Method for regulating and controlling growth of lactic acid bacteria

Abstract

The invention belongs to the technical field of microorganisms, and relates to a method for regulating and controlling growth of lactic acid bacteria. On one hand, the invention provides a method for mining a microbial growth metabolism regulation factor, and the method comprises the following steps: (1) analyzing the correlation between a target microorganism and other microorganisms in a fermentation system to obtain a correlation analysis result of the target microorganism and the other microorganisms; (2) on the basis of the correlation analysis result, screening microorganisms having a significant negative correlation relationship with the target microorganisms; and (3) performing function prediction on the biosynthetic gene cluster of the microorganism having the significant negative correlation with the target microorganism based on a biosynthetic gene cluster prediction technology, and mining the growth metabolism regulation factor of the target microorganism based on a prediction result. A new thought is provided for exploration and mining of growth metabolism regulation factors of microorganisms in a fermentation system.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a method for regulating the growth of lactic acid bacteria. Background Art

[0002] Lactic acid is produced by the metabolism of lactic acid bacteria during the liquor brewing process. Since lactic acid is a non-volatile acid, it accumulates continuously during the brewing process as the fermentation time increases, and directly affects the evolution of the microbial community and the direction of the fermentation process. Therefore, the regulation of lactic acid bacteria growth and organic acid metabolism in the brewing system is of great significance to the production of liquor. In fact, lactic acid plays an important role in the flavor of liquor and the microecological balance of the brewing microbial community structure. On the one hand, lactic acid is an important contributor to the sour and fragrant dimension of liquor. It directly gives liquor its sour and fragrant flavor characteristics, and can work together with other flavor substances in liquor to form the unique flavor quality of liquor. On the other hand, the presence of lactic acid establishes an acidic brewing environment that helps inhibit the growth of miscellaneous bacteria. Although acidity is crucial to maintaining product quality and promoting normal fermentation, however, too high acidity may lead to adverse consequences. For example, excessive lactic acid accumulation will affect the enrichment of wine-producing yeast, and ultimately affect the production and quality of liquor. In the fermentation process of Luzhou-flavor liquor, lactic acid bacteria multiply in large numbers at the beginning of fermentation. Excessive lactic acid will inhibit the growth of caproic acid bacteria and the content of caproic acid in the mash, causing the content of caproic acid and ethyl caproate to decrease, resulting in deterioration of liquor quality.

[0003] Acetilactobacillμsjinshanensis is a new genus of Lactobacillaceae, and it is the only representative of the genus. Studies have shown that Lactobacillμsjinshanensis is widely present in the traditional liquor brewing process in my country and maintains a high abundance. By regulating the growth and metabolism of Lactobacillμsjinshanensis during liquor brewing, lactic acid can be kept in an appropriate concentration range, which has important theoretical and practical significance for the production of liquor. Biosynthetic gene cluster (BGC) refers to a group of genes in an organism that encodes the synthesis of a complex natural product and can be used to predict biosynthetic ability. The development of BGC prediction technology provides a basis for the mining of microbial growth and metabolic regulatory factors. Summary of the invention

[0004] The purpose of the present invention is to provide a method for mining microbial growth metabolism regulatory factors and a method for regulating the growth of lactic acid bacteria.

[0005] According to a first aspect of the present invention, the present invention provides a method for mining microbial growth and metabolism regulatory factors, the method comprising the following steps:

[0006] (1) analyzing the correlation between the target microorganism and other microorganisms in the fermentation system to obtain a correlation analysis result between the target microorganism and the other microorganisms;

[0007] (2) Based on the correlation analysis results, screening microorganisms that are significantly negatively correlated with the target microorganism;

[0008] (3) Predicting the function of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism based on the biosynthetic gene cluster prediction technology, and mining the growth and metabolic regulatory factors of the target microorganism based on the prediction results.

[0009] In some embodiments of the present invention, in step (1), the method for analyzing the correlation between Lactobacillus jinshanensis and other microorganisms in the fermentation system comprises: analyzing based on the Spearman correlation analysis method.

[0010] In some embodiments of the present invention, in step (3), predicting the function of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism based on the biosynthetic gene cluster prediction technology comprises the following steps:

[0011] The microorganism that is significantly negatively correlated with the target microorganism is subjected to metagenomic sequencing to obtain metagenomic raw sequencing data; the raw sequencing data is subjected to quality control processing to obtain high-quality sequencing data; contigs are assembled for the high-quality sequencing data; metagenomic binning analysis is performed on the assembled contigs; functional annotation is performed on the obtained metagenomic assembled genome to obtain a function prediction result of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism.

[0012] In some embodiments of the present invention, the prediction of the function of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism based on the biosynthetic gene cluster prediction technology also includes: performing metagenomic binning analysis on the assembled contigs, reassembling and quality evaluating the obtained metagenomic assembled genome, and functionally annotating the metagenomic assembled genome after the quality evaluation.

[0013] In some embodiments of the present invention, mining the growth metabolic regulatory factors of the target microorganism based on the prediction results includes the following steps: based on the biosynthetic gene cluster function of the microorganism that is significantly negatively correlated with the target microorganism, analyzing the metabolites that may be synthesized by the microorganism that is significantly negatively correlated with the target microorganism, and the metabolites are the growth metabolic regulatory factors of the target microorganism.

