A strain of Lactobacillus brevis and its antibacterial optimized culture method and application

Abstract

The present invention belongs to the field of microbial technology, and discloses a strain of Lactobacillus brevis and its antibacterial optimization culture method and application. The strain is Lactobacillus brevis (Lactobacillus brevis) L1612, which has been deposited in Guangdong Provincial Microbial Culture Collection Center on August 4, 2022, with a deposit number of GDMCC 62642. The strain of the present invention has broad-spectrum, efficient antibacterial properties, has the potential to produce new bacteriocins, and is safe and non-resistant. It can be used in the preparation of intestinal antibacterial drugs and in functional foods containing lactic acid bacteria. The present invention also provides an antibacterial optimization culture method for the bacteria, which uses MRS culture medium as a basal culture medium, optimizes the composition ratio and culture conditions in the basal culture medium, and makes the diameter of the inhibition zone of the bacterial fermentation broth increase significantly, which is 25.21% higher than before optimization, thereby facilitating the bacteria to be widely used in the fields of antibacterial drugs and functional food production.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to a strain of Lactobacillus brevis L1612 having a broad-spectrum and highly effective antibacterial effect, being safe and non-resistant, and an antibacterial optimized culture method and application thereof. Background Art

[0002] Lactobacillus brevis, a member of the genus Lactobacillus, is a lactic acid heterofermentative microorganism widely found in nature. It plays important roles in human health, such as promoting nutrient digestion and absorption, lowering blood sugar and cholesterol, providing antioxidant benefits, enhancing immunity, and exerting antibacterial properties. Lactobacillus brevis has been shown to produce unique flavors and effectively degrade nitrites in food fermentation, leading to its frequent use in the fermentation, pharmaceutical, and food industries.

[0003] As a recognized GRAS (Generally Recognized As Safe) strain, Lactobacillus brevis has long been used in various traditional fermented foods. However, although O'Sullivan et al. and Collins et al. mention Lactobacillus brevis in their lists of strains currently used in probiotic products, it is still not included in the "List of Probiotic Species Acceptable for Food Use." Furthermore, the European Food Safety Authority (EFSA) recommends that antimicrobial resistance be a decisive factor in safety assessments of bacteria introduced into the food chain. Therefore, to verify the potential of a Lactobacillus brevis strain for food industry application, it must undergo a series of safety evaluation tests, including nitroreductase testing, indole testing, hemolytic testing, acute oral toxicity testing in animals, and antimicrobial resistance assessment.

[0004] Lactobacillus brevis is a dominant species in the normal microbial flora of the human and animal digestive tracts, playing a vital role in maintaining digestive microbial balance and normal digestive function. Numerous studies have confirmed that Lactobacillus brevis fermentation products contain antibacterial substances. With advances in clinical microbiology, Lactobacillus brevis has been increasingly used in live bacterial preparations internationally, demonstrating potent inhibitory effects against pathogens such as Staphylococcus aureus, Salmonella enteritidis, and Escherichia coli.

[0005] Optimizing fermentation strains using inhibition diameter as the primary indicator can improve the antibacterial capacity of fermentation broths, thereby saving costs, shortening production cycles, and improving production efficiency in the production of antibacterial drugs. Optimizing optimal culture formulations and conditions through response surface methodology is a key technology in the development of functional products and lays the foundation for industrialization and large-scale production. Summary of the Invention

[0006] The present invention aims to provide a Lactobacillus brevis L1612 strain and a method for optimizing its antibacterial culture, thereby providing a theoretical basis for the research and practical application of Lactobacillus brevis.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a strain of Lactobacillus brevis, which is Lactobacillus brevis L1612, which was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC for short) on August 4, 2022, with a deposit number of GDMCC62642.

[0009] The present invention also provides the above-mentioned bacteriostatic optimization culture method of Lactobacillus brevis, which is carried out according to the following steps:

[0010] (1) Lactobacillus brevis L1612 was inoculated into MRS culture medium for activation to obtain seed solution;

[0011] (2) Inoculate the seed solution into the antibacterial optimized culture medium and culture for 34 to 48 hours.

[0012] Preferably, the antibacterial optimized culture medium is formulated as follows: in parts by mass, 1 to 3 parts of carbon source, 0.4 to 2 parts of yeast extract powder, 0.1 to 0.5 parts of dipotassium hydrogen phosphate, 0.2 to 1 part of triammonium citrate, 0.3 to 1.1 parts of sodium acetate, 0.02 to 0.1 parts of magnesium sulfate heptahydrate, 0.005 to 0.025 parts of manganese sulfate tetrahydrate, 1 to 5 parts of beef extract powder, 1 to 5 parts of bacteriological peptone, 0.05 to 0.15 parts of Tween 80, and the volume is made up to 100 parts with distilled water.

[0013] Preferably, the antibacterial optimized culture medium formula is: in parts by mass, 2 parts of glucose, 1.2 parts of yeast extract powder, 0.24 parts of dipotassium hydrogen phosphate, 0.8 parts of triammonium citrate, 0.53 parts of sodium acetate, 0.04 parts of magnesium sulfate heptahydrate, 0.015 parts of manganese sulfate tetrahydrate, 4 parts of beef extract powder, 3 parts of bacteriological peptone, 0.108 parts of Tween 80, and the volume is made up to 100 parts with distilled water.

