Method for constructing and using an artificial bacterial flora for vinegar fermentation

Abstract

The present invention belongs to the technical field of food fermentation and relates to a rational construction method and use of an artificial bacterial flora for vinegar fermentation. The artificial bacterial flora construction method provided in the present invention is based on metatranscriptome sequencing technology and constructs an artificial bacterial flora for the first time by grouping cluster analysis, which has a similarity of more than 90% with the active microbial community in the process of in situ acetic acid fermentation of vinegar, and the metabolic characteristics of the bacterial flora are more than 80%, so that the rational construction of the artificial bacterial flora can be realized. In addition, the present invention can fully improve the stability of vinegar fermentation, and can also be used as a reference for other conventional fermented food bacterial flora construction, analysis and reproducible flavor synthesis technologies, and has high scientific and application value.

Description

[Technical Field]

[0001] The present invention relates to the technical field of food fermentation, and more particularly to a rational method for constructing and using an artificial bacterial consortium for vinegar fermentation. [Background technology]

[0002] Vinegar production in China primarily uses grain vinegar and fruit vinegar as raw materials, employing either traditional multi-strain solid-state fermentation or pure-bacterial (acetic acid bacteria) liquid fermentation. The former produces a mellow flavor but slow fermentation, while the latter produces a fast fermentation but monotonous flavor. Traditional vinegar is primarily produced through an open fermentation process, with the acetic acid fermentation stage being the primary stage for the formation and accumulation of flavor substances. A natural fermentation agent, "starter mash," provides abundant microorganisms for the acetic acid fermentation stage of vinegar. However, in actual production processes, the microbial communities abundant in "starter mash" are susceptible to various influences and fluctuations, resulting in instability in the vinegar fermentation process and product quality, presenting a common technical challenge in the industry that urgently needs to be resolved. With the development of science and technology and the continued increase in research on microbial communities, a shift from natural fermentation to artificial microbial communities capable of fermentation is essential to ensure the quality of fermented foods.

[0003] The food fermentation process relies on the activity of a limited number of microbial genera. Microorganisms not only produce flavor compounds but also interact with other microorganisms to stabilize fermentation and ensure the normal progression of food fermentation. For example, acetic acid produced by acetic acid bacteria is the main flavor substance in vinegar, while lactic acid produced by lactic acid bacteria significantly reduces the sourness of vinegar and can also produce various beneficial factors and bacteriocins. Furthermore, bacillus bacteria can cooperate with lactic acid bacteria to produce more flavor substances. While research has been conducted on vinegar production by simply combining microorganisms with different functions, this method lacks scientific basis and cannot effectively guarantee the quality of the vinegar.

[0004] Thanks to the development of high-throughput sequencing technology, preliminary research has been conducted on the construction and use of artificial microbial consortia based on the analysis of microbial consortia structure. However, the microbial consortia obtained by this method do not necessarily have high metabolic activity and cannot reflect the true metabolic status and function of the colony during the fermentation process. Summary of the Invention

[0005] The present invention provides a rational method for constructing and using an artificial microbial consortium for vinegar fermentation. The method for constructing an artificial microbial consortium provided by the present invention uses metatranscriptome sequencing technology to obtain a core microbial population through grouping cluster analysis to construct an artificial microbial consortium. Using this technology in the vinegar fermentation process can improve the flavor and quality of vinegar while achieving stable production. This technology solves the problem that artificially constructed microbial consortium structures do not necessarily have relatively high metabolic activity and do not reflect the true metabolic status and function of colonies during the fermentation stage. This technology uses metatranscriptome sequencing technology to obtain a core microbial population through grouping cluster analysis to construct an artificial microbial consortium. Furthermore, based on microbial functional activity analysis, the metabolic network of active microorganisms is reconstructed to construct an artificial microbial consortium for vinegar fermentation. This effectively ensures the stability of the fermentation process and product quality.

[0006] A rational method for constructing an artificial bacterial consortium for vinegar fermentation includes the following steps.

[0007] (1) Functional and species annotation of microbial communities: Vinegar mash samples to be simulated at different fermentation stages were collected, impurities were removed, and total microbial RNA was extracted and subjected to metatranscriptome sequencing. Functional and species annotation was performed on the sequencing results.

[0008] (2) Removal of contaminants and pathogens: For microorganisms whose corresponding functions are annotated, contaminants and pathogens are removed.

[0009] (3) Determining the initial core microorganisms: Based on the abundance of all microorganisms (N species) at each fermentation stage in the metatranscriptome results, sort them in order of the gradual decrease in the average abundance of each species throughout the fermentation process. The first two species are combined into the first group, and one species is added and combined in turn to finally obtain (N-1) microbial groups. If the microbial community and the original microbial community at each stage are all successfully clustered and have similarities of ≥90%, the minimum number of species required is determined as the initial core microbial community. Furthermore, the species abundance data for each microbial group was imported into the software Minitab, and cluster analysis was performed by calculating the similarity between each microbial group and the original microbial flora at each stage using the longest distance method and Euclidean distance. The species abundance includes the type of microorganism and the corresponding concentration. Preferably, all similarities are >95%.