[0014] In some embodiments of the present invention, the microorganism that is significantly negatively correlated with the target microorganism is Bacillus lysinicus.

[0015] In some embodiments of the present invention, the target microorganism is Lactobacillus aceticus.

[0016] In some embodiments of the present invention, the possible synthetic metabolite is β-propiolactone.

[0017] According to a second aspect of the present invention, the present invention provides an application of a growth metabolism regulatory factor mined by any method described in the first aspect of the present invention in regulating the growth of lactic acid bacteria and / or lactic acid metabolism.

[0018] In some embodiments of the present invention, the growth metabolism regulator is a metabolite of Bacillus lysinicus.

[0019] In some embodiments of the present invention, the growth metabolism regulator is β-propiolactone.

[0020] In some embodiments of the present invention, the lactic acid bacteria is Lactobacillus aceticus.

[0021] In some embodiments of the present invention, the added concentration of β-propiolactone is 0.01% or more compared to the total weight of the fermentation system. Specifically, the added concentration of β-propiolactone can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more compared to the total weight of the fermentation system, or a range consisting of any two of the above values. It should be noted that β-propiolactone is commonly used for sterilization of plasma and vaccines. It is very easy to hydrolyze without residue. It disappears after hydrolysis in a 37°C water bath for 2 hours. The hydrolysis product is non-toxic and harmless and can be safely used for the growth regulation of lactic acid bacteria and the regulation of lactic acid metabolism.

[0022] According to the third aspect of the present invention, the present invention further provides a method for regulating the growth of lactic acid bacteria, wherein the method uses β-propiolactone, a metabolite of Bacillus lysinicus, as a regulatory factor to regulate the growth of lactic acid bacteria.

[0023] In some embodiments of the invention, the method comprises:

[0024] After adding β-propiolactone to the fermentation system, lactic acid bacteria seed solution is introduced to perform anaerobic fermentation, thereby regulating the growth of the lactic acid bacteria during the fermentation process; and / or,

[0025] After the lactic acid bacteria grow to the logarithmic growth phase, β-propiolactone is added to the fermentation system containing the lactic acid bacteria to carry out anaerobic fermentation, thereby achieving regulation of the growth of the lactic acid bacteria during the fermentation process.

[0026] In some embodiments of the present invention, the lactic acid bacteria is Lactobacillus aceticus.

[0027] In some embodiments of the present invention, the added concentration of β-propiolactone is 0.01% or more compared to the total weight of the fermentation system. Specifically, the added concentration of β-propiolactone can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more compared to the total volume of the fermentation system, or a range consisting of any two of the above values.

[0028] In some embodiments of the present invention, the temperature condition of the anaerobic fermentation is 28-35° C. Specifically, the temperature condition of the anaerobic fermentation can be 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C. or a range consisting of any two of the above values.

[0029] In some embodiments of the present invention, the inoculation amount of the Jinshan Lactobacillus acetic acid is 5-15% of the total weight of the fermentation system. Specifically, the inoculation amount of the Jinshan Lactobacillus acetic acid can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more of the range consisting of any two of the above values ​​compared to the total weight of the fermentation system.

[0030] In some embodiments of the present invention, the fermentation system includes a culture medium for culturing lactic acid bacteria.

[0031] In some embodiments of the present invention, the formula of the culture medium includes the following components: tryptone 8-15.0 g / L, anhydrous sodium acetate 8-15.0 g / L, cysteine ​​0.5-1.5 g / L, beef extract 1.5-2.5 g / L, saline 80-120 mL / L, acetic acid 8-15 g / L, glucose 15-25 g / L, diammonium hydrogen citrate 1.5-2.5 g / L, yeast extract 4.5-5.5 g / L, Tween 80 0.5-1.5 mL / L, fermented grains extract 300-400 mL / L, lactic acid 25-35 g / L, and balance water.

[0032] In some embodiments of the present invention, the method for extracting the fermented grains extract comprises: mixing the fermented grains and water in a weight ratio of 1:1.5-3, shaking for 5-15 minutes, ultrasonicating for 5-15 minutes, filtering with gauze, centrifuging for 10-20 minutes at a speed of 8000-12000 rpm, and taking the supernatant as the fermented grains extract.

[0033] In some embodiments of the present invention, the salt solution includes the following components: KH2PO4 0.3-0.7 g / L, K2HPO4 0.3-0.7 g / L, FeSO4·7H2O 0.005-0.015 g / L, MgSO4·7H2O 0.05-0.3 g / L, MnSO4·H2O 0.05-0.1 g / L, and the solvent is water.

[0034] According to a fourth aspect of the present invention, the present invention further provides a method for regulating lactic acid metabolism of lactic acid bacteria, wherein the method utilizes β-propiolactone, a metabolite of Bacillus lysinicus, as a regulatory factor to regulate the lactic acid metabolism of lactic acid bacteria.