[0014] Preferably, the carbon source is one or more of sucrose, starch, glycerol, lactose and maltose.

[0015] Preferably, Lactobacillus brevis L1612 is inoculated into the antibacterial optimized culture medium at an inoculum volume ratio of 1% to 5%; the initial pH of the culture of Lactobacillus brevis L1612 in the antibacterial optimized culture medium is 4.0 to 7.0; and the culture temperature is 32 to 42°C.

[0016] Preferably, the Lactobacillus brevis L1612 is inoculated into the antibacterial optimized culture medium at an inoculum volume ratio of 4%; the initial pH of the culture of the Lactobacillus brevis L1612 in the antibacterial optimized culture medium is 5.0; and the culture temperature is 37°C.

[0017] The present invention provides the use of the Lactobacillus brevis in antibacterial application.

[0018] The invention provides a functional food containing the Lactobacillus brevis.

[0019] The invention provides an intestinal antibacterial drug containing the Lactobacillus brevis.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] (1) The present invention discloses a strain of Lactobacillus brevis L1612, which has been proven to be safe for consumption through safety experiments and drug resistance experiments; and has been proven to have broad-spectrum and highly effective antibacterial properties through antibacterial experiments, and has the potential to produce new bacteriocins.

[0022] (2) The present invention discloses for the first time the complete genome sequence of Lactobacillus brevis L1612. The complete genome sequence of the strain of the present invention was determined by combining third-generation ONT and second-generation Illumina technologies, and it contains one chromosome (2,518,301 bp) and two plasmids (59,967 bp and 46,081 bp). The complete genome sequence was submitted to the GenBank database of NCBI, and the chromosome and plasmid accession numbers are CP102751, CP102752, and CP102753, respectively.

[0023] (3) Under the antibacterial optimized culture medium and culture conditions disclosed in this invention, the diameter of the inhibition zone of the fermentation broth of Lactobacillus brevis L1612 increased significantly, by 25.21% compared to the pre-optimization level. This fermentation optimization method significantly improves the antibacterial ability of the fermentation broth of this strain, laying a foundation for the subsequent research and production of intestinal antibacterial drugs and lactic acid bacteria-containing functional foods using this strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : The bacterial morphology of Lactobacillus brevis L1612 after Gram staining in Example 1.

[0025] Figure 2 : Phylogenetic tree of Lactobacillus brevis L1612 strain in Example 1.

[0026] Figure 3 : The whole genome circle map of Lactobacillus brevis L1612 in Example 2.

[0027] Figure 4: The nitroreductase experimental results of Lactobacillus brevis L1612 in Example 4 are shown in the figure. The left test tube is the experimental group with the addition of the quality control strain Escherichia coli, the middle test tube is the experimental group with the addition of Lactobacillus brevis L1612, and the right test tube is the blank control group.

[0028] Figure 5 : Indole experiment result figure of Lactobacillus brevis L1612 in Example 4, the left test tube is the experimental group with the addition of quality control strain Escherichia coli, the middle test tube is the experimental group with the addition of Lactobacillus brevis L1612, and the right test tube is the blank control group.

[0029] Figure 6 : The growth of Lactobacillus brevis L1612 on Columbia blood agar plates in Example 4, (a) is the growth of the quality control strain Escherichia coli on blood agar plates; (b) is the growth of Lactobacillus brevis L1612 on blood agar plates.

[0030] Figure 7 : The antiSMAH secondary metabolism database prediction results of Lactobacillus brevis L1612 in Example 6.

[0031] Figure 8: Effects of different single factors on the diameter of the inhibition zone of the fermentation broth of Lactobacillus brevis L1612 in the antibacterial experiment in Example 7. (a) is the effect of different carbon sources (sucrose, starch, glycerol, lactose, maltose, glucose) on the diameter of the inhibition zone of the fermentation broth of Lactobacillus brevis L1612 in the antibacterial experiment; (b) is the effect of adding glucose with a mass fraction of 1%, 1.5%, 2%, 2.5%, and 3% on the diameter of the inhibition zone of the fermentation broth of Lactobacillus brevis L1612 in the antibacterial experiment; (c) is the effect of adding potassium dihydrogen phosphate with a mass fraction of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% on the diameter of the inhibition zone of the fermentation broth of Lactobacillus brevis L1612 in the antibacterial experiment. (d) is the effect of adding 0.3%, 0.5%, 0.7%, 0.9% and 1.1% of sodium acetate on the diameter of the inhibition zone of the fermentation broth of Lactobacillus brevis L1612 in the antibacterial experiment; (e) is the effect of adding 0.02%, 0.04%, 0.06%, 0.08% and 0.1% of magnesium sulfate heptahydrate on the diameter of the inhibition zone of the fermentation broth of Lactobacillus brevis L1612 in the antibacterial experiment; (f) is the effect of adding 0.005%, 0.01%, 0.015%, 0.02% and 0.025% of manganese sulfate tetrahydrate on the diameter of the inhibition zone of Lactobacillus brevis L1612 fermentation broth in the antibacterial experiment; (g) is the effect of adding 0.2%, 0.4%, 0.6%, 0.8% and 1% of ammonium citrate trihydrate on the diameter of the inhibition zone of Lactobacillus brevis L1612 fermentation broth in the antibacterial experiment; (h) is the effect of adding 0.4%, 0.8% and 1% of ammonium citrate trihydrate on the diameter of the inhibition zone of Lactobacillus brevis L1612 fermentation broth in the antibacterial experiment; (i) is the effect of adding 1%, 2%, 3%, 4% and 5% of beef extract powder on the diameter of the inhibition zone of Lactobacillus brevis L1612 fermentation broth in the antibacterial experiment; (j) is the effect of adding 1%, 2%, 3%, 4% and 5% of bacteriological peptone on the diameter of the inhibition zone of Lactobacillus brevis L1612 fermentation broth in the antibacterial experiment.