[0010] (4) Verification of metabolic activity of core microbial communities a. Determine the major flavor substances and corresponding metabolic genes in samples at the end of fermentation. b. Calculate the ratio of the transcription expression level of the metabolic genes in the initial core microbial community to the transcription expression level of the metabolic genes in the original microorganisms at each fermentation stage. If the transcription rates of the major flavor metabolic genes at each stage are all ≥ 80%, proceed to the next step. If the transcription rates of the major flavor metabolic genes at a certain stage are < 80%, add one species in order according to the abundance of the species in step (3) and repeat step (4) until the transcription rates of the major flavor metabolic genes at each stage are all ≥ 80%, thereby determining the core microbial community. Preferably, the transcription rate of metabolic genes in the core microbial community is 85% or more. Furthermore, the composition of the major flavor substances in samples taken at the end of fermentation was detected using devices such as high-performance liquid chromatography and gas chromatography mass spectrometry. The flavor intensity of these substances was analyzed along with the threshold and content of the flavor substances, and organic acids and amino acids with a TAV>1 (umami intensity value) were defined as major umami substances. These have an important contribution to the umami tastes of vinegar, such as sourness, sweetness, and bitterness. Volatile aroma compounds with an ROAV>0.1 (relative aroma activity value) were defined as major aroma compounds. These two together constitute the major flavor substances of vinegar. The determination of the metabolic genes for the main flavor substances at each fermentation stage refers to the determination by comparing them with the KEGG database and screening the results of metatranscriptome sequencing.

[0011] (5) Screening of artificial bacterial flora strains: Based on the composition of the core microbial community, we screen samples such as koji, sake mash, and vinegar mash to obtain strains with superior performance. Furthermore, if necessary, different selective media are used to perform primary screening of microorganisms in the vinegar mash sample, followed by secondary screening of the strains based on conditions such as acid tolerance, temperature tolerance, and alcohol tolerance. Finally, fermentation function verification is performed according to different experimental strains, thereby determining the optimal strain and finally determining the specific microbial composition of the core microbial community. Furthermore, by using a selective medium containing different substrates such as starch, cellulose, glucose, ethanol, and acetic acid, it is possible to screen for microorganisms that have properties such as the hydrolysis of starch or cellulose, and the conversion of glucose or ethanol to produce organic acids, alcohols, esters, and aldehydes in samples such as vinegar mash. Furthermore, the proportions of the strains in the artificial bacterial flora are mixed together in proportion to the average biomass of the corresponding microorganisms in the core microbial community in the original fermentation sample at each stage. Furthermore, even when the artificial bacterial consortium strains are mixed and cultured, no obvious inhibitory effect is observed, and the original physiological activity properties can be maintained even under moisture conditions of 20% to 40%.

[0012] The present invention further provides the use of the artificial bacterial flora, in particular the use of the artificial bacterial flora in vinegar fermentation instead of conventional "seed mash".

[0013] Furthermore, after each strain of the artificial flora is purely cultured to prepare a pure bacterial agent or bran koji, the addition ratio of each strain is determined based on the average ratio of the microbial composition at each fermentation stage in the results of metatranscriptome sequencing, and the strains are mixed in the corresponding ratio and further used in the acetic acid fermentation stage of vinegar.

[0014] Furthermore, the mixed bacterial agent is used in the fermentation process of liquid or solid grain vinegar or fruit vinegar.

[0015] Furthermore, the mixed bran koji is used in the fermentation process of liquid or solid grain vinegar or fruit vinegar.

[0016] Furthermore, the inoculation amount of the artificial flora was 10 viable bacteria per 1 kg of raw material. 8 ~10 12 Add cfu.

[0017] Advantages and positive effects of the present invention (1) In this invention, for the first time, an artificial microbial consortium is constructed by group cluster analysis based on metatranscriptome sequencing technology, and the similarity with the active microbial community in the in situ acetic acid fermentation process of vinegar is >90%, and the metabolic characteristics of the microbial consortium are >80%, realizing the rational construction of an artificial microbial consortium. Furthermore, the present invention can significantly improve the stability of vinegar fermentation and can serve as a reference for other conventional fermented food microbial consortium construction, analysis, and reproducible flavor synthesis techniques, and has great scientific and application value. (2) The present invention constructs a microbial community based on metatranscriptome sequencing, which has a more accurate analysis of key functional microorganisms and has a more theoretical significance. (3) In the present invention, grain vinegar fermentation or fruit vinegar fermentation is performed using an artificial bacterial flora instead of conventional starter mash, and at the end of fermentation, the artificial bacterial flora has a faster fermentation rate as a fermenting agent, the fermentation period is shortened by 2 to 8 days, the raw material utilization rate is improved by 6% to 13%, nonvolatile acid, reducing sugar, amino acid nitrogen, and total esters are increased by 30% to 150%, 10% to 40%, 10% to 25%, and 10% to 50%, respectively, and the nonvolatile acid / volatile acid ratio is improved by 50% to 180%. The softness of the vinegar is improved and the quality of the vinegar is improved. [Brief explanation of the drawings]