[0035] In some embodiments of the invention, the method comprises:

[0036] After adding β-propiolactone to the fermentation system, lactic acid bacteria seed solution is introduced to perform anaerobic fermentation, thereby regulating the lactic acid metabolism of lactic acid bacteria during the fermentation process; and / or,

[0037] After the lactic acid bacteria grow to the logarithmic growth phase, β-propiolactone is added to the fermentation system containing the lactic acid bacteria to carry out anaerobic fermentation, thereby achieving regulation of the lactic acid metabolism of the lactic acid bacteria during the fermentation process.

[0038] In some embodiments of the present invention, the lactic acid bacteria is Lactobacillus aceticus.

[0039] In some embodiments of the present invention, the temperature condition of the anaerobic fermentation is 28-35° C. Specifically, the temperature condition of the anaerobic fermentation can be 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C. or a range consisting of any two of the above values.

[0040] In some embodiments of the present invention, the added concentration of β-propiolactone is 0.01% or more compared to the total weight of the fermentation system. Specifically, the added concentration of β-propiolactone can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more compared to the total weight of the fermentation system, or a range consisting of any two of the above values.

[0041] In some embodiments of the present invention, the inoculation amount of the Jinshan Lactobacillus acetic acid is 5-15% of the total weight of the fermentation system. Specifically, the inoculation amount of the Jinshan Lactobacillus acetic acid can be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more of the range consisting of any two of the above values ​​compared to the total weight of the fermentation system.

[0042] In some embodiments of the present invention, the fermentation system includes a culture medium for culturing lactic acid bacteria.

[0043] In some embodiments of the present invention, the formula of the culture medium includes the following components: tryptone 8-15.0 g / L, anhydrous sodium acetate 8-15.0 g / L, cysteine ​​0.5-1.5 g / L, beef extract 1.5-2.5 g / L, saline 80-120 mL / L, acetic acid 8-15 g / L, glucose 15-25 g / L, diammonium hydrogen citrate 1.5-2.5 g / L, yeast extract 4.5-5.5 g / L, Tween 80 0.5-1.5 mL / L, fermented grains extract 300-400 mL / L, lactic acid 25-35 g / L, and balance water.

[0044] In some embodiments of the present invention, the method for extracting the fermented grains extract comprises: mixing the fermented grains and water in a weight ratio of 1:1.5-3, shaking for 5-15 minutes, ultrasonicating for 5-15 minutes, filtering with gauze, centrifuging for 10-20 minutes at a speed of 8000-12000 rpm, and taking the supernatant as the fermented grains extract.

[0045] In some embodiments of the present invention, the salt solution includes the following components: KH2PO4 0.3-0.7 g / L, K2HPO4 0.3-0.7 g / L, FeSO4·7H2O 0.005-0.015 g / L, MgSO4·7H2O 0.05-0.3 g / L, MnSO4·H2O 0.05-0.1 g / L, and the solvent is water.

[0046] In summary, the present application includes at least one of the following beneficial technical effects:

[0047] The present invention provides a method for mining microbial growth and metabolism regulatory factors based on metagenomic data, clarifies the interaction relationship between microorganisms in the community through correlation analysis, splices the whole genome of the target strain, and predicts and mines a certain substance that can affect the growth and metabolism of microorganisms through biosynthetic gene clusters (BGC), which provides a new idea for the exploration and mining of the growth regulation and growth influencing factors of microorganisms in the system. As described in the embodiment of the present invention, based on the method provided by the present invention, with Jinshan Acetobacterium aceticum as the target microorganism, it is found through correlation analysis that Lysinibacillus and Jinshan Acetobacterium aceticum are significantly negatively correlated, the whole genome of Lysinibacillus is spliced, and it is predicted through biosynthetic gene clusters (BGC) that Lysinibacillus may affect the growth and metabolism of Jinshan Acetobacterium aceticum through synthesized β-propiolactone, and finally after experimental verification, it is predicted that the metabolite β-propiolactone of Lysinibacillus aceticum has a significant effect on the growth and metabolism of Jinshan Acetobacterium aceticum, further characterizing the reliability and accuracy of the mining method. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The results of the Spearman correlation analysis of the relationship between microbial communities in the fermentation system in Example 1 of the present invention;

[0049] Figure 2 The predicted result of the biosynthetic gene cluster (BGC) of Lysinibacillus in Example 1 of the present invention;

[0050] Figure 3 The predicted result of the biosynthetic gene cluster (BGC) of Lysinibacillus in Example 1 of the present invention;

[0051] Figure 4 The results of the effects of different concentrations of β-propiolactone addition conditions (0%, 5%, 1%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.001%) on the growth of Lactobacillus aceticus in Example 2 of the present invention are shown;

[0052] Figure 5 The results of the effects of different concentrations of β-propiolactone addition (0.01%, 0.05%, 0.1%) on the growth of Lactobacillus aceticus in Example 3 of the present invention are shown;

[0053] Figure 6 The results of the effects of different concentrations of β-propiolactone added (0.01%, 0.05%, 0.1%) on lactic acid metabolism in Example 3 of the present invention are shown;

[0054] Figure 7 The results of the effects of different concentrations of β-propiolactone addition (0.01%, 0.05%, 0.1%) on the growth of Lactobacillus aceticus in Example 4 of the present invention are shown;

[0055] Figure 8 The results of the effects of different concentrations of β-propiolactone added (0.01%, 0.05%, 0.1%) on lactic acid metabolism in Example 4 of the present invention are shown;