[0032] Figure 9: 3D response surface and contour plots of the interaction effects of various factors in the BBD design in Example 7. (a) and (b) are the response surface and contour plots for sodium acetate and glucose; (c) and (d) are the response surface and contour plots for dipotassium hydrogen phosphate and glucose; (e) and (f) are the response surface and contour plots for dipotassium hydrogen phosphate and sodium acetate. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below with reference to specific examples, but the embodiments of the present invention are not limited thereto. For process parameters not particularly noted, conventional techniques may be used.

[0034] Example 1 A strain of Lactobacillus brevis L1612

[0035] This example is a strain of Lactobacillus brevis L1612, and the preservation instructions are as follows:

[0036] Strain name: Lactobacillus brevis

[0037] Latin name: Lactobacillus brevis

[0038] Strain ID: L1612

[0039] Depository: General Microbiology Center of China Culture Collection Administration

[0040] Address: Building 59, No. 100, Xianlie Middle Road, Guangzhou

[0041] Deposit date: August 4, 2022

[0042] The registration number of the deposit center is GDMCC 62642.

[0043] The biological characteristics of the Lactobacillus brevis strain L1612 are as follows: when streaked on an MRS medium plate and cultured at 37°C for 24 to 72 hours, the colonies are round, milky white, with a flat, moist surface and neat edges. Gram staining microscopy reveals that the bacteria are Gram-positive, with short rods with rounded ends, arranged singly or in chains (e.g., Figure 1 As shown). The results of physiological and biochemical tests showed that the strain was facultative anaerobic, negative in catalase test, negative in hydrogen sulfide test, did not liquefy gelatin, and negative indole test. It could utilize glucose, fructose, sucrose, maltose, D-ribose, arabinose, and melibiose, but could not utilize trehalose, rhamnose, melezitose, sorbitol, and mannitol. Based on the 16S rDNA oligonucleotide sequence (as shown in SEQ ID NO: 1) and physiological and biochemical characteristics, the strain L1612 was identified as Lactobacillus brevis (as shown). Figure 2 shown).

[0044] Example 2 Determination of the complete genome sequence of Lactobacillus brevis L1612

[0045] Whole-genome sequencing was performed using a combination of third-generation ONT and second-generation Illumina technologies. Library construction was completed using third-generation Pacbio / ONT sequencing (SQK-LSK109 ligation kit). Third-generation sequencing reads were assembled using Flye, and quality control of the Illumina sequencing data was performed using Fastp software. Third-generation sequencing reads are longer but less accurate than second-generation sequencing reads. Pilon (version 1.23) was used to calibrate the genome assembly to ensure more accurate and reliable results. One circular chromosome and two circular plasmids (0gaps) were identified. Gene prediction was performed using the NCBI (National Center for Biotechnology Information Search database).

[0046] The whole genome composition of Lactobacillus brevis L1612 is as follows: the chromosome is 2,518,301 bp in size with an average GC content of 45.98%; the plasmid 1 is 59,967 bp in size with an average GC content of 42.49%; the plasmid 2 is 46,081 bp in size with an average GC content of 42.81% ( Figure 3 The whole genome contains 2525 protein-coding genes, 73 tRNA genes, and 15 rRNA genes. The specific statistics are shown in Table 1.

[0047] Table 1 Genomic characteristics of Lactobacillus brevis L1612

[0048]

[0049] Example 3 Gene Function Annotation of Lactobacillus brevis L1612

[0050] The obtained whole-genome DNA sequence of Lactobacillus brevis L1612 was compared with five databases, including Nr (Non-Redundant Protein Database), Swiss-Prot, KEGG (the Kyoto Encyclopedia of Genes and Genomes), GO (Gene Ontology), and COG (Cluster of Orthologous Groups of proteins), to perform BLAST comparison and functional annotation of the corresponding genes.

[0051] A total of 2525 genes were BLAST-aligned and functionally annotated, of which 2392 genes (94.73%) were annotated to the species Lactobacillus brevis in the Nr database; a total of 55 GO categories were annotated; 1620 genes (64.16%) obtained COG annotation; and 1287 genes (50.97%) obtained KEGG annotation.

[0052] Table 2 Nr classification of protein coding gene function prediction of Lactobacillus brevis L1612

[0053]

[0054] Table 3 GO classification of protein coding gene function prediction of Lactobacillus brevis L1612

[0055]

[0056] Table 3 GO categories of protein-coding gene function prediction of Lactobacillus brevis L1612 (continued)

[0057]

[0058] Table 4 COG classification of protein coding gene function prediction of Lactobacillus brevis L1612

[0059]

[0060]

[0061] Table 5 KEGG classification of protein coding gene function prediction of Lactobacillus brevis L1612

[0062]

[0063] Example 4 Safety Evaluation of Lactobacillus brevis L1612

[0064] In this example, Lactobacillus brevis L1612 was activated using MRS culture medium. The formula of the MRS culture medium is as follows: by weight: 2 parts glucose, 0.4 parts yeast extract powder, 0.2 parts dipotassium hydrogen phosphate, 0.2 parts triammonium citrate, 0.5 parts sodium acetate, 0.02 parts magnesium sulfate heptahydrate, 0.005 parts manganese sulfate tetrahydrate, 1 part beef extract powder, 1 part bacteriological peptone, 0.108 parts Tween 800, and the volume was adjusted to 100 parts with distilled water. The pH before sterilization was 5.5-5.9.