[0018] [Figure 1] Dynamic changes in species classification, composition, and their relative abundance based on metatranscriptome sequencing results are shown. [Figure 2] The top 10 species (ordered from left to right in descending order) with the highest mean abundance in the fermentation stage based on metatranscriptome sequencing are shown. [Figure 3] 1 shows the grouping cluster analysis of microorganisms, i.e., the determination of the initial core microbial genera. In the figure, d indicates the original fermentation sample, d-1 indicates clustering using the top five abundant microorganisms, and d-2 indicates clustering using the top six abundant microorganisms. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described below with reference to specific embodiments. Unless otherwise specified, all technical means used in the present invention are methods known to those skilled in the art. Furthermore, the embodiments are merely for the purpose of illustrating the present invention and do not limit the scope of the present invention. The essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments without departing from the essence and scope of the present invention are also included in the protection scope of the present invention.

[0020] Unless otherwise specified, the percent symbol "%" described in the examples refers to the mass percent of the solid, the percent of the solution refers to the number of grams of solute contained in 100 mL, and the percent of the liquid refers to the volume ratio of the solution at 25°C.

[0021] The TAV (taste intensity) described in the present invention is an evaluation of the sensory contribution rate of non-volatile flavor substances by the taste intensity value method. The TAV value is the ratio of the content of each taste substance to its threshold value.

[0022] TAV i =C i / T i In the formula: C i represents the content of each taste substance. T i represents the corresponding sensory threshold of this taste substance.

[0023] When the TAV value is greater than 1, this substance is judged to contribute to the taste. On the other hand, when the TAV value is less than 1, this substance is judged not to contribute to the taste.

[0024] The ROAV (relative aroma activity value) of the present invention: The determination of characteristic volatile flavor compounds evaluates the contribution of each volatile component to the aroma of the entire sample by the relative aroma activity value method. That is, JPEG0007796441000001.jpg5170In the formula: [[ID=​​​​​​​​​​​​​The sensory thresholds for the calculation process of TAV and ROVA may be determined based on the thresholds disclosed in the prior art, and reference may be made to "Flavor Analysis and Quality Improvement of Shanxi Lao Chen Vinegar" [D]. Cheng Cheng. Tianjin University of Science and Technology, 2018.

[0025] The present invention will be further described below with reference to specific examples.

[0026] Example 1: Composition analysis of active communities in conventional fermentation processes (1) Metatranscriptome sequencing: Using Shanxi Laochen Vinegar as an example, equal amounts of vinegar mash samples were collected on the 1st, 3rd, 5th, 7th, and 9th days of the acetic acid fermentation stage, impurities were removed, and total RNA of the sample microbial community at each stage was extracted. Metatranscriptome sequencing was performed on the total RNA at each stage. (2) Microbiota composition analysis: Unigene sequences from samples at each stage were compared with bacterial, archaeal, fungal, and viral sequences in the NCBI-NT database via BLASTN to obtain the compositional distribution of the active microbiota at the family, genus, and species taxonomic levels for each sample. The microbiota composition from DNA-targeted metagenomic sequencing was used as a control. Table 1 shows the distribution of the top 10 microbiota with the highest average relative abundance throughout the fermentation stage. As can be seen from Table 1, the abundance of Lactobacillus and Acetobacter based on metagenomic sequencing was relatively high, at 52.32% and 10.83%, respectively, with Unclassified accounting for 29.52%. Based on metatranscriptome sequencing, the abundance of Lactobacillus and Acetobacter was 66.21% and 11.05%, respectively, with Unclassified accounting for 5.75%. This indicates a relatively large discrepancy in the sequencing results under different conditions. RNA-targeted metatranscriptome sequencing is used to analyze the transcriptional status of functional genes in microbial communities, which can reveal the metabolic activity of microorganisms during the fermentation process and reflect the actual contribution of microorganisms to the fermentation process. Therefore, in this study, we performed subsequent microbial community analysis using RNA-targeted metatranscriptome sequencing.