[0056] Fig. 9 The results are as follows: the effects of potential substances (γ-valerolactone, caprolactone, phenylethyl alcohol, roxithromycin, sodium diacetate, 2-methyl-1-butanol, tetracycline, genistein, and vanillin) in Comparative Example 1 of the present invention on the growth of Lactobacillus aceticus in Jinshan;

[0057] Fig.10 The results are the effects of different concentrations (0.1%, 0.01%) of potential substances (γ-valerolactone, caprolactone, roxithromycin, phenylethyl alcohol) in comparative example 1 of the present invention on the growth of Lactobacillus aceticus in Jinshan;

[0058] Fig.11 The results are the effects of adding 0.05% β-propiolactone on the growth of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bread, and Lactobacillus plantarum seed liquid under the condition of comparative example 2 of the present invention. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0060] Example 1

[0061] This embodiment provides a method for mining microbial growth and metabolism regulatory factors, comprising the following steps:

[0062] 1. Microbial correlation analysis

[0063] Microbial communities always interact with each other. Studies have shown that Bacillus can inhibit the growth of other microorganisms through its metabolites. However, there are many types of Bacillus, and some Bacillus are difficult to separate. It is unknown which type of Bacillus can have a regulatory effect on the target strain. Based on this, this embodiment analyzes the interaction relationship between microbial communities in the fermentation system based on Spearman correlation. Specifically, taking Jinshan Lactobacillus acetate as the target microorganism, the correlation between microorganisms in the fermentation system is analyzed based on the abundance information of each microorganism in the fermentation system through the Spearman correlation analysis method. The results are as follows: Figure 1 As shown, according to Figure 1 The results shown in the figure show that there is a significant negative correlation between Lysinibacillus and Lactobacillus aceticus in the microbial community. The possible reason is that Lysinibacillus and Lactobacillus aceticus inhibit each other's growth metabolism.

[0064] 2. Biosynthetic Gene Cluster (BGC) Prediction

[0065] After clarifying the interaction relationship between Lysinibacillus and the target microorganism Jinshan Lactobacillus acetate based on the method of step one, the biosynthetic gene cluster function of the Lysinibacillus of the microorganism that is significantly negatively correlated with the target microorganism Jinshan Lactobacillus acetate is further predicted based on the biosynthetic gene cluster technology, and the metabolites synthesized by Lysinibacillus that have inhibitory and regulatory effects on Jinshan Lactobacillus acetate are further explored. The specific steps are as follows:

[0066] (1) Raw sequencing data acquisition and preprocessing: The raw metagenomic data of Bacillus lysinicus was obtained based on Illumina sequencing. FastQC (v0.11.6) was first used for evaluation, and then Trimmomatic (v0.38) was used to trim low-quality reads and adapters to eliminate reads less than 150 bp in length, adapters, leading or trailing bases with a Phred base quality (BQ) score of <20, and strings with an average BQ score of <25 for every five bases. After quality control, we obtained 8.64 Tbp of clean data, including 3.79 billion paired-end sequences;

[0067] (2) Contig assembly: MEGAHIT (v1.1.2) was used to assemble high-quality reads of each sample into contigs with the parameter “--min-contig-len 1000”, and other parameters were set as default;

[0068] (3) Metagenome binning analysis: 1) Using the “binning” module in MetaWRAP software, the contigs assembled based on step (3) were grouped into metagenome-assembled genomes (MAGs) using the maxbin2, metabat2, and concoct algorithms. The outputs of these three algorithms were integrated through MetaWRAP software to obtain an optimized set of MAGs. 2) Reassembly: To further improve the quality of MAGs, the “bin_refinement” module was used to evaluate the MAGs, and the “reassemble_bins” module was used to reassemble them. 3) Purification: The reassembled MAGs were further purified using the “bin_refinement” module in MetaWRAP software. 4) Quality assessment: The completeness and contamination of MAGs assembled by the maxbin2, metabat2, and concoct algorithms were evaluated using CheckM software to assess the quality of the binned MAGs. 5) Functional annotation of MAGs: The “metawrap” module in MetaWRAP software was used to evaluate the functional annotation of MAGs. The “annotate_bins” module calls Prokka software to annotate the functions of MAGs;

[0069] (4) Analysis of microbial metabolic potential: 1) Annotation and analysis of BGCs: For all obtained MAGs, antiSMASH (v6.0.0) with default settings was used to predict BGCs of contigs ≥5 kb in MAGs; 2) BGC classification: BGCs were also divided into eight categories using BiG-SCAPE (v1.1.5), namely: “PKSI”, “PKS-NPR_Hybrids”, “PKSothers”, “NRPS”, “RiPPs”, “Terpene”, “Saccharides” and “Orthers”.

[0070] The prediction results of the biosynthetic gene cluster (BGC) of Lysinibacillus obtained by the above method are as follows: Figure 2 , Figure 3 As shown, according to Figure 2 , Figure 3The results shown in the figure show that there are only two BGCs on the genome of Bacillus lysininus, one BGC (ctg43_3) is a gene cluster for synthesizing β-propiolactone (betalactone), and the other BGC (ctg43_6) is unknown. Based on the above results, it is further predicted that the metabolite β-propiolactone synthesized by Bacillus lysininus may have an effect on the growth and lactic acid metabolism of Lactobacillus jinshanensis, that is, the metabolite β-propiolactone synthesized by Bacillus lysininus is preliminarily confirmed as a growth and metabolic regulatory factor of Lactobacillus jinshanensis.

