Bacterial co-culture metabolite for improving yield of bacteriocin and application of bacterial co-culture metabolite

Abstract

The invention discloses a bacterial co-culture metabolite for improving the yield of bacteriocin and application thereof, and is characterized in that the bacterial co-culture metabolite comprises at least one of L-chorolactone, N-acetyl aspartic acid, butyryl trihexyl citrate, N-acetyl-L-glutamic acid and ascorbic acid; the invention also provides an application of the bacterial co-culture metabolite in preparation of a fermentation medium for improving the yield of bacteriocin, and the fermentation medium is prepared by adding 0.3% by mass of L-chorolactone, 0.1% by mass of N-acetylaspartic acid, 0.1% by mass of butyryl trihexyl citrate, 0.1% by mass of L-aspartic acid, 0.1% by mass of N-acetylaspartic acid, 0.1% by mass of citric acid butyryl trihexyl ester, 0.1% by The culture medium is an MRS liquid culture medium containing 0.2% of N-acetyl-L-glutamic acid and 0.2% of ascorbic acid or an MRS liquid culture medium containing 0.1% of N-acetyl aspartic acid, 0.1% of L-chorolactone, 0.3% of ascorbic acid and 0.3% of butyryl trihexyl citrate in mass concentration. The culture medium has the advantages that the yield of bacteriocin of co-culture and single bacteria is increased.

Description

Technical Field

[0001] The present invention relates to a method for improving the yield of bacteriocin, in particular to a bacterial co-culture metabolite for improving the yield of bacteriocin and an application thereof. Background Art

[0002] Diseases caused by pathogenic and spoilage microorganisms are serious public health issues in my country. Antibiotics and food preservatives can play an effective role in prevention and control. Long-term intake of chemical preservatives in food can cause serious harm to the human body. Lactic acid bacteria bacteriocins are a class of peptides or proteins with antibacterial activity synthesized by bacterial ribosomes. Bacteriocins can inhibit the growth of various spoilage bacteria and pathogenic bacteria, and are widely used in food biological preservation and pharmaceutical industries, becoming an ideal substitute for antibiotics and chemical preservatives.

[0003] my country is rich in lactic acid bacteria resources, and most lactic acid bacteria produce bacteriocins. However, the yield of bacteriocins is low and the molecular weight is small, so it is difficult to separate and purify them to obtain pure proteins. Currently, Nisin and Pediocin PA-1 are mainly used in the market. Although there are many reports on bacteriocins at home and abroad, the key problem of low yield has not yet been solved. The synthesis of bacteriocins is regulated by quorum sensing (QS), and the communication between cells within and between species is affected by different induction signals. In addition to genetic engineering and changing fermentation conditions, co-culture of closely related lactic acid bacteria can effectively induce the production of bacteriocins, and the yield of bacteriocins is generally higher than that of non-closely related species. The related metabolites secreted by lactic acid bacteria during co-culture play a key role in the synthesis of bacteriocins. At present, there are no relevant research reports on bacterial co-culture metabolites and their application in increasing bacteriocin yield. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a bacterial co-culture metabolite for increasing bacteriocin production and its application in preparing a fermentation medium for increasing bacteriocin production.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a bacterial co-culture metabolite for increasing the yield of bacteriocin, characterized by comprising at least one of L-gulolactone, N-acetylaspartic acid, butyryl trihexyl citrate, N-acetyl-L-glutamic acid and ascorbic acid.

[0006] Preferably, the bacterial co-culture metabolites consist of L-gulolactone, N-acetylaspartic acid, butyryl trihexyl citrate, N-acetyl-L-glutamic acid and ascorbic acid.

[0007] Preferably, the bacterial co-culture metabolites consist of L-gulolactone, N-acetylaspartic acid, butyryl trihexyl citrate and ascorbic acid.

[0008] Furthermore, the bacteria mentioned above are Lactobacillus plantarum and Lactobacillus mucilaginosus fermentans.

[0009] The present invention also provides the use of the bacterial co-culture metabolites in preparing a fermentation medium for increasing the yield of bacteriocin.

[0010] Preferably, the fermentation medium is an MRS liquid medium supplemented with 0.3% L-gulolactone, 0.1% N-acetylaspartic acid, 0.1% butyryl trihexyl citrate, 0.2% N-acetyl-L-glutamic acid and 0.2% ascorbic acid, which increases the diameter of the co-culture inhibition zone of the two lactic acid bacteria by 2.15 times.

[0011] Preferably, the fermentation medium is an MRS liquid medium added with 0.1% N-acetylaspartic acid, 0.1% L-gulolactone, 0.3% ascorbic acid and 0.3% butyryl trihexyl citrate, which can increase the diameter of the inhibition zone of Lactobacillus plantarum fermentation liquid by 1.9 times.