[0027] Table 1: Major microbial composition in metagenomic and metatranscriptomic sequencing results JPEG0007796441000002.jpg69170

[0028] Example 2: Rational construction of an artificial bacterial flora using the traditional fermentation of Shanxi Lao Chen vinegar as an example (1) Metatranscriptome sequencing: Equal amounts of vinegar mash samples were collected on days 1, 3, 5, 7, and 9 (AAF1d, AAF3d, AAF5d, AAF7d, and AAF9d) during the acetic acid fermentation stage of vinegar. Impurities were removed, and total RNA of the microbial community was extracted. Metatranscriptome sequencing was performed on the total RNA. (2) Composition and functional annotation of the active microbial flora: The unigene sequences of each sample were compared with bacterial, archaeal, fungal, and viral sequences in the NCBI-NT database via BLASTN to obtain the species-level compositional distribution of the active microbial flora for each sample. Figure 1 and Table 2 show the abundance ranking (top) of active microorganisms in each vinegar fermentation process. Functional annotation was performed on the unigene sequences in the sequencing results using the KEGG database.

[0029] Table 2: Taxonomic composition and dynamic changes of species JPEG0007796441000003.jpg69170JPEG0007796441000004.jpg96170

[0030] (3) Removal of contaminating and pathogenic microorganisms: Contaminating and pathogenic microorganisms such as Alternaria alternata and Pantoea ananatis were removed from the microorganisms in the annotation results.

[0031] (4) Determination of core microorganisms: Based on the abundance of active microorganisms (N species) in the metatranscriptome results for the entire fermentation stage, the species were sorted in order of decreasing abundance (Figure 2 shows the top 10 species, sorted from left to right in order of decreasing species abundance). The top two species were combined to form the first group, and each species was added and combined in order to obtain N-1 microbial groups. Cluster analysis was performed using Minitab software, calculating the similarity between each microbial group and the original microbial groups at each stage using the longest distance method and Euclidean distance. The initial core microbial group was determined as the minimum species combination required for successful clustering of each microbial group and the original microbial composition at each stage with a similarity of ≥90%. The results are shown in Figure 3. Here, crossover phenomenon was observed in the clustering results between the top five abundant microbial combinations and the original microbial combinations (Figure 3-A), indicating that clustering was not successful. The clustering results for the top six microbial combinations and the original microbial combination were relatively good (Figure 3-B). The similarities to the original fermentation sample microorganisms on days 1, 3, 5, 7, and 9 of fermentation were 91.04%, 98.87%, 92.39%, 94.37%, and 93.84%, respectively. The similarities between the samples in each group were all >90%. Therefore, the six microorganisms identified as the initial core microbial group were Lactobacillus acetotolerans, Acetobacter pasteurianus, Lactobacillus helveticus, Lactobacillus kunkeei, Lactobacillus fermentum, and Streptococcus lactis.

[0032] (5) Metabolic activity verification of core microbial communities a. Determination of major flavor substances: Using instruments such as high-performance liquid chromatography and gas chromatography mass spectrometry, the composition and content of flavor substances in samples at the end of vinegar fermentation were detected, and the flavor intensity of each flavor substance was calculated based on the threshold and content of the flavor substance. Taste substances with TAV>1 and aroma substances with ROVA>0.1 were determined to be the major flavor substances in vinegar.

[0033] As shown in Table 3, the main taste substances in the sample are oxalic acid, succinic acid, citric acid, lactic acid, tartaric acid, acetic acid, glutamic acid, histidine, alanine, and valine. As shown in Table 4, the main aroma substances in the sample are acetic acid (which acts as both an aroma substance and a taste substance), 3-methylbutyric acid, benzaldehyde, furfural, ethyl acetate, 2,3-butanedione, 3-hydroxy-2-butanone, phenethyl acetate, isopentyl acetate, 2,3,5-trimethylpyrazine, and furfuryl alcohol.

[0034] Table 3: Taste thresholds and taste activity values ​​(TAV) of tastants JPEG0007796441000005.jpg86170

[0035] Table 4: Threshold values ​​and relative odor activity values ​​(ROVA) of odorants JPEG0007796441000006.jpg115170

[0036] b. Determination of key metabolic genes: By comparing using the KEGG database, one or more metabolic genes that are most important for the formation of key flavor substances were obtained, and the corresponding species were obtained by screening from the results of metatranscriptome sequencing, and then calculated separately for different species corresponding to the same (or different) genes.

[0037] Take the synthesis of citric acid as an example. 1) Search for genes important for citrate formation: We searched for citric acid in KEGG, clicked on C00158 (i.e., citric acid), and then clicked on pathways to obtain a series of metabolic pathways involving citrate. We clicked on map00020 (TCA cycle) to obtain genes important for citrate formation: gltA (EC: 2.3.3.1), ACLY (EC: 2.3.3.8), EC: 2.3.3.3, and acnA (EC: 4.2.1.3). We then clicked on the second pathway, map00250, which was skipped because it did not involve citrate formation (no genes for citrate formation). We then clicked on the next pathway, map00630 (glyoxylate metabolism), to obtain the genes important for citrate formation, gltA and acnA. In this way, we found genes important for citrate formation in all metabolic pathways (key genes specifically refer to genes for the final step of citrate synthesis). In summary, the genes important for citrate formation were gltA, ACLY, EC:2.3.3.3, and acnA.