[0065] (1) Nitroreductase assay

[0066] The activated Lactobacillus brevis L1612 and the positive control strain Escherichia coli were inoculated into the nitroreductase culture medium at a rate of 4% (v / v), and a blank control group was set up at the same time. After constant temperature cultivation at 37°C for 72 hours, 8 to 10 drops of α-naphthylamine solution and p-aminobenzenesulfonic acid solution were added to the culture medium, shaken and allowed to stand, and the color change of the culture medium was observed and the results were recorded. If the culture medium turns red, the nitroreductase test result is positive, otherwise it is negative. The results are as follows Figure 4 As shown, the culture medium inoculated with E. coli turned red, indicating a successful positive control. The culture medium inoculated with Lactobacillus brevis L1612 did not change color, indicating a negative result for the nitroreductase test. This indicates that Lactobacillus brevis L1612 lacks active nitroreductase and does not reduce nitrate to nitrite. The formula for the nitroreductase test medium is: by weight, 1 part sodium chloride, 0.5 part potassium nitrate, 1 part bacteriological peptone, add distilled water to 100 parts, and adjust the pH to 6.8.

[0067] (2) Indole experiment

[0068] The activated Lactobacillus brevis L1612 seed liquid and the positive control strain Escherichia coli were inoculated into a peptone water culture medium at a 4% (v / v) inoculation rate, and a blank control was set up at the same time. After constant temperature cultivation at 37°C for 72 hours, 8 to 10 drops of indole reagent were added and the experimental results were observed. If a red ring appeared at the interface of the culture medium, it was positive, otherwise it was negative. The results are as follows Figure 5 As shown, a red ring appears at the interface of the culture medium inoculated with E. coli, indicating a positive indole test result and a successful positive control. The color of the interface of the culture medium inoculated with Lactobacillus brevis L1612 remains unchanged, indicating a negative indole test result. The formula for the peptone water medium is: 0.5 parts sodium chloride, 1 part bacteriological peptone, distilled water to 100 parts, and the pH is adjusted to 7.6.

[0069] (3) Hemolytic test

[0070] The activated Lactobacillus brevis L1612 and the quality control strain Escherichia coli were streaked on Columbia blood agar using a fully sterilized inoculation loop and incubated at 37°C for 48 hours. The presence of obvious hemolysis zones around the colonies was observed and recorded. Figure 6 (a)), indicating that it has hemolytic properties; compared with the quality control strain, no obvious hemolytic zone was formed around Lactobacillus brevis L1612 ( Figure 6 (b)), indicating that the strain is non-hemolytic and will not cause hemolytic damage to the human body.

[0071] (4) Animal acute oral toxicity test

[0072] This experiment was conducted and the results interpreted using the limit method according to the "National Food Safety Standard Acute Oral Toxicity Test" (GB 15193.3-2014). The test dose was set at 10,000 mg / kg body weight. Twenty SPF Kunming mice, weighing 20.0 to 22.0 g, half male and half female, were enrolled. L1612 fermentation broth, cultured to 1×109 CFU / mL, was administered orally once via gavage at a rate of 0.2 mL / 10 g body weight. The animals were fasted for 4 hours prior to gavage and had free access to water. One hour after gavage, they were given a normal diet. The mice were observed for 14 days, and their weights and mortality were recorded. The results are shown in Table 6. No mice experienced poisoning or mortality during the experimental period. At the end of the observation period, surviving mice were humanely euthanized and autopsied, revealing no abnormalities. The test results confirmed that the acute oral toxicity LD50 of the oral solution containing L1612 is greater than 10,000 mg / kg body weight, which is classified as practically non-toxic according to GB15193.3-2014.

[0073] Table 6 Results of acute oral toxicity test in animals

[0074]

[0075] The present invention provides a safety evaluation of the Lactobacillus brevis L1612, which includes a nitroreductase test, an indole test, a hemolytic test, an animal acute oral toxicity test, and a drug resistance test of the strain. The experiments show that the Lactobacillus brevis L1612 is negative in the nitroreductase test, negative in the indole test, is non-hemolytic, and has an actual non-toxic acute oral toxicity level in animals, indicating that the strain is safe.

[0076] Example 5 Evaluation of drug resistance of Lactobacillus brevis L1612

[0077] The reference method of this embodiment and the critical value of each antibiotic are derived from the "Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance" promulgated by the European Food Safety Authority in 2012. Using activated Lactobacillus brevis L1612 as an indicator bacteria, the minimum inhibitory concentration (MIC) of each antibiotic was determined by serial dilution method, and the drug resistance of the strain was evaluated by comparing the size of the MIC and the critical value. When the minimum inhibitory concentration is equal to or less than the established critical value, the strain is considered to be sensitive to the antibiotic; when the minimum inhibitory concentration is greater than the established critical value, the strain is considered to be resistant to the antibiotic. As shown in Table 7, it can be seen that Lactobacillus brevis L1612 is sensitive to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, tetracycline, and chloramphenicol, and has the potential for application in the food industry.