[0012] Compared with the prior art, the advantages of the present invention are as follows: the present invention discloses a bacterial co-culture metabolite for increasing bacteriocin production and its application, the differential metabolite pure product obtained by metabolomics analysis screening has low cost and high safety, the addition of five metabolites can effectively stimulate the enzymes that regulate, process and release bacteriocins, and can also enhance the absorption of nutrients by cells, thereby promoting the synthesis of co-cultured and single bacterial bacteriocins and increasing bacteriocin production. This method helps to solve the problem of low bacteriocin production, provides theoretical guidance for mixed fermentation of lactic acid bacteria, promotes the application of bacteriocin-producing lactic acid bacteria, and ensures people's health.

[0013] The above-mentioned Lactobacillus plantarum ( Lactiplantibacillus plantarum ) ZY-1 strain, with the deposit number CGMCC NO. 29542, was deposited on January 8, 2024 at the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0014] The above fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) RC4 strain, with the deposit number CGMCC NO. 30124, was deposited on March 25, 2024 at the General Microbiology Center of China Culture Collection Administration, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The results of antibacterial activity test of co-culture group, non-contact co-culture group and control group under different seed age conditions. Note: Groups 1, 2, 3, and 4 represent samples of seed age 6, 10, 16, and 20 h, respectively; a, b, and c in the figure have no specific meanings, and are only used to distinguish whether they are the same. Different letters are marked on the columns of each group. There are significant differences between the values ​​(P<0.05), otherwise there are no significant differences; Figure 2 Genes related to bacteriocin synthesis in co-culture group, non-contact co-culture group and single-bacteria culture group under different seed age conditions plnE / F and plnJ The expression results of A are the fermentation broth of 6 h old, B is the fermentation broth of 10 h old, C is the fermentation broth of 16 h old, and D is the fermentation broth of 20 h old. * is P < 0.05; Figure 3 The principal component analysis (PCA) results of metabolomics of the co-culture group, non-contact co-culture group, and control group; Figure 4 Partial least squares discriminant analysis (PLS-DA) results of metabolomics of the co-culture group, non-contact co-culture group, and control group; Figure 5 Comparison of the number of differential metabolites between the co-culture group and the control group, the non-contact co-culture group and the control group, and the co-culture group and the non-contact co-culture group; Figure 6 The difference statistics and volcano plots of significantly different metabolites between the co-culture group and the control group, and the non-contact co-culture group and the control group, where A is the co-culture group and the control group, and B is the non-contact co-culture group and the control group; Figure 7 HMDB classification pie charts of different metabolites in the co-culture group and the control group, and the non-contact co-culture group and the control group; Figure 8 Filtered heat map analysis of the five key differential metabolites screened for metabolomics analysis in each group; Fig. 9 The average relative abundance of the five key differential metabolites screened for metabolomics analysis in each group, where A is butyryl trihexyl citrate, B is ascorbic acid, C is L-gulolactone, D is N-acetyl-L-glutamate, and E is N-acetylaspartate, * is P < 0.05, ** is P < 0.01, and *** is P < 0.001; Fig.10 The results of antibacterial assays with five different concentrations of differential metabolites added to the co-culture group; Fig.11 The results of antibacterial activity test of adding five different concentrations of differential metabolites to the single bacteria ZY-1 culture group; Fig.12This is a bar chart showing the antibacterial activity of different metabolites with different optimal concentrations added to the co-culture group. * indicates P < 0.05. Fig.13 This is a bar chart showing the antibacterial changes of the differential metabolites with different optimal concentrations added to the single bacteria ZY-1 group. In the figure, * indicates P<0.05. DETAILED DESCRIPTION

[0016] The present invention is further described in detail below with reference to the accompanying drawings.

[0017] Specific Example 1: Screening of bacterial co-culture metabolites.

[0018] 1. Preparation of fermentation broth: The Lactobacillus plantarum with the deposit number CGMCC NO. 29542 ( Lactiplantibacillus plantarum ) ZY-1 was inoculated into 100 ml of MRS liquid medium and cultured at 37°C for 16 hours for activation. Then, Lactobacillus plantarum seed liquids of different ages were obtained after culture at 37°C for 6, 10, 16, and 20 hours respectively. The fermented mucus lactobacillus (CGMCC NO. 30124) Limosilactobacillus fermentum ) RC4 was inoculated into 100 ml of MRS liquid medium and cultured at 37°C for 16 h for activation. Then, fermented mucus Lactobacillus seed liquids of different ages were obtained after culture at 37°C for 6, 10, 16, and 20 h, respectively; The plant lactobacillus seed liquid was inoculated into 30 ml of MRS liquid culture medium at an inoculation rate of 2% by volume, and the mixture was cultured at 37°C for 24 h to obtain the plant lactobacillus fermentation liquid; at the same time, the fermented mucinous Lactobacillus seed liquid was inoculated into 30 ml of MRS liquid culture medium at an inoculation rate of 2% by volume, and the mixture was cultured at 37°C for 24 h to obtain the fermented mucinous Lactobacillus fermentation liquid, and then 15 ml of the plant lactobacillus fermentation liquid and the fermented mucinous Lactobacillus fermentation liquid of the same age were respectively taken and mixed as the control group, recorded as ZY-1 (RC).