[0071] Example 2 Investigating the effect of β-propiolactone on the growth of Lactobacillus aceticus

[0072] Based on the factors predicted by the method of Example 1 that affect the growth and lactic acid metabolism of Lactobacillus jinshanensis, the metabolite β-propiolactone of Bacillus lysininensis affects the growth and lactic acid metabolism of Lactobacillus jinshanensis. Based on the prediction results of the biosynthetic gene cluster (BGC) of Bacillus lysininensis obtained in step 2 above, this example further verifies the effect of β-propiolactone, a metabolite of Bacillus lysininensis, on the growth and lactic acid metabolism of Lactobacillus jinshanensis, as follows:

[0073] (1) preparing Jinshan Lactobacillus acetate culture medium; wherein, the formula of Jinshan Lactobacillus acetate culture medium is shown in Table 1:

[0074] Table 1 Culture medium components

[0075]

[0076]

[0077] The extraction method of the fermented grains extract, a component in the Jinshan Lactobacillus acetate culture medium formula, is as follows: fermented grains and water (1:2), shake for 10 minutes, ultrasonicate for 10 minutes, filter through gauze, centrifuge for 15 minutes (10000 rpm), and freeze the supernatant for later use; the components of the salt solution, a component in the Jinshan Lactobacillus acetate culture medium formula, are shown in Table 2:

[0078] Table 2 Salt solution composition

[0079]

[0080] The specific preparation method of the Jinshan Lactobacillus acetate culture medium is to add culture medium components and salt solution in sequence, and add lactic acid and acetic acid after sterilization to obtain the Jinshan Lactobacillus acetate culture medium;

[0081] (2) Preparing Jinshan Lactobacillus acetate seed liquid: inoculating Jinshan Lactobacillus acetate into the Jinshan Lactobacillus acetate liquid culture medium prepared above for culturing to obtain Jinshan Lactobacillus acetate seed liquid;

[0082] (3) A series of β-propiolactone concentrations were set, namely: 0%, 5%, 1%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, and 0.001%, and the above different concentrations of β-propiolactone were added to the Jinshan Lactobacillus acetate culture medium. After adding β-propiolactone, the Jinshan Lactobacillus acetate seed liquid was inoculated at an inoculum of 10%, and anaerobically cultured at 30°C for 48 hours, and the bacterial OD was detected. 600 , characterize the bacterial concentration, and further investigate the effect of the above-mentioned control concentration on the growth of Lactobacillus aceticus Jinshanensis; among them, bacterial OD 600 The detection steps are as follows: centrifuge the centrifuge tube containing 2mL of bacterial solution at 12000r / min for 5min, discard the supernatant, add an equal volume of ultrapure water to resuspend the bacteria, repeat the above steps 3 times, and draw 200μL for bacterial concentration detection.

[0083] The results obtained according to the above verification method are as follows Figure 4 As shown, according to Figure 4 The results shown in the figure show that, in general, with the increase of β-propiolactone stress concentration, the OD of Lactobacillus jinshanensis increased. 600 Gradually decreased, the higher the β-propiolactone concentration, the more obvious the inhibitory effect on the strain. Specifically, the inhibition range was 2.56%-91.50%. β-propiolactone concentration above 0.03% could show strong inhibition, and the inhibition efficiency on Jinshan Lactobacillus acetate was higher than 83.37%. β-propiolactone concentration of 0.01% still had a slight inhibitory effect on the growth of Jinshan Lactobacillus acetate.

[0084] Example 3 Investigating the effect of β-propiolactone on the growth and lactic acid metabolism of Lactobacillus aceticus

[0085] Based on the inhibitory effect of different concentrations of β-propiolactone on the growth of Lactobacillus aceticus obtained in Example 2, this example further verifies the effect of β-propiolactone, a metabolite of Bacillus lysinicus, on the growth and lactic acid metabolism of Lactobacillus aceticus, as follows:

[0086] (1) Preparation of Jinshan Lactobacillus acetate seed solution: same as in Example 2;

[0087] (2) Preparation of Jinshan Lactobacillus acetate culture medium: same as in Example 2;