[0038] 2) The transcriptome sequencing results were used to search for the gltA, ACLY, EC:2.3.3.3, and acnA genes, and the transcript expression levels and corresponding microorganisms were obtained. The results are shown in Table 5, but no transcription results were found for the EC:2.3.3.3 gene. (Transcriptome data allows for a one-to-one correspondence between a gene and a species. In the previous step, the corresponding gene name and species name were mapped using species and functional annotations for each gene ID in the sequencing results. Metatranscriptome analysis allows for the acquisition of microbial community composition data, the one-to-one correspondence between a gene and a source microorganism, and the relative transcription concentration of a gene.)

[0039] Table 5: Statistics of transcription results of key enzymes using the example of citrate formation JPEG0007796441000007.jpg108170

[0040] c. Statistics of transcription results of important genes: The percentage of the sum of transcriptions of genes important for the formation of major flavor substances at each fermentation stage of microorganisms in the initial core microbial community accounted for by the transcription of genes important for the formation of major flavor substances at each stage.

[0041] Taking citric acid as an example: as can be seen from step b, in the vinegar acetic acid fermentation process, the genes important for the formation of citric acid are gltA, ACLY, and acnA. The transcription expression levels of the three genes corresponding to the core microorganisms at each stage in Table 5 were summed up, and the transcription expression levels of the three genes at each fermentation stage were summed up and calculated. As a result, the transcription levels of the genes important for the formation of citric acid in the microorganisms in the initial core microbial group (Lactobacillus acetotolerans, Acetobacter pasteurianus, Lactobacillus helveticus, Lactobacillus kunkeei, Lactobacillus fermentum, Streptococcus lactis) on the 1st, 3rd, 5th, 7th, and 9th days of fermentation were 46.15%, 48.28%, 84.06%, 98.20%, and 100%, respectively, of the microorganisms at each stage.

[0042] Using a similar method, we calculated the sum of the transcription expression levels of genes important for the formation of other flavor substances at each fermentation stage of the initial core microbial group, added up the expression levels of genes important for flavor substances at each fermentation stage, and calculated the ratio of the expression levels of the initial core microbial group to the transcription of genes important for flavor substances at each stage.

[0043] As a result, on the 1st, 3rd, 5th, 7th, and 9th days of fermentation, the transcription of genes involved in the formation of the main flavor substances of the initial core microbial community was 72.03%, 87.54%, 73.15%, 77.78%, and 82.17% of the transcription of the original microbial community genes at each stage (days 1, 3, 5, 7, and 9), respectively.

[0044] (6) Verifying metabolic activity of core microbial communities and improving artificial flora Because the transcription rates of metabolic genes in the initial core microbial community were <80% on days 1, 5, and 7 of fermentation, we added Lactobacillus plantarum (ranked 7) to the initial core microbial community according to the abundance of the microbial species in steps (2)–(4). Following step (5), we calculated the transcription rates of genes important for the formation of major flavor substances in the new microbial community. The transcription rates of genes important for the formation of major flavor substances in this new microbial community were 73.39%, 89.68%, 73.96%, 78.17%, and 82.74%, respectively, on days 1, 3, 5, 7, and 9 of fermentation, which did not meet the requirements. Similarly, we added Bacillus amyloliquefaciens (ranked 8). The transcription rates of genes important for the formation of major flavor substances in the resulting microbial community were 78.77%, 94.38%, 79.08%, 82.23%, and 87.24%, respectively, on days 1, 3, 5, 7, and 9 of fermentation, which did not meet the requirements. Using the same method, we subsequently added Pediococcus pentosaceus (rank 9). The transcription rates of genes important for the formation of major flavor substances in the resulting microbial community were 83.51%, 96.07%, 83.87%, 85.17%, and 91.47%, respectively, on days 1, 3, 5, 7, and 9 of fermentation. All of the transcription rates were >80%, meeting the requirements. Therefore, the nine core microorganisms, Lactobacillus acetotolerans, Acetobacter pasteurianus, Lactobacillus helveticus, Lactobacillus kunkeei, Lactobacillus fermentum, Streptococcus lactis, Lactobacillus plantarum, Bacillus amyloliquefaciens, and Pediococcus pentosaceus, were used as the starting species for the artificial microbiota.