[0019] The seed liquid of Lactobacillus plantarum and the seed liquid of Lactobacillus fermentans of the same age were inoculated into 30 ml of MRS liquid culture medium at a volume ratio of 2%, and the fermentation liquid obtained by culturing at 37°C for 24 h was used as the co-culture group, recorded as ZY-1+RC.

[0020] Take the seed liquid of Lactobacillus plantarum and the seed liquid of Lactobacillus mucilaginosus of the same age, inoculate the seed liquid of Lactobacillus plantarum into the upper layer of the cell migration experiment chamber (transwell culture dish, cells cannot pass through the membrane but can exchange metabolites) at a volume ratio of 2%, and inoculate the seed liquid of Lactobacillus mucilaginosus into the lower layer of the above cell migration experiment chamber at a volume ratio of 2%. The fermentation liquid obtained by culturing at 37°C for 24h is used as the non-contact co-culture group, recorded as ZY-1+RC(T).

[0021] 2. Detection of antibacterial activity: Preparation of antibacterial plates: Escherichia coli ATCC 25922 was used as the indicator bacteria. 2 mL of activated Escherichia coli ATCC25922 seed solution was inoculated into 100 mL of LB broth. After shaking culture at 37°C for 10 h, the bacterial solution was diluted to 10 -2 First, pour a thin layer of sterilized LB agar on the culture dish. After solidification, lower the temperature to about 45-50°C. Mix 4.5 mL of sterilized LB soft agar with 0.5 mL of indicator bacteria solution, pour it on the agar layer to form a soft agar layer, and place it at 4°C for later use after solidification.

[0022] Fermentation broth treatment: centrifuge the fermentation broth of each group prepared in 1 above to obtain the supernatant (4 ℃, 8000 rpm, 10 min), freeze-dry each group of supernatant to obtain a concentrated crude product, then add 2 ml of sterile water to reconstitute the concentrated crude product, adjust the pH to 6.5-7.0 with 5 M NaOH solution, filter through a sterile 0.2 μm filter membrane to sterilize and remove impurities, add filtered and sterilized catalase solution to a concentration of 1 mg / mL, and incubate in a water bath at 37 ℃ for 2 h to obtain the fermentation supernatant that has been deacidified and dehydrogen peroxideed.

[0023] The antibacterial activity was determined by the Oxford cup agar well diffusion method: take the antibacterial plate, place a sterile Oxford cup in it, take 200 μL of the fermentation supernatant after removing acid and hydrogen peroxide, add it to the Oxford cup, place it in a 4℃ refrigerator for 4 hours, and measure the diameter of the inhibition zone after culturing at 37℃ for 10 hours. At the same time, add LB broth adjusted to pH 6.5-7.0 as a blank group. Each group has 3 parallels.

[0024] The results are as follows Figure 1 As shown, when the inoculum amount and other factors such as carbon source were controlled to be the same, the diameter of the inhibition zone of the co-culture group was the largest when the strain age was 16 hours, which was significantly larger than that of the control group and the non-contact co-culture group, indicating that the bacteriocin production of the co-culture group reached its peak at this time, and the maximum diameter of the inhibition zone was 18.20 ± 0.19 mm.

[0025] In summary, the optimal fermentation age was 16 h. After optimization, the diameter of the inhibition zone in the co-culture group increased from 14.18 ± 0.07 mm to 18.20 ± 0.19 mm, an increase of 1.28 times, and 5.28 mm higher than that in the control group.

[0026] 3. Bacteriocin-related genes plnE / F and plnJ Expression assay validation: Take the fermentation broth of each group prepared in 1 above, extract RNA according to the OMEGA bacterial RNA kit (Nanjing Novozyme Biotechnology Co., Ltd.), adjust the concentration of RNA extracted from each group to the same, and then reverse it into stable cDNA using a reverse transcription kit (Nanjing Novozyme Biotechnology Co., Ltd.), and then design important genes for bacteriocin synthesis plnE / F and plnJ Specific fluorescent quantitative primers were used for qPCR verification.

