentrations of bile salt(0%～0.30%)decreased with the increase of the concentration.When the bile salt concentration was 0.3%,the viable counts of SC25,SC26 and SC114 strains were 3.3 log CFU/mL.0.9 log CFU/mL and 0.8 log CFU/mL,indicating that SC25 strain was more tolerant to bile salt.The results showed that SC26 was resistant to 5 antibiotics(vancomycin,streptomycin,cephalexin,gentamicin and ciprofloxacin),and SC25 and SC114 were resistant to 3 antibiotics(vancomycin,streptomycin and ciprofloxacin).The growth curve showed that SC25 strain could enter the logarithmic growth phase after 3 hours of culture,while SC26 and SC114 strains needed 6 hours of culture.The stability of GABA production ability of the selected strains was determined.The results showed that the GAB A content of the three strains fluctuated between 2～3 mg/mL in each generation,among which strain SC25 had the strongest GABA production ability with an average value of 2.60±0.25 mg/mL.In summary,the strain SC25 was screened out,which could grow rapidly in both aerobic and anaerobic environments,tolerate artificial intestinal fluid and bile salt environment,have high safety,and have the strongest GABA production ability.The strain was identified as Limosilactobacillus fermentum by sequencing.3.The whole genome of SC25 strain was obtained by second and third generation sequencing.The genome of the strain was circular DNA,which consisted of 2081217 bp,GC content was 61.65%,and contained 2087 protein coding sequences.The number of genes annotated by COG,KEGG,Swiss-Port and GO databases were 1855,1217,1566 and 1645,respectively.Among them,the number of genes for carbohydrate and amino acid metabolism was higher.The 4-aminobutyrate aminotransferase gene related to GABA metabolism was found in the NR database,suggesting that it may be related to the synthesis of GABA in SC25 strain.VFDB and CARD database predicted that the strain contained 6 virulence factors and 23 genes related to antibiotic resistance.The virulence factors were mainly related to bacterial adhesion,immune regulation and stress stress,but had no pathogenicity to the host.Antibiotic resistance genes are mainly related to resistance to fluoroquinolones,vancomycin,rifamycin and lipopeptide antibiotics.ConclusionCompared with other brands of vinegars,traditional sun-dried vinegar has advantages in main flavor and nutrient contents,especially rich in amino acids and the highest content of GABA.The microbial community was characterized by high abundance of Lactobacillus,Acetobacter,Zygosaccharomyces and Schwanniomyces during the SS and AF stages,but Lactobacillus became the dominant bacterium during the AAF stage,and Zygosaccharomyces was the most abundant yeast.The genera Lactobacillus and Zygosaccharomyces may play the most important roles in the production of flavor and nutrients during the fermentation of traditional sundried vinegar.A strain of Limosilactobacillus fermentum with strong sugar and amino acid metabolism and high GABA production was successfully screened out.It has the characteristics of high safety,rapid growth in both aerobic and anaerobic environments,and good survival performance in digestive environment,which is consistent with the basic characteristics of probiotics and can be used for the future research and development of healthy vinegar and functional food.Innovation points1.This study for the first time explored the correlation between flavor substances and microbial succession during the fermentation process of traditional sun-dried vinegar,one of the representatives of intangible cultural heritage in Sichuan Province,and provided an important reference for further exploration of its health effects.2.In this study,the Lactobacillus strain with strong carbohydrate and amino acid metabolism ability,rapid growth in aerobic and anaerobic environments,good survival in digestive environment,and high GABA production was successfully obtained,which is of great significance for the subsequent research and development of healthy vinegar and functional food.3.Zygosaccharomyces was found for the first time to be the dominant yeast in the middle and late stages of the fermentation of traditional sun-dried vinegar,which might be related to the high content of free amino acids during the fermentation. |
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