[0045] Example 3: Use of mixed rice bran koji with artificial bacterial flora in the acetic acid fermentation process of homemade grain vinegar (1) Construction of an artificial bacterial flora: An artificial bacterial flora was determined by the method of Example 2. (2) Screening of artificial bacterial flora strains a. Primary screening of microorganisms: Koji, sake mash, vinegar mash, etc. involved in the vinegar fermentation process were used as screening samples, and different media were used to screen for Lactobacillus acetotolerans, Acetobacter pasteurianus, Lactobacillus helveticus, Lactobacillus kunkeei, Lactobacillus fermentum, Streptococcus lactis, Lactobacillus plantarum, Bacillus amyloliquefaciens, and Pediococcus pentosaceus in the samples. Colonies that grew relatively quickly, had relatively large colonies, and were distributed at a relatively wide density but had different morphologies were selected, purified, and then preserved. b. Secondary screening of microorganisms: The pure culture microorganisms obtained by the screening were sequenced, and their fermentation performance and tolerance were compared with those of the same species. The microorganisms with better fermentation performance and tolerance (acid tolerance, temperature tolerance, alcohol tolerance) were designated as the dominant microorganisms of the same species. If no dominant microorganism was identified in this step, the screening and identification were repeated according to the microbial screening method.

[0046] In the case of acetic acid bacteria and lactic acid bacteria, fermentation performance refers to the ability of the strain to produce acetic acid and lactic acid, respectively. In the case of Bacillus, fermentation performance refers to the ability of the strain to produce amylase, protease, and cellulase (raw material degradation). In the first screening, a screening medium containing different substrates such as starch, cellulose, glucose, ethanol, or acetic acid is selected or set based on the fermentation performance. Microorganisms with properties such as the ability to hydrolyze starch or cellulose and convert glucose or ethanol to produce organic acids, alcohols, esters, and aldehydes in vinegar mash samples are screened.

[0047] Here, tolerance mainly refers to the tolerance of microorganisms to acetic acid, ethanol, and temperature. The solid vinegar fermentation medium was set up with different acetic acid gradients (0%, 1%, 2%, 3%, 4%), different ethanol gradients (0%, 2%, 4%, 6%, 8%), and different temperature gradients (30°C, 35°C, 40°C, 45°C, 50°C). After inoculation and cultivation, the growth of the microorganisms cultivated for 24 to 36 hours was measured by the plate count method.

[0048] Simulation of the composition of solid-state fermentation medium for vinegar: 30% rice bran, 10% rice husk, 1.2% glucose, 0.6% peptone, 0.6% beef paste, 0.3% yeast extract, 0.3% anhydrous sodium acetate, 0.06% Tween 80, 0.12% triammonium citrate, 0.12% dipotassium hydrogen phosphate, 0.035% magnesium sulfate, 0.015% manganese sulfate, and the remainder is water.

[0049] (3) Preparation of mixed rice bran koji a. Preparation of pure-bacterial rice bran koji: 450 g of rice bran was weighed, 360 mL of water was added, and the mixture was stirred uniformly. 100 g was dispensed into 500 mL Erlenmeyer flasks at 100 g per flask. After sterilization, the microbial culture solution obtained by screening, activating, and growing in step (2) was inoculated into the rice bran koji at 1% to 10%. The mixture was then cultured at 30 to 37°C for 60 to 65 hours and crushed to obtain rice bran koji. The culture was expanded to produce shallow disk-shaped koji, and the viable bacteria were counted. b. Preparation of mixed rice bran koji: Based on the results of metatranscriptome sequencing in Example 2, the average ratio of bacterial species throughout the fermentation stage (samples from each stage were mixed in equal amounts and then the ratio of bacterial species was measured) was used as the basis. Based on the calculation results for the pure rice bran koji in a, the pure rice bran koji was uniformly mixed in a ratio of Lactobacillus acetotolerans: Lactobacillus helveticus: Lactobacillus kunkeei: Lactobacillus fermentum: Acetobacter pasteurianus: Streptococcus lactis: Lactobacillus plantarum: Bacillus amyloliquefaciens: Pediococcus pentosaceus = 60:10:5:2:15:2:1:1:1 to obtain mixed rice bran koji.

[0050] (4) Use of mixed rice bran koji in the acetic acid fermentation process of grain vinegar a. Alcoholic fermentation: After crushing, the sorghum was soaked in warm water for 4-8 hours. It was liquefied at 90°C or higher for 30 minutes using a thermostable α-amylase at 0.1% of the sorghum mass. After the temperature was lowered to 60°C, saccharification was carried out at 58-60°C for 1 hour using a solid saccharifying enzyme at 2% of the sorghum mass. Next, crushed koji (62.5% of the sorghum mass) was added, water was replenished, and activated dry yeast was added. The sorghum was then placed in a sake bottle for alcoholic fermentation, with the first three days of open fermentation and the next four days of closed fermentation (7 days total). The fermentation temperature was maintained at 28-30°C. b. Use of mixed rice bran koji in acetic acid fermentation: After alcoholic fermentation was completed, rice bran and rice husk were added in a ratio of sake mash: rice bran: rice husk = 5:1.1:0.6, mixed evenly, and dispensed into vinegar bottles. 9 ~10 10 The mixed rice bran koji was added to the raw material at a concentration of cfu / kg (concentration of core microorganisms in the original sample) and repeatedly mixed uniformly with the moromi. The bottle neck was covered with a straw mat and the moromi was turned upside down (by turning the bottle upside down) every day. Fermentation was monitored and sampled, and stopped when the alcohol content dropped to 0.5% and the acidity and reducing sugars remained unchanged. Three parallel experimental groups were set up. c) Under the same conditions, solid-state fermentation of vinegar was carried out using conventional "seed mash" as a control, and the physical and chemical indicators of the vinegar mash at the end of fermentation were measured. The results are shown in Table 6.