[0027] pln The nucleotide sequence of the forward primer for quantitative PCR amplification of gene E is shown in SEQ ID NO.1: 5'-ACAATATTCCAGGTTGCCGCAA-3', pln The nucleotide sequence of the reverse primer for fluorescent quantitative PCR amplification of the E gene is shown in SEQ ID NO.2: 5'-CCACGAATGCCTGCAACTGAA-3'.

[0028] pln The nucleotide sequence of the forward primer for F gene fluorescence quantitative PCR amplification is shown in SEQ ID NO.3: 5'-TCCTCGGAGAGCGCTAATGA-3', pln The nucleotide sequence of the reverse primer for fluorescent quantitative PCR amplification of the F gene is shown in SEQ ID NO.4: 5'-GGTGGCGTTTTCCATGCCTA-3'.

[0029] pln The nucleotide sequence of the forward primer for fluorescent quantitative PCR amplification of the J gene is shown in SEQ ID NO.5: 5'-TGTAGTAGATGCATTTGCACCT-3', pln The nucleotide sequence of the reverse primer for fluorescent quantitative PCR amplification of the J gene is shown in SEQ ID NO.6: 5'-TTAACGACGGATTGCTCTGC-3'.

[0030] pln The nucleotide sequence of the forward primer for K gene fluorescence quantitative PCR amplification is shown in SEQ ID NO.7: 5'-AGAATTAACTGCTGACGCTGAA-3', pln The nucleotide sequence of the reverse primer for fluorescent quantitative PCR amplification of the K gene is shown in SEQ ID NO.8: 5'-CTTGAACCACCAAGCACGG-3'.

[0031] The nucleotide sequence of the forward primer for 16S rDNA fluorescence quantitative PCR amplification is shown in SEQ ID NO.9: 5'-GGCGTGCTATTCATACCAGTC-3', and the nucleotide sequence of the reverse primer for 16S rDNA fluorescence quantitative PCR amplification is shown in SEQ ID NO.10: 5'-CAGGTGTTATCCCGTGCTTC-3'.

[0032] The SYBR Green method was used, and the thermal cycling protocol included denaturation at 94°C for 30 seconds, followed by annealing for 30 seconds, and amplification and extension for 30 cycles. Finally, the relative expression level of each related gene was calculated according to the -2^ΔΔCt method.

[0033] The results are as follows Figure 2 As shown, Figure 2 A in the middle represents the expression levels of bacteriocin-related genes in each group at 6 h of seed age. Compared with the control group, the expression levels in the co-culture group and the non-contact co-culture group were upregulated, and the expression level in the co-culture group was the highest; Figure 2 B in the middle represents the expression levels of bacteriocin-related genes in each group at 10 h of seed age. Compared with the control group, the expression levels in the co-culture group and the non-contact co-culture group were similar to those at 6 h, and the expression level in the co-culture group was the highest; Figure 2 C in the middle represents the expression level of bacteriocin-related genes in each group at 16h of incubation. Compared with the control group, the expression level of bacteriocin-related genes in the co-culture group plnE / F and plnJ The expression level was significantly upregulated, with the highest increase being 6.8 times; Figure 2 D in the middle represents the expression levels of bacteriocin-related genes in each group at 20 h of seed age. Compared with the control group, the expression levels in the co-culture group and the non-contact co-culture group were also upregulated, but the expression level in the co-culture group was higher than that in the non-contact co-culture group, but lower than that at 16 h.

[0034] In summary, the bacteriocin-related genes in the co-culture group plnE / F and plnJ The expression level was higher than that of the control group and the non-contact co-culture group, and the highest expression level was at 16h of seed age, which was significantly upregulated compared with the control group. F The expression trend of bacteriocin-related genes was consistent with the measurement results of the inhibition zone.

[0035] 4. Metabolomics analysis to screen key differential metabolites, including the following steps: Step 1. Preparation of samples to be tested: The best seed age seed solution was cultured for 24 hours according to method 1 to obtain three groups of fermentation broth, namely the control group ZY-1 (RC4), the co-culture group ZY-1 + RC4 and the non-contact co-culture group ZY-1 + RC4 (T). The fermentation broth was centrifuged at 8,000 × g for 10 minutes at 4°C, and the bacteria were discarded and filtered through a 0.22 μm sterile filter. 100 μL of the supernatant was taken, and then 400 μL of cold methanol (-20°C) was added and vortexed at maximum speed for 1 minute. The supernatant was then concentrated by vacuum freeze drying and resuspended in 150 μL of 80% methanol solution containing 0.04 g / L 2-chlorobenzylamine, and the sample to be tested was obtained through a 0.22 μm filter membrane and evaluated by LC-MS / MS.