[0088] (3) A series of β-propiolactone concentrations were set, namely: 0.01%, 0.05%, and 0.1%, and the above different concentrations of β-propiolactone were added to the Jinshan Lactobacillus acetate culture medium. After adding β-propiolactone, the Jinshan Lactobacillus acetate seed liquid was inoculated at an inoculum of 10%, and anaerobically cultured at 30°C for 48 hours to detect the bacterial OD 600 and lactic acid concentration, and further investigated the effect of the above-mentioned regulated concentration of β-propiolactone on the growth and lactic acid production of Lactobacillus jinshanensis. 600 The detection steps are as follows: centrifuge a centrifuge tube containing 2 mL of bacterial liquid at 12000 r / min for 5 min, discard the supernatant, add an equal volume of ultrapure water to resuspend the bacteria, repeat the above steps 3 times, and take 200 μL for bacterial concentration detection; the detection steps of the lactic acid concentration of the fermentation broth are as follows: take 1 mL of fermentation broth and dilute it 50 times; take 2 mL of the diluted fermentation broth, centrifuge it at 6000 r / min for 5 min, filter it through a 0.22 μm filter membrane, and obtain the supernatant; transfer the supernatant obtained after filtration to a 2 mL sample bottle for sampling and analysis; Detection and analysis: Ultra high performance liquid chromatography was used to detect the lactic acid content of the fermentation broth in the sample bottle, wherein the detection and analysis conditions were as follows: AcquityHSST3 chromatographic column (100 mm×2.1 mm×1.8 μm) was used for separation; 0.02 mol / L potassium dihydrogen phosphate was used as the mobile phase, the flow rate was 0.1 mL / min, the injection volume was 1 μL, the diode array detector was used, and the detection wavelength was 208 nm; injection: the sample solution was injected with an automatic injector, and the chromatogram was recorded; data analysis: the lactic acid content was calculated based on the peak area in the chromatogram.

[0089] The results of the effect of the above-mentioned controlled concentration of β-propiolactone on the growth of Lactobacillus aceticus obtained by the above-mentioned verification method are as follows: Figure 5 As shown, according to Figure 5 The results shown are consistent with the inhibition results of Example 2. 0.05% and 0.1% β-propiolactone have obvious inhibitory effects on the growth of Lactobacillus jinshanensis, and 0.01% β-propiolactone has a slight inhibitory effect on the growth of Lactobacillus jinshanensis. At the same time, the results of the effects of the above-mentioned controlled concentrations of β-propiolactone on lactic acid metabolism obtained according to the above verification method are as follows Figure 6 As shown, according to Figure 6 The results show that the lactic acid accumulation corresponding to β-propiolactone concentrations of 0.01%, 0.05%, and 0.1% were 6.13 g / L, 1.57 g / L, and 1.63 g / L, respectively. Compared with the control, the lactic acid accumulation decreased by 44.60%, 85.79%, and 85.23%, respectively. Figure 5The results show that the growth regulation of Lactobacillus aceticus in Jinshan is consistent with that of β-propiolactone, and the higher the concentration of β-propiolactone, the stronger the inhibitory effect on lactic acid metabolism. In summary, the addition of β-propiolactone has a significant inhibitory and regulatory effect on the growth and lactic acid metabolism of Lactobacillus aceticus in Jinshan.

[0090] Example 4 Investigating the effect of β-propiolactone on the growth and lactic acid metabolism of Lactobacillus aceticus

[0091] Generally speaking, the regulation of microbial growth and metabolism mainly focuses on the lag phase and the logarithmic phase. In order to further verify the regulatory effect of β-propiolactone on the growth of Lactobacillus aceticus, this example uses different methods to explore the effects of β-propiolactone on the growth and lactic acid metabolism of Lactobacillus aceticus, as follows:

[0092] (1) Preparation of Jinshan Lactobacillus acetate seed solution: same as in Example 2;

[0093] (2) Preparation of Jinshan Lactobacillus acetate culture medium: same as in Example 2;

[0094] (3) A series of β-propiolactone concentrations were set, namely: 0.01%, 0.05%, and 0.1%, and the Jinshan Lactobacillus acetic acid seed liquid was first inoculated into the Jinshan Lactobacillus acetic acid culture medium at an inoculation rate of 10%. After the Jinshan Lactobacillus acetic acid grew to the logarithmic growth phase (cultured for about 24 hours), the above-mentioned different concentrations of β-propiolactone were added respectively, and anaerobically cultured at 30° C. At the culture time of 0 h, 12 h, 24 h, 30 h, 36 h, 42 h, 48 h, and 54 h, the OD of the bacteria in the fermentation liquid was detected accordingly. 600 and lactic acid concentration to characterize the bacterial concentration, and further investigate the effect of the above-mentioned regulated concentration of β-propiolactone on the growth and lactic acid production of Lactobacillus jinshanensis. 600 The detection method of and the detection method of the lactic acid concentration of the fermentation broth are the same as those in Example 3.

[0095] The results of the effects of the above-mentioned controlled concentration of β-propiolactone on the growth of Lactobacillus aceticus in Jinshan obtained by the above-mentioned research method are as follows: Figure 7 As shown, according to Figure 7The results shown in the figure are that 0.01%, 0.05% and 0.1% β-propiolactone were added after 24h of fermentation, i.e. when Lactobacillus jinshanensis grew to the logarithmic phase. In general, it is consistent with the results of Example 3 that as the concentration of β-propiolactone increases, the inhibitory effect on the growth of the strain becomes more obvious. After adding β-propiolactone, the addition of 0.01% β-propiolactone and the growth of CK both showed an upward trend, indicating that this concentration of β-propiolactone had a weak inhibitory effect on the growth of Lactobacillus aceticus in the logarithmic phase. After 48 hours, the growth of Lactobacillus aceticus in Jinshan entered a stable period, while the growth of Lactobacillus aceticus in Jinshan showed a significant downward trend under the stress of 0.05% and 0.1% β-propiolactone. At 54 hours, the addition of 0.01% β-propiolactone was consistent with the bacterial concentration of CK, and the bacterial concentration of the 0.05% treatment group was slightly higher than that of the 0.1% β-propiolactone treatment group. The inhibitory effects of 0.01%, 0.05%, and 0.1% β-propiolactone on Lactobacillus aceticus in Jinshan were 1.22%, 47.87%, and 50.49%, respectively. In summary, β-propiolactone can effectively inhibit and regulate the growth of Lactobacillus aceticus in the logarithmic growth period.