[0051] Table 6: Physical and chemical indices of the artificial bacterial flora used for acetic acid fermentation of grain vinegar (unit: g / 100 g vinegar mash) JPEG0007796441000008.jpg53170

[0052] As can be seen from Table 6, compared to conventional starter mash, producing vinegar using an artificial bacterial flora shortened the fermentation period by 22.22%, and at the end of fermentation, non-volatile acids, reducing sugars, amino acid nitrogen, total esters, and raw material utilization rates increased by 33.33% to 41.95%, 35.77% to 39.43%, 15.79% to 21.05%, 20.87% to 25.51%, and 10.46% to 12.54%, respectively. Non-volatile acids / volatile acids increased by 49.89% to 61.13%, and the softness of the vinegar was improved.

[0053] Example 4: Use of a mixed bacterial agent using an artificial bacterial flora in the liquid fermentation process of fruit vinegar (1) Construction of an artificial microbial flora: Using naturally fermented grape vinegar as the research subject, an artificial microbial flora was constructed using the method described in Example 2. The core microbial flora was determined to be Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracase, Lactobacillus fermentum, and Acetobacter pasteurianus. Metatranscriptome sequencing revealed that the ratio of each species was Lactococcus lactis:Lactobacillus plantarum:Lactobacillus casei:Lactobacillus paracase:Lactobacillus fermentum:Acetobacter pasteurianus = 10:25:25:25:25:1.

[0054] (2) Screening of strains of artificial bacterial flora: Strains were screened by the method of Example 3.

[0055] (3) Preparation of mixed bacterial agent a. Preparation of pure bacterial preparations: The activated strains were inoculated into growth medium at an inoculum level of 1% to 10% and cultured until the stationary growth phase. Then, pure bacterial preparations of Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracase, Lactobacillus fermentum, and Acetobacter pasteurianus were prepared by spray freeze-drying. b. Preparation of mixed bacterial preparation: Viable cell count of pure bacterial preparation: Lactococcus lactis: Lactobacillus plantarum: Lactobacillus casei: Lactobacillus paracase: Lactobacillus fermentum: Acetobacter pasteurianus = 10 9 :2.5×10 9 :2.5×10 9 :2.5×10 9 :2.5×10 9 :10 8 The mixture was mixed in the ratio of 1 to 10 to obtain a mixed fungicide.

[0056] (4) Use of mixed microbial agents in the liquid fermentation process of grape vinegar a. Raw material pretreatment and alcoholic fermentation: Fresh grapes are destemmed and crushed, then 0.2% SO2 (potassium metabisulfite) is added and sterilized for 1 hour, followed by 0.1% pectinase (10,000 U / g enzyme activity) and enzymatic decomposition at 50°C for 4 hours. After cooling to room temperature, the sugar content is adjusted to 18°C, and finally activated dry yeast is added to the fermentation tank and fermented at 30°C. Alcoholic fermentation is complete when the specific gravity and sugar content no longer change (approximately 3 to 5 days). b. Acetic acid fermentation: Grape juice was used to reduce the original alcohol content to approximately 8%, and the feed volume to the fermentation tank was adjusted to 75%-80%, the aeration rate to 0.15 vvm, and the rotation speed to 2000 r / min. After the temperature reached 30°C, the dissolved oxygen electrode was calibrated to zero using saturated anhydrous sodium sulfite. The dissolved oxygen electrode was then inserted into the fermentation broth and calibrated to 100%. A 0.4% (v / w) mixed bacterial agent solution in water (0.4 g mixed bacterial agent added to 100 mL of raw material) was inoculated and monitored for sampling. Fermentation was stopped when the alcohol content fell below 0.5% and the acidity no longer increased. The number of microbial cells per 1 g of the bacterial agent was 10: Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracase, Lactobacillus fermentum, and Acetobacter pasteurianus. 9 :2.5×10 9 :2.5×10 9 :2.5×10 9 :2.5×10 9 :10 8 Three experimental groups were set up as parallel controls. c) Using the naturally fermented grape vinegar under the above conditions as a control, the physical and chemical indicators of the sample at the end of fermentation were measured. The results are shown in Table 7.