[0036] Step 2. Metabolomics analysis: Chromatographic separation was performed using a Thermo UltiMate 3000 system and a Thermo Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific, Austin, TX, USA). Samples were analyzed in positive and negative ion modes using an ACQUITY UPLC® HSS T3 column (150 × 2.1 mm, 1.8 μm, Waters Corporation, MA, USA). The column temperature was maintained at 40 °C, the mobile phase flow rate was 0.25 mL / min; the injection volume was 2.0 μL. The mobile phase was 0.1% formic acid aqueous solution or ammonium formate aqueous solution as phase A, and acetonitrile solution as phase B. The program was 0-0.5 min, 2% B (v / v); 0.5-7 min, 2%-50% B (v / v); 7-9 min, 40%-95% B (v / v); 9-12 min, 50%-98% B (v / v); 12-13.5 min, 98% B (v / v); 13.5 ~ 14 min, 98% ~ 2% B (v / v); 14-20 min, 2% B (v / v). For mass spectrometry data acquisition, a Thermo Q-Exactive Plus mass spectrometer equipped with an electrospray ionization source (ESI) was used. To obtain more detailed information of fragment ions and metabolites, all data were acquired in MSE mode with the following parameters: full scan resolution of 60,000, scan range of 70–1050 m / z, and normalized collision energy of 35 eV.

[0037] Step 3, screening of key differential metabolites: Multivariate statistical analysis was performed using the Bioconductor R software package (version 1.6.2) on the Majorbio cloud platform. The analysis included principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) to identify various metabolites. R² and Q² were used to evaluate the effectiveness of the model, ensure interpretability to prevent overfitting, and determine differential metabolites based on variable importance value (VIP) ≥ 1. The differential metabolites identified by LC-MS / MS were associated with multiple pathways, visualized using heat maps, and analyzed by metabolomics pathway analysis. The KEGG database was used to further elucidate the location and function of differential metabolites in various metabolic pathways.

[0038] The results are shown in Figure 3. Analysis of the PCA score plot showed that PCA showed two principal components. PC1 explained 29.80% of the inter-group differences, while PC2 accounted for 18.70%. The co-culture group, non-contact co-culture group, and control group were located in different quadrants, and there were differences between the samples in the group and within the confidence interval, which was in line with expectations, indicating that there were significant differences in metabolism between the co-culture group, non-contact co-culture group, and control group under the optimal seed age conditions.

[0039] To optimize the separation between groups based on storage intervals, supervised partial least squares (PLS) analysis was used. Based on the traditional PLS regression model, partial least squares discriminant analysis (PLS-DA) was used to classify and distinguish the problems. The results are shown in Figure 4. The PLS-DA score plot shows significant differences between different categories, with component 1 explaining 34.3% of the variance and component 2 accounting for 22.4%. The differential metabolites representing the co-culture group, non-contact co-culture group, and control group showed different clustering patterns and were distributed in different quadrants, indicating that there were significant differences in the characteristics of the differential metabolites.

[0040] After analyzing the differences in samples from each group, differential metabolites were analyzed for the co-culture group, non-contact co-culture group, and control group according to the screening criteria for differential metabolites (VIP>1, P<0.05). Figure 5 As shown in Figure 2, compared with the control group, 577 significantly different metabolites were found in the co-culture experimental group and 585 significantly different metabolites were found in the non-contact co-culture experimental group. Figure 6 As shown, Figure 6 Middle A is the difference volcano plot between the co-culture group and the control group. Among the 577 differential metabolites, 261 metabolites were up-regulated and 316 metabolites were down-regulated. Figure 6Middle B is the difference volcano plot between the non-contact co-culture group and the control group. Among the 585 differential metabolites, 438 metabolites were up-regulated and 147 metabolites were down-regulated. Red represents up-regulated and blue represents down-regulated. Figure 6 The significance of the differential metabolites between the co-culture experimental group and the non-contact co-culture experimental group and the control group can be obtained, and the names of the differential metabolites can be obtained by combining the ion peak numbers analyzed by the metabolite cloud platform. Figure 7 As shown, it mainly includes organic acids and their derivatives, lipids and lipid-like molecules, vitamins, nucleic acids and antibiotics.