[0096] At the same time, the results of the effects of the above-mentioned regulated concentrations of β-propiolactone on lactic acid metabolism obtained according to the above-mentioned verification method are as follows Figure 8 As shown, according to Figure 8 The results shown in the figure show that the production of lactic acid is consistent with the trend of bacterial concentration changes. After the addition of β-propiolactone, the effects of low concentration (0.01%) and high concentration stress groups (0.05%, 0.1%) on the lactic acid metabolism of the strain showed significant differences. It is worth noting that the stress of β-propiolactone did not completely inhibit the lactic acid metabolism of the strain. The lactic acid concentration of the three stress treatments increased. In general, with the increase of β-propiolactone concentration, the inhibitory effect on the lactic acid metabolism of Lactobacillus aceticus in Jinshan became more obvious. The lactic acid production after low concentration β-propiolactone stress was significantly higher than that of the high concentration stress group. Compared with the control group, the lactic acid accumulation of the strain added with 0.01%, 0.05% and 0.1% β-propiolactone was reduced by 4.61%, 25% and 26.63%, respectively, indicating that β-propiolactone can inhibit the lactic acid metabolism of Lactobacillus aceticus in Jinshan during the logarithmic growth period.

[0097] Comparative Example 1

[0098] In order to further explore substances that have a regulatory effect on Lactobacillus aceticus Jinshan, a series of potential regulatory substances were further screened out by consulting a large amount of information, including γ-valerolactone, caprolactone, phenylethyl alcohol, roxithromycin, sodium diacetate, 2-methyl-1-butanol, tetracycline, genistein, vanillin, etc. Based on a similar method in Example 2, the above substances were used to explore the growth inhibition regulation and lactic acid metabolism of Lactobacillus aceticus Jinshan. The specific steps are as follows:

[0099] (1) Preparation of Jinshan Lactobacillus acetate seed solution: same as in Example 2;

[0100] (2) Preparation of Jinshan Lactobacillus acetate culture medium: same as in Example 2;

[0101] (3) The concentrations of the potential regulatory substances were set to 5% and 1%, respectively. The potential substances (γ-valerolactone, caprolactone, phenylethyl alcohol, roxithromycin, sodium diacetate, 2-methyl-1-butanol, tetracycline, genistein, and vanillin) at different concentrations (5% and 1%) were added to the Jinshan Lactobacillus acetate culture medium. After adding the above substances, the Jinshan Lactobacillus acetate seed liquid was inoculated at a 10% inoculation rate, and anaerobically cultured at 30°C for 48 hours to detect the bacterial OD. 600 , further investigate the inhibitory regulation of the above-mentioned potential substances on the growth of Lactobacillus aceticus; among them, the bacterial OD 600 The detection method is the same as in Example 3.

[0102] The results are as follows Fig. 9 As shown, according to Fig. 9 The results shown show that except for γ-valerolactone, caprolactone, roxithromycin and phenylethanol which have slight inhibitory effects, other substances have no antibacterial activity.

[0103] Based on the above research results, the antibacterial activity of lower concentrations (0.1%, 0.01%) of γ-valerolactone, caprolactone, roxithromycin, and phenylethanol against Lactobacillus aceticus was further investigated. Fig.10 As shown, according to Fig.10 The results shown show that γ-valerolactone, caprolactone, erythromycin and phenylethanol below 0.1% have no inhibitory activity on Lactobacillus jinshanensis. In summary, the growth metabolic regulatory factors of Lactobacillus jinshanensis are mined by the method provided by the present invention, and the growth metabolic regulatory factors of Lactobacillus jinshanensis can be regulated based on the growth metabolic regulatory factors obtained by mining.

[0104] Comparative Example 2

[0105] This comparative example explores the regulatory effect of β-propiolactone on other types of lactic acid bacteria (Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bread, Lactobacillus plantarum) in the fermentation system. The specific steps are as follows:

[0106] (1) MRS medium was prepared by adding 10 g of tryptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 1 mL of Tween 80, 0.5 g of magnesium sulfate, and 0.25 g of manganese sulfate to 1 L of deionized water. The pH was adjusted to 6.2-6.4 with 1 mol / L NaOH, and the medium was sterilized by steam sterilization at 121° C. under high pressure for 15 min to obtain MRS medium;

[0107] (2) preparing seed solutions of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bread, and Lactobacillus plantarum, respectively, by the specific steps of: adding the Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bread, and Lactobacillus plantarum to the above-mentioned MRS liquid culture medium for culturing to obtain seed solutions;

[0108] (3) The concentration of β-propiolactone was set to 0.05%, and β-propiolactone of the above concentration was added to the MRS medium. After adding the above concentration of substances to the MRS medium, the seed liquid of Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus bread, and Lactobacillus plantarum were inoculated at an inoculum rate of 10%, and the culture was anaerobically cultured at 30°C for 48 hours, and the bacterial OD was detected. 600 , further investigate the above-mentioned regulated concentration of β-propiolactone on the growth inhibition of other types of lactic acid bacteria in the fermentation system; among them, the bacterial OD 600 The detection method is the same as in Example 3.