[0078] Table 7 Drug resistance evaluation test results

[0079]

[0080]

[0081] Example 6 Evaluation of the antibacterial ability of Lactobacillus brevis L1612

[0082] (1) Antibacterial spectrum experiment

[0083] The activated Lactobacillus brevis L1612 seed liquid was inoculated into MRS medium at a 4% (v / v) inoculation rate, cultured on a 37°C shaking table for 34 hours, extracted with 1:1 volume of ethyl acetate, and rotary evaporated until there was no odor of organic reagents. The liquid was placed in a 45°C vacuum drying oven overnight and reconstituted with sterile water at 1 / 20 of the original volume to prepare a crude fermentation liquid extract. The antibacterial activity of the crude fermentation liquid extract was tested using 9 Gram-positive and 9 Gram-negative strains as indicator bacteria to explore the antibacterial spectrum of the Lactobacillus brevis L1612 fermentation liquid. The results are shown in Table 8. The experimental results show that the L1612 fermentation liquid has a good antibacterial effect on Gram-negative bacteria and most Gram-positive bacteria, especially on Gram-negative bacteria such as Escherichia coli. As a natural new type of biological food preservative, it has a broad range of applications and development prospects.

[0084] Table 8 Antibacterial spectrum experimental results

[0085]

[0086] (2) Anti-SMAH secondary metabolism database prediction

[0087] The activated Lactobacillus brevis L1612 seed liquid was inoculated into MRS medium at a 4% (v / v) inoculation rate and cultured in a shaking incubator at 37°C for 34 hours. The Lactobacillus brevis L1612 genome was extracted using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver3.0 kit according to the instructions. The concentration and quality of the genome sample were tested and sent to Guangzhou Kidio for whole genome sequencing. The bacteriocin biosynthesis gene cluster analysis was predicted using the antiSMASH bacterial version (https: / / antismash.secondarymetabolites.org / #! / start). The results are as follows Figure 7 As shown, a total of three bacteriocin biosynthesis gene clusters were predicted, and the similarity predictions were all 0, indicating that Lactobacillus brevis L1612 has the potential to produce new bacteriocins.

[0088] Example 7 Culture medium optimization of Lactobacillus brevis L1612

[0089] The formula of the MRS culture medium described in this example is the same as that in Example 2.

[0090] (1) Single-factor test and results

[0091] In a single-factor carbon source experiment, MRS medium was used as the base, with two portions of sucrose, starch, glycerol, lactose, and maltose replacing the carbon source (glucose) in the MRS medium. MRS medium served as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in different carbon source media. The results are shown in Figure 8(a). The largest inhibition zone diameter was obtained when glucose was the carbon source, followed by maltose and lactose.

[0092] In a single-factor glucose experiment, MRS medium was used as the base, with glucose concentrations varied to 1, 1.5, 2.5, and 3 parts. MRS medium supplemented with 2 parts glucose served as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in medium containing different glucose concentrations. The results are shown in Figure 8(b). The inhibition zone diameter was largest when glucose was present at 2 parts, so 2 parts glucose was selected as the optimal concentration for subsequent studies.

[0093] In a single-factor experiment with potassium hydrogen phosphate (KHPO), MRS medium was used as the base, with KHPO concentrations varied to 0.1, 0.3, 0.4, and 0.5 parts. MRS medium supplemented with 0.2 parts KHPO served as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in medium containing different KHPO concentrations. The results are shown in Figure 8(c). The inhibition zone diameter was largest when KHPO was added at 0.2 parts, so 0.2 parts KHPO was selected as the optimal concentration for subsequent studies.

[0094] In a single-factor sodium acetate experiment, MRS medium was used as the base, with sodium acetate added at 0.3, 0.7, 0.9, and 1.1 parts. MRS medium supplemented with 0.5 parts sodium acetate served as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in medium containing different sodium acetate contents. The results are shown in Figure 8(d). The diameter of the inhibition zone was the largest when sodium acetate was added at 0.5 parts, so 0.5 parts sodium acetate was selected as the optimal concentration for subsequent studies.

[0095] In the magnesium sulfate heptahydrate single factor test, based on MRS culture medium, magnesium sulfate heptahydrate was changed to 0.04 part, 0.06 part, 0.08 part, and 0.1 part, with MRS culture medium containing 0.02 part of magnesium sulfate heptahydrate as the control. Lactobacillus brevis L1612 in the logarithmic growth phase was inoculated and cultured at 37°C for 48h. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for subsequent use. Lactobacillus brevis L1612 fermentation broth samples using different magnesium sulfate heptahydrate portions of culture medium were subjected to antibacterial tests. As shown in Figure 8(e), when magnesium sulfate heptahydrate was 0.04 part, the inhibition zone diameter was the largest, so selecting 0.04 part of magnesium sulfate heptahydrate was the optimal subsequent study.