[0041] Lactobacillus plantarum ( Lactiplantibacillus plantarum ) ZY-1 and Lactobacillus mucilaginosus fermentum ( Limosilactobacillus fermentum The metabolites found in different amounts during the co-culture of Lactobacillus plantarum ZY-1 and RC4 were mainly involved in the metabolic pathways of alanine, aspartate and glutamate, ascorbic acid, histidine and ABC transporters. In the metabolic pathways of alanine, aspartate and glutamate, α-ketoglutarate, γ-aminobutyric acid and l-glutamine were the key metabolites. γ-aminobutyric acid is a non-protein amino acid that helps maintain the stability of intracellular pH. Glutamate plays an important role in protein metabolism, including the synthesis of bacteriocins in various organisms. Glutamine is a key hub in carbon and nitrogen metabolism in Lactobacillus plantarum ZY-1 cells and may be involved in the synthesis of substances required for bacteriocin production. Amino acid biosynthesis, aminoacyl-tRNA biosynthesis and ABC transporters are three important pathways for the synthesis of proteins and peptides. Changes in amino acid metabolism are crucial for the synthesis of bacteriocins because amino acids play a vital role in stress resistance, metabolic processes, redox energy generation and the regulation of intracellular pH. In addition, the addition of amino acids can also increase the production of bacteriocins. Amino acids also serve as substrates and perform various functions, such as inducing enzymes involved in the regulation, processing, and release of bacteriocins. ABC transporter proteins facilitate the passage of mature bacteriocins across the cytoplasmic membrane into the extracellular space. High expression of these proteins enhances the cellular uptake of nutrients, especially glucose, amino acids, and peptides, thereby promoting the synthesis of bacteriocins.

[0042] Differences in the abundance of organic acids observed in the co-culture system with the inducer compared to the monoculture. The production of organic acids by yeast fermentation is a result of chemical processes such as lactose fermentation, lipolysis, and proteolysis. Under acidic stress, Lactobacillus acidophilus NCFM significantly increased the accumulation of AI-2 and upregulated the luxS gene. In the co-culture system, the relative concentrations of ascorbic acid, N-acetyl-L-glutamate, trihexyl butyrate citrate, N-acetylaspartic acid, L-gulolactone, and histidine showed an increasing trend. In contrast, the relative concentrations of uric acid and tenuazonic acid decreased significantly. Ascorbic acid is a gluconolactone extracted from glucuronic acid and belongs to the water-soluble ketolactone, which contains two ionizable hydroxyl groups.

[0043] In summary, based on the results of metabolomics analysis and combined with the review of relevant literature, five key differential metabolites were screened and identified by comparing the different differential metabolites between the co-culture group and the control group, and the non-contact co-culture group and the control group. The metabolite names are ascorbic acid, N-acetyl-L-glutamic acid, butyryl trihexyl citrate, N-acetylaspartic acid and L-gulolactone. Their enrichment heat maps and response values ​​in the culture group, non-contact co-culture group and control group are shown in Figure 2. Figure 8 and Fig. 9 shown. Figure 8 The results showed that compared with the green control group, the five differential metabolites were detected at the highest levels in the co-culture group, and their expression levels were also higher in the non-contact co-culture group but lower than the co-culture group. Red represents upregulation and blue represents downregulation. Fig. 9 Middle A, Fig. 9 Middle B, Fig. 9 Middle C, Fig. 9 Middle D and Fig. 9 E in the figure are the response values ​​of butyryl trihexyl citrate, ascorbic acid, L-gulolactone, N-acetyl-L-glutamate and N-acetylaspartic acid in the co-culture group, non-contact co-culture group, control group and single bacteria ZY-1 culture group, respectively. Figure 8 Consistent.

[0044] Specific Example 2: Study on the effects of the five key differential metabolites screened in Specific Example 1 on co-culture and single bacterial bacteriocin production.

[0045] 1. Individual verification of key metabolites Activation of Lactobacillus plantarum under optimal seed age conditions ( Lactiplantibacillus plantarum ) ZY-1 and Lactobacillus mucilaginosus fermentum ( Limosilactobacillus fermentum )RC4, and obtained plant lactobacillus seed liquid and fermented mucus lactobacillus seed liquid respectively.

[0046] The seed liquid of Lactobacillus plantarum and the seed liquid of Lactobacillus fermentans were inoculated into 30 ml of MRS liquid culture medium containing a differential metabolite at a volume ratio of 2%, and the fermentation liquid obtained by culturing at 37°C for 24 hours was used as the co-culture group. The seed liquid of Lactobacillus plantarum was inoculated into 30 ml of MRS liquid culture medium containing a differential metabolite at a volume ratio of 2%, and the fermentation liquid obtained by culturing at 37°C for 24 hours was used as the single bacteria ZY-1 culture group. The differential metabolites were ascorbic acid, N-acetyl-L-glutamic acid, trihexyl citrate butyryl, N-acetylaspartic acid and L-gulolactone.

[0047] Take the fermentation broth of each group and centrifuge to obtain the supernatant (4 ℃, 8000 rpm, 10 min), filter it through a 0.22 μm sterile filter, freeze-dry the supernatant for 48 hours to obtain the concentrated crude product, reconstitute the concentrated crude product with 2 ml sterile water, adjust the pH to 6.5-7.0 with 5 M NaOH solution, and filter it through a sterile 0.2 μm filter membrane to remove bacteria and impurities, then add the filtered and sterilized catalase solution to the fermentation supernatant to a concentration of 1 mg / mL, and water bath at 37 ℃ for 2 hours. Take the antibacterial plate, place a sterile Oxford cup in it, take 200 μL of the supernatant that has been deacidified and dehydrogenated to the Oxford cup, place it in a 4 ℃ refrigerator for 4 hours, and measure the diameter of the inhibition zone after culturing at 37 ℃ for 10 hours. At the same time, add LB broth adjusted to pH 6.5-7.0 as a blank group. Each group has 3 parallels.

[0048] The results are as follows Fig.10As shown in the figure, different mass percentage concentration gradients (0.1% to 0.4%) of differential metabolites were added to the co-culture group, and 0% was the co-culture group without any metabolites, that is, the control group. After the addition of metabolites, the five curves all showed an upward trend, which shows that the addition of five differential metabolites at different concentrations can promote the increase of the diameter of the inhibition zone. This shows that these five differential metabolites can promote the synthesis of bacteriocins in the co-culture group to a certain extent. Among the five differential metabolites, L-gulolactone has the most significant effect on the production of bacteriocins. When the concentration of L-gulolactone is 0.3%, the diameter of the inhibition zone of the co-culture group reaches the maximum value, which is 1.8 times that of the control group. However, when the concentrations of N-acetyl-L-glutamic acid and N-acetylaspartic acid exceed 0.1% and 0.2%, respectively, the diameter of the inhibition zone of the co-culture group gradually decreases, indicating that the production of bacteriocins decreases. The reason may be that low concentrations of amino acids can act as inducers for enzymes involved in regulating, processing and releasing bacteriocins, thereby promoting the synthesis of bacteriocins. In contrast, as the concentration of amino acids increased, their decomposition would lead to increased lactic acid production, thereby inhibiting the growth of the microorganisms and the production of bacteriocin. Metabolomics analysis showed that the levels of all five metabolites were significantly higher in the co-culture group compared with the control group. In summary, the addition of these five metabolites under co-culture conditions could significantly promote the synthesis of bacteriocin, indicating that they play a vital role in the synthesis of bacteriocin during the co-culture of Lactobacillus plantarum ZY-1 and Lactobacillus fermentans RC4.

[0049] The results are as follows Fig.11 As shown in the figure, the antibacterial activity of differential metabolites with different mass percentage gradients (0.1% to 0.4%) was added to the single culture group of Lactobacillus plantarum ZY-1. The results showed that 0% was the single bacterial culture group without any metabolites added, that is, the control group, and the diameter of the inhibition zone was 13.21 mm. Compared with the control group, after adding different concentrations of N-acetyl-L-glutamic acid, there was no significant difference in the diameter of the inhibition zone of the single bacterial ZY-1 culture group, which indicated that the metabolite N-acetyl-L-glutamic acid had little effect on the synthesis of bacteriocin by Lactobacillus plantarum ZY-1 and could not promote the synthesis of bacteriocin. In contrast, the change curves of the addition of the remaining four differential metabolites were generally higher than those of the control group, indicating that these four differential metabolites can promote the synthesis of bacteriocin by Lactobacillus plantarum ZY-1. Among them, the concentration of 0.1% N-acetylaspartic acid had the most significant effect, and the diameter of the inhibition zone of the single bacterial culture group increased by 1.4 times compared with the control group.

[0050] 2. Mixed validation of key metabolites According to the individual validation results of key metabolites, the metabolites added to the co-culture group were ranked from high to low according to their influence: L-gulolactone, N-acetylaspartic acid, butyryl trihexyl citrate, N-acetyl-L-glutamic acid, and ascorbic acid. Their optimal mass percentage concentrations were 0.3%, 0.1%, 0.1%, 0.2%, and 0.2%, respectively. In the single-bacteria ZY-1 culture group, after removing N-acetyl-L-glutamic acid, which had no promoting effect, the influence of the differential metabolites was ranked as follows: N-acetylaspartic acid, L-gulolactone, ascorbic acid, and butyryl trihexyl citrate. Their optimal mass percentage concentrations were 0.1%, 0.1%, 0.3%, and 0.3%, respectively.