[0109] The results are as follows Fig.11 As shown, according to Fig.11 The results show that 0.05% β-propiolactone has a slight inhibitory effect on Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus bread, and Lactobacillus plantarum, and has almost no inhibitory effect on Lactobacillus paracasei.

[0110] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A method for mining microbial growth and metabolic regulatory factors, characterized in that: The method comprises the following steps: (1) analyzing the correlation between the target microorganism and other microorganisms in the fermentation system to obtain a correlation analysis result between the target microorganism and the other microorganisms; (2) Based on the correlation analysis results, screening microorganisms that are significantly negatively correlated with the target microorganism; (3) Based on the biosynthetic gene cluster prediction technology, the function of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism is predicted, and the growth and metabolic regulatory factors of the target microorganism are mined based on the prediction results.

2. The method according to claim 1, characterized in that In step (3), the predicting of the function of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism based on the biosynthetic gene cluster prediction technology includes: Performing metagenomic sequencing on the microorganism that is significantly negatively correlated with the target microorganism to obtain metagenomic raw sequencing data; performing quality control processing on the raw sequencing data to obtain high-quality sequencing data; assembling contigs on the high-quality sequencing data; performing metagenomic binning analysis on the assembled contigs; performing functional annotation on the obtained metagenomic assembled genome to obtain a function prediction result of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism; Preferably, the prediction of the function of the biosynthetic gene cluster of the microorganism that is significantly negatively correlated with the target microorganism based on the biosynthetic gene cluster prediction technology also includes: after grouping the assembled contigs into metagenomic assembly genomes, reassembling and quality evaluating the obtained metagenomic assembly genomes, and functionally annotating the metagenomic assembly genomes after the quality evaluation.

3. The method according to claim 1, characterized in that The mining of the target microbial growth and metabolism regulatory factors based on the prediction results includes: Based on the biosynthetic gene cluster function of the microorganism that is significantly negatively correlated with the target microorganism, analyzing the metabolites that may be synthesized by the microorganism that is significantly negatively correlated with the target microorganism, wherein the metabolites are growth metabolic regulatory factors of the target microorganism; Preferably, the microorganism that is significantly negatively correlated with the target microorganism is Bacillus lysinicus; Preferably, the target microorganism is Lactobacillus acetate of Jinshan; Preferably, the possible synthesized metabolite is β-propiolactone.

4. Application of the growth metabolism regulatory factor mined by the method according to any one of claims 1 to 3 in regulating the growth of lactic acid bacteria and / or lactic acid metabolism.

5. The use according to claim 4, characterized in that The growth metabolism regulatory factor is a metabolite of Bacillus lysinicus; Preferably, the growth metabolism regulatory factor is β-propiolactone; The lactic acid bacteria is Lactobacillus aceticus.

6. The use according to claim 4, characterized in that: The added concentration of the β-propiolactone is 0.01% or more compared to the total weight of the fermentation system.

7. A method for regulating the growth of lactic acid bacteria, characterized in that: The method uses the metabolite beta-propiolactone of lysine bacillus as a regulating factor to regulate the growth of lactic acid bacteria.

8. The method according to claim 7, characterized in that The method comprises: After adding β-propiolactone to the fermentation system, lactic acid bacteria seed solution is introduced to perform anaerobic fermentation, thereby regulating the growth of the lactic acid bacteria during the fermentation process; and / or, After the lactic acid bacteria grow to a logarithmic growth phase, β-propiolactone is added to the fermentation system containing the lactic acid bacteria to perform anaerobic fermentation, thereby regulating the growth of the lactic acid bacteria during the fermentation process; Preferably, the lactic acid bacteria is Lactobacillus aceticus.

9. A method for regulating lactic acid metabolism of lactic acid bacteria, characterized in that: The method uses β-propiolactone, a metabolite of lysine Bacillus, as a regulatory factor to regulate the lactic acid metabolism of lactic acid bacteria; Preferably, the method comprises: After adding β-propiolactone to the fermentation system, lactic acid bacteria seed solution is introduced to perform anaerobic fermentation, thereby regulating the lactic acid metabolism of lactic acid bacteria during the fermentation process; and / or, After the lactic acid bacteria grow to a logarithmic growth phase, β-propiolactone is added to the fermentation system containing the lactic acid bacteria to perform anaerobic fermentation, thereby regulating the lactic acid metabolism of the lactic acid bacteria during the fermentation process; Preferably, the lactic acid bacteria is Lactobacillus aceticus.

10. The method according to any one of claims 7 to 9, characterized in that: The added concentration of β-propiolactone is 0.01% or more compared to the total weight of the fermentation system; preferably, the inoculation amount of the lactic acid bacteria is 5-15%; preferably, the fermentation system includes a culture medium for culturing lactic acid bacteria.

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