[0096] In a single-factor experiment using MRS medium as the base, MRS medium was modified with 0.01, 0.015, 0.02, and 0.025 parts of MRS tetrahydrate. MRS medium supplemented with 0.005 parts of MRS tetrahydrate served as a control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in medium containing different amounts of MRS tetrahydrate. The results are shown in Figure 8(f). The diameter of the inhibition zone was the largest when MRS tetrahydrate was 0.015 parts, so 0.015 parts of MRS tetrahydrate was selected as the optimal concentration for subsequent studies.

[0097] In a single-factor experiment with ammonium citrate tribasic, MRS medium was used as the base, with ammonium citrate tribasic concentrations varied to 0.4, 0.6, 0.8, and 1 part, with MRS medium supplemented with 0.2 part ammonium citrate tribasic as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in culture medium with varying amounts of ammonium citrate tribasic. The results are shown in Figure 8(g). The diameter of the inhibition zone was largest when 0.8 part ammonium citrate tribasic was used, so 0.8 part ammonium citrate tribasic was selected as the optimal concentration for subsequent studies.

[0098] In a single-factor experiment with yeast extract powder, MRS medium was used as the base, with yeast extract powder concentrations varied to 0.8, 1.2, 1.6, and 2 parts. MRS medium supplemented with 0.4 parts yeast extract powder served as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in medium containing different amounts of yeast extract powder. The results are shown in Figure 8(h). The diameter of the inhibition zone was the largest when the yeast extract powder concentration was 1.2 parts. Therefore, the 1.2 parts yeast extract concentration was selected as the optimal concentration for subsequent studies.

[0099] In a single-factor experiment using beef extract powder, MRS medium was used as the base, with 2, 3, 4, and 5 portions of beef extract powder added. MRS medium supplemented with 1 portion of beef extract powder served as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in medium containing different amounts of beef extract powder. The results are shown in Figure 8(i). The diameter was largest when the beef extract powder was added in 4 portions, so the 4-portion beef extract powder was selected as the optimal medium for subsequent studies.

[0100] In a single-factor experiment using bacteriological peptone, MRS medium was used as the base, with the peptone content varied to 2, 3, 4, and 5 parts. MRS medium supplemented with 1 part peptone was used as the control. Lactobacillus brevis L1612 in its logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were conducted on Lactobacillus brevis L1612 fermentation broth samples grown in medium containing different peptone content ratios. The results are shown in Figure 8(j). The diameter was largest when the peptone content was 3 parts, so the peptone content of 3 parts was selected as the optimal medium for subsequent studies.

[0101] (2) Plackett-Burman test screened significant influencing factors

[0102] A Plackett-Burman design was performed using glucose, yeast extract powder, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, magnesium sulfate heptahydrate, manganese sulfate tetrahydrate, beef extract powder, and bacteriological peptone to screen for factors that most significantly affect the diameter of the inhibition zone of the supernatant of Lactobacillus brevis L1612 in an antibacterial experiment. The factor numbers and levels are shown in Table 9.

[0103] Table 9 Factor numbers and levels in Plackett-Burman experimental design

[0104]

[0105] The experimental design and response values ​​are shown in Table 10.

[0106] Table 10Plackett-Burman experimental design and response values

[0107]

[0108] The test analysis table is shown in Table 11.

[0109] Table 11Plackett-Burman test analysis table

[0110]

[0111] Using Design Expert software for analysis, glucose, sodium acetate and dipotassium hydrogen phosphate were significant influencing factors, with contributions of 20.79%, 16.07% and 19% respectively, and the model was significant.

[0112] (3) Box-Behnken response surface design and verification

[0113] A Box-Benhnken experimental design was conducted using glucose, sodium acetate, and dipotassium hydrogen phosphate. Three levels were selected for each factor: glucose at 1.5, 2, and 2.5 parts; sodium acetate at 0.3, 0.5, and 0.7 parts; and dipotassium hydrogen phosphate at 0.1, 0.2, and 0.3 parts. The response value was the diameter (in mm) of the inhibition zone of Lactobacillus brevis L1612 fermentation broth in the antibacterial experiment. The experimental design and results are shown in Table 12.

[0114] Table 12BBD design scheme and results

[0115]

[0116] The regression equation of the number of viable bacteria for glucose (A), sodium acetate (B) and dipotassium hydrogen phosphate (C) was obtained by software analysis: Inhibition zone diameter (mm) = -27.70988 + 23.016A + 81.4375B + 17.565C - 7.575AB + 9AC + 9.625BC - 5.184A 2 -64.8375B 2 -84.6C 2 The F value of the model is 5.53, p = 0.0173 < 0.05, and the lack of fit term is not significant. The coefficient of determination R 2 =0.8767, indicating that the model's predicted values ​​fit the actual values ​​well. The 3D response surface plot of the experimental design results is shown in Figure 9.

[0117] The results show that when the content of glucose is 2.05 parts, the content of sodium acetate is 0.53 parts, and the content of dipotassium hydrogen phosphate is 0.24 parts, the diameter of the inhibition zone can reach the theoretical maximum value of 19.4026 mm.

[0118] Verification tests using the optimized culture medium revealed an inhibition zone diameter of 19.37 mm, consistent with the predicted value. The optimized culture medium composition, by mass, consisted of: 2.05 parts glucose, 1.2 parts yeast extract powder, 0.24 parts dipotassium hydrogen phosphate, 0.8 parts triammonium citrate, 0.53 parts sodium acetate, 0.04 parts magnesium sulfate heptahydrate, 0.015 parts manganese sulfate tetrahydrate, 4 parts beef extract powder, 3 parts bacteriological peptone, and 0.108 parts Tween 80. The volume was adjusted to 100 parts with distilled water. The final inhibition zone diameter reached 19.37 mm, a 25.21% increase compared to the pre-optimization value.

[0119] Example 8

[0120] MRS liquid culture medium was prepared for activating strain L1612. The formula was as follows: by mass: 2 parts of glucose, 0.4 parts of yeast extract powder, 0.2 parts of dipotassium hydrogen phosphate, 0.2 parts of triammonium citrate, 0.5 parts of sodium acetate, 0.02 parts of magnesium sulfate heptahydrate, 0.005 parts of manganese sulfate tetrahydrate, 1 part of beef extract powder, 1 part of bacteriological peptone, and 0.108 parts of Tween 80. The volume was adjusted to 100 parts with distilled water. The pH before sterilization was 5.5-5.9.

[0121] Prepare LB liquid culture medium for activating Escherichia coli with the following formula: by mass ratio, 1 part bacteriological peptone, 1 part sodium chloride, 0.5 part yeast extract powder, and dilute to 100 parts with distilled water.

[0122] Prepare LB solid medium for preparing E. coli plates with the following formula: by mass ratio, 1 part bacteriological peptone, 1 part sodium chloride, 0.5 part yeast extract powder, 0.75 part agar, and distilled water to 100 parts.

[0123] Prepare an E. coli inhibition plate by inoculating E. coli into LB liquid medium at 4% (v / v), shaking at 37°C to an OD600 of approximately 1.0, and adjusting the plate to an OD600 of 0.5 with LB medium. Inoculate E. coli at an OD600 of 0.5 into sterile LB solid medium at 1.6 μL / mL, mix thoroughly, pour onto the plate, seal, and store in a refrigerator at 4°C until needed.

[0124] The Lactobacillus brevis L1612 in the logarithmic phase is inoculated in the antibacterial optimized culture medium with an inoculum size of 4% (v / v), and supernatant is taken after 37 ℃ of shaking cultures for 48h. After centrifugation, pH is adjusted to 6.0, and filtration is performed for subsequent use. Take out 4 ℃ of refrigerated antibacterial plates, and after the punch (10mm in diameter) is fully sterilized with an alcohol lamp, holes are punched on the antibacterial plates, and picked out with a sterile toothpick. Every hole adds 100 μ L bacterial strain L1612 fermented liquid, and after drying in a clean bench, is placed on 37 ℃ of constant temperature cultures for 8h, and the inhibition zone diameter is measured. The optimized antibacterial culture medium is formulated as follows: by mass: 2 parts glucose, 1.2 parts yeast extract powder, 0.2 parts dipotassium hydrogen phosphate, 0.8 parts triammonium citrate, 0.5 parts sodium acetate, 0.04 parts magnesium sulfate heptahydrate, 0.015 parts manganese sulfate tetrahydrate, 4 parts beef extract powder, 3 parts bacteriological peptone, and 0.108 parts Tween 80. The volume is adjusted to 100 parts with distilled water, and the pH before sterilization is 5. The final inhibition zone diameter can reach 18.80 mm, an increase of 21.53% compared to the pre-optimization level.

[0125] Example 9

[0126] The method for preparing MRS liquid culture medium, LB liquid culture medium, LB solid culture medium, and E. coli antibacterial plate is the same as that in Example 6.

[0127] The Lactobacillus brevis L1612 in the logarithmic phase is inoculated in the antibacterial optimized culture medium with an inoculum size of 4% (v / v), and supernatant is taken after 37 ℃ of shaking cultures for 48h. After centrifugation, pH is adjusted to 6.0, and filtration is performed for subsequent use. Take out 4 ℃ of refrigerated antibacterial plates, and after the punch (10mm in diameter) is fully sterilized with an alcohol lamp, holes are punched on the antibacterial plates, and picked out with a sterile toothpick. Every hole adds 100 μ L bacterial strain L1612 fermented liquid, and after drying in a clean bench, is placed on 37 ℃ of constant temperature cultures for 8h, and the inhibition zone diameter is measured. The optimized antibacterial culture medium is formulated as follows: by mass: 1.5 parts glucose, 1.2 parts yeast extract powder, 0.3 parts dipotassium hydrogen phosphate, 0.8 parts triammonium citrate, 0.5 parts sodium acetate, 0.04 parts magnesium sulfate heptahydrate, 0.015 parts manganese sulfate tetrahydrate, 4 parts beef extract powder, 3 parts bacteriological peptone, and 0.108 parts Tween 80. The volume is adjusted to 100 parts with distilled water, and the pH before sterilization is 5. The final inhibition zone diameter can reach 17.77 mm, an increase of 14.42% compared to the pre-optimization level.

[0128] Example 10

[0129] The method for preparing MRS liquid culture medium, LB liquid culture medium, LB solid culture medium, and E. coli antibacterial plate is the same as that in Example 6.

[0130] The Lactobacillus brevis L1612 in the logarithmic phase is inoculated in the antibacterial optimized culture medium with an inoculum size of 4% (v / v). After 37 ℃ of shaking cultures for 48h, the supernatant is taken, and after centrifugation, pH is adjusted to 6.0, and the filter is standby. Take out the antibacterial plates for standby use at 4 ℃ of refrigerations, and after the punch (10mm in diameter) is fully sterilized with an alcohol lamp, the plate is punched and picked out with a sterile toothpick. Every hole adds 100 μ L bacterial strain L1612 fermented liquid, and after drying in a clean bench, is placed at 37 ℃ of constant temperature cultures for 8h, and the inhibition zone diameter is measured. The optimized antibacterial culture medium is formulated as follows: by mass: 2.5 parts glucose, 1.2 parts yeast extract powder, 0.3 parts dipotassium hydrogen phosphate, 0.8 parts triammonium citrate, 0.5 parts sodium acetate, 0.04 parts magnesium sulfate heptahydrate, 0.015 parts manganese sulfate tetrahydrate, 4 parts beef extract powder, 3 parts bacteriological peptone, and 0.108 parts Tween 80. The volume is adjusted to 100 parts with distilled water, and the pH is 5 before sterilization. The final inhibition zone diameter can reach 18.57 mm, a 20.04% increase compared to the pre-optimization level.

[0131] Example 11 Optimization of culture conditions for Lactobacillus brevis L1612

[0132] In the pH single-factor experiment, the initial pH of the MRS culture medium was changed to 4, 5, 6, and 7, and the MRS culture medium pH = 5.7 was used as a control. Lactobacillus brevis L1612 in the logarithmic growth phase was inoculated and cultured at 37°C for 48 hours. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for later use. Antibacterial tests were performed on Lactobacillus brevis L1612 fermentation broth samples using culture media with different initial pH values. The results are shown in Table 13. By comparison, the diameter of the inhibition zone was the largest at pH = 5, which was 22.6 ± 1.01 mm (P < 0.05). Therefore, pH = 5 was selected as the optimal initial pH for subsequent experiments.

[0133] Table 13 Effect of pH on inhibition diameter

[0134]

[0135] In the inoculum size single factor test, changing the seed liquid inoculum size is 1%, 2%, 3%, 4%, 5% (v / v).Inoculation is in the short lactobacillus L1612 of logarithmic growth phase, cultivates 48h at 37 ℃, gets supernatant centrifugal, transfers pH6.0, filters standby.The short lactobacillus L1612 fermented liquid sample of different inoculum sizes is carried out antibacterial test, the result is as shown in table 14, and contrast can be known, inoculum size is 4% when diameter is maximum, is 21.3 ± 0.26mm (P<0.05), therefore selects 4% for use as optimal inoculum size to carry out subsequent research.

[0136] Table 14 Effect of inoculation amount on inhibition diameter

[0137]

[0138] In the fermentation temperature single factor experiment, changing the fermentation temperature was 32 ℃, 37 ℃, 40 ℃, and 42 ℃. Inoculation was performed on Lactobacillus brevis L1612 in the logarithmic growth phase, and the cells were cultured at 37 ℃ for 48 h. The supernatant was centrifuged, adjusted to pH 6.0, and filtered for subsequent use. The Lactobacillus brevis L1612 fermentation broth samples of different inoculum sizes were subjected to an antibacterial test. The results are shown in Table 15. By contrast, the diameter was the largest at 37 ℃, at 31.93 ± 0.50 mm (P < 0.05). Therefore, 37 ℃ was selected as the optimal fermentation temperature for subsequent research.

[0139] Table 15 Effect of fermentation temperature on inhibition diameter

[0140]

Claims

1. A strain of Lactobacillus brevis L1612, characterized in that Lactobacillus brevis L1612 was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 4, 2022, with the deposit number GDMCC NO: 62642.

2. The antibacterial optimization culture method of Lactobacillus brevis L1612 according to claim 1, characterized in that Follow these steps: (1) Lactobacillus brevis L1612 was inoculated into MRS culture medium for activation to obtain seed solution; (2) Inoculate the seed solution into the antibacterial optimized culture medium and culture for 34 to 48 hours; The antibacterial optimized culture medium is formulated as follows: by mass: 2 parts of glucose, 1.2 parts of yeast extract powder, 0.24 parts of dipotassium hydrogen phosphate, 0.8 parts of triammonium citrate, 0.53 parts of sodium acetate, 0.04 parts of magnesium sulfate heptahydrate, 0.015 parts of manganese sulfate tetrahydrate, 4 parts of beef extract powder, 3 parts of bacteriological peptone, 0.108 parts of Tween 80, and the volume is adjusted to 100 parts with distilled water.

3. The method according to claim 2, characterized in that Lactobacillus brevis L1612 is inoculated into an antibacterial optimized culture medium at an inoculation rate of 1% to 5% by volume; the initial pH value of the culture of the Lactobacillus brevis L1612 in the antibacterial optimized culture medium is 4.0 to 7.0; and the culture temperature is 32 to 42°C.

4. The method according to claim 3, characterized in that The Lactobacillus brevis L1612 is inoculated into an antibacterial optimized culture medium at an inoculum volume ratio of 4%; the initial pH of the culture of the Lactobacillus brevis L1612 in the antibacterial optimized culture medium is 5.0; and the culture temperature is 37°C.

5. Use of the Lactobacillus brevis according to claim 1 in preparing a product for inhibiting Gram-positive or Gram-negative bacteria, wherein the Gram-positive bacteria are Staphylococcus aureus RN4220 and Listeria monocytogenes ATCC 19115; and the Gram-negative