[0051] The best-aged Lactobacillus plantarum seed liquid and the best-aged Lactobacillus fermentative mucus seed liquid were inoculated at a volume ratio of 2% into 30 ml of MRS liquid culture medium containing 1-5 differential metabolites, and the fermented liquid was cultured at 37°C for 24 hours to obtain a co-culture group. The metabolite with the highest degree of influence was preferentially added in the co-culture group - L-gulolactone with a concentration of 0.3% was recorded as the added metabolite quantity group 1; L-gulolactone with a concentration of 0.3% and N-acetylaspartic acid with a concentration of 0.1% was recorded as the added metabolite quantity group 2; L-gulolactone with a concentration of 0.3%, N-acetylaspartic acid with a concentration of 0.1% and butyryl trihexyl citrate with a concentration of 0.1% was recorded as the added metabolite quantity group 3; L -gulolactone, 0.1% N-acetylaspartic acid, 0.1% butyryl trihexyl citrate and 0.2% N-acetyl-L-glutamic acid were recorded as the added metabolite quantity group 4; the added concentration of 0.3% L-gulolactone, 0.1% N-acetylaspartic acid, 0.1% butyryl trihexyl citrate, 0.2% N-acetyl-L-glutamic acid and 0.2% ascorbic acid was recorded as the added metabolite quantity group 5; the group without added metabolites was the control group, recorded as 0.

[0052] The metabolite mixing verification results of the co-culture group are as follows Fig.12 As shown in the figure, the diameter of the inhibition zone of the co-culture group increases with the increase in the number of key metabolites added, which indicates that the production of bacteriocin also gradually increases. This phenomenon shows that the increase in the number of simple metabolites can significantly promote the synthesis of bacteriocin during co-culture. When five differential metabolites were added, the diameter of the inhibition zone reached 34.12 mm, which was 1.9 times higher than that of the control group. The more differential metabolites there are, the stronger the interaction between them, which significantly increases the production of bacteriocin.

[0053] The Lactobacillus plantarum seed liquid was inoculated with an inoculum volume ratio of 2% into 30 ml of MRS liquid medium containing 1-4 differential metabolites, and the fermentation liquid obtained by culturing at 37°C for 24 hours was used as the single-bacterium ZY-1 culture group. The concentration of 0.1% N-acetylaspartic acid was recorded as the added metabolite quantity group 1; the concentration of 0.1% N-acetylaspartic acid and 0.1% L-gulolactone was recorded as the added metabolite quantity group 2; the concentration of 0.1% N-acetylaspartic acid, 0.1% L-gulolactone and 0.3% ascorbic acid was recorded as the added metabolite quantity group 3; the concentration of 0.1% N-acetylaspartic acid, 0.1% L-gulolactone, 0.3% ascorbic acid and 0.3% butyryl trihexyl citrate was recorded as the added metabolite quantity group 4, and the group without added metabolites was the control group, recorded as 0.

[0054] The metabolite mixing verification results of the single strain ZY-1 culture group are as follows Fig.13 As shown in the figure, the diameter of the inhibition zone of the single-bacteria ZY-1 culture group also increases with the increase in the number of key metabolites added. This phenomenon shows that the increase in the number of simple metabolites can not only significantly promote the synthesis of bacteriocins during co-culture, but also significantly promote the synthesis of bacteriocins during the culture of individual strains. When four different metabolites were added, the diameter of the inhibition zone reached 28.43 mm, which was 2.1 times higher than that of the control group. This provides a theoretical basis for the mechanism of action of metabolites in the synthesis of bacteriocins in other strains in the future.

[0055] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A bacterial co-culture metabolite for increasing bacteriocin production, characterized in that The invention comprises at least one of L-gulolactone, N-acetylaspartic acid, butyryl trihexyl citrate, N-acetyl-L-glutamic acid and ascorbic acid.

2. A bacterial co-culture metabolite according to claim 1, characterized in that It is composed of L-gulolactone, N-acetylaspartic acid, butyryl trihexyl citrate, N-acetyl-L-glutamic acid and ascorbic acid.

3. The bacterial co-culture metabolite for increasing bacteriocin production according to claim 1, characterized in that It is composed of L-gulolactone, N-acetylaspartic acid, butyryl trihexyl citrate and ascorbic acid.

4. The bacterial co-culture metabolite for increasing bacteriocin production according to claim 1, characterized in that The bacteria are plant lactobacillus and fermented mucus lactobacillus.

5. Use of the bacterial co-culture metabolite according to any one of claims 1 to 4 in preparing a fermentation medium for increasing bacteriocin production.

6. Use of the bacterial co-culture metabolite according to claim 5 in preparing a fermentation medium for increasing bacteriocin production, characterized in that: The fermentation medium is an MRS liquid medium added with 0.3% L-gulolactone, 0.1% N-acetylaspartic acid, 0.1% butyryl trihexyl citrate, 0.2% N-acetyl-L-glutamic acid and 0.2% ascorbic acid.

7. Use of the bacterial co-culture metabolite according to claim 5 in preparing a fermentation medium for increasing bacteriocin production, characterized in that: The fermentation medium is an MRS liquid medium added with 0.1% N-acetylaspartic acid, 0.1% L-gulolactone, 0.3% ascorbic acid and 0.3% butyryl trihexyl citrate.

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