[0057] Table 7: Physical and chemical indices of the artificial flora used in the liquid fermentation of grape vinegar (unit: g / 100 mL) JPEG0007796441000009.jpg69170

[0058] The grape vinegar obtained by fermentation using the artificial bacterial flora had a purple-red appearance, a clear, glossy finish, a sweet and sour taste, a rich grapefruit aroma, and no off-flavors. As seen in Table 7, compared with the grape vinegar obtained by natural fermentation, the total acid content increased by 14.44%-16.14%, the nonvolatile acid content increased by 159.57%-176.60%, and the nonvolatile acid / volatile acid ratio increased by 155.87%-180.01%. The contents of acetic acid, lactic acid, malic acid, and total esters increased by 7.88%-10%, 282.61%-313.04%, and 33.33%-46.67%, respectively. The fermentation time was shortened by 53.33%, and the acetic acid formation rate increased by 70.21%-74.47%. The content of non-volatile acids, especially lactic acid, increased significantly, making the acidity of the grape vinegar softer and reducing the harsh acidity. At the same time, the addition of lactic acid bacteria increased the content of total esters, enhancing the richness of the fruit vinegar and improving the quality of fresh grape vinegar.

[0059] The above examples merely represent some embodiments of the present invention, and although the descriptions are relatively specific and detailed, they do not limit the scope of the present invention. Those skilled in the art may make various modifications, combinations, and improvements to the above embodiments without departing from the spirit of the present invention, and all such modifications and improvements are within the scope of the present invention. Therefore, the scope of the present invention is defined by the claims.

Claims

1. A method for constructing an artificial bacterial consortium for vinegar fermentation, comprising the following steps (1) to (5): (1) Functional and species annotation of microbial communities: Vinegar mash samples from different stages of fermentation were collected, impurities were removed, and the total RNA of each microorganism was extracted. Metatranscriptome sequencing was performed, and functional and species annotation was performed on the sequencing results. (2) Removal of contaminants and pathogens: For microorganisms whose corresponding functions are annotated, removal of contaminants and pathogens; (3) Determining the initial core microorganisms: Based on the abundance of all microorganisms (N species) at each fermentation stage in the metatranscriptome results, the microorganisms are sorted in order of decreasing average abundance of each species throughout the fermentation process, and the first two species are combined into the first group, and one species is added and combined in turn, finally obtaining N-1 microbial groups; A cluster analysis is performed to calculate the similarity between each microbial group and the original microbial group at each stage using the longest distance method and Euclidean distance, and the minimum number of species required when the calculated similarities are all ≥ 90% are determined as the initial core microbial group; (4) Verification of metabolic activity of core microbial communities a. determining the major flavor substances and corresponding metabolic genes in samples at the end of fermentation; b. Calculate the ratio of the transcription expression level of the metabolic genes of the initial core microbial community to the transcription expression level of the metabolic genes of the original microorganisms at each fermentation stage; if the transcription rates of the main flavor metabolic genes at each stage are all ≧80%, proceed to the next step; if the transcription rates of the main flavor metabolic genes at a certain stage are <80%, add one species in order according to the abundance of the species in step (3) and repeat step (4) until the transcription rates of the main flavor metabolic genes at each stage are all ≧80%, thereby determining the core microbial community; Here, the term "major flavor substances" refers to the detection and analysis of the major flavor substance composition in a sample at the end of fermentation, whereby organic acids and amino acids with a TAV>1 are defined as major umami substances, and volatile aroma compounds with a ROAV>0.1 are defined as major aroma compounds, and both together constitute the major flavor substances of vinegar; and the determination of metabolic genes corresponding to the major flavor substances refers to the determination by comparing with a database and screening from the results of metatranscriptome sequencing; (5) Screening of artificial bacterial flora strains: Screening samples such as koji, sake mash, and vinegar mash based on the composition of the core microbial community to obtain strains with excellent performance, and then blending them at the average biomass ratio of the corresponding microorganisms in the core microbial community in the original fermentation samples at each stage to finally obtain the composition of the artificial bacterial flora. This method for constructing an artificial bacterial flora for vinegar fermentation involves the following steps: first screening the microorganisms in the vinegar mash sample using different selective media; second screening of the strains based on conditions such as acid tolerance, temperature tolerance, and alcohol tolerance; and finally verifying the fermentation function according to different experimental strains, thereby determining the optimal strain and finally determining the composition of the artificial bacterial flora.

2. The method for constructing an artificial bacterial flora for vinegar fermentation according to claim 1, characterized in that the microbial population and the original microbial population at each stage are successfully clustered and have a similarity of ≥ 90%.

3. The method for constructing an artificial bacterial flora for vinegar fermentation described in claim 1, characterized in that the transcriptional expression level of the main flavor substance metabolic genes in the core microbial group accounts for more than 85% of the transcriptional expression level of the main flavor substance metabolic genes in the original microorganisms at each fermentation stage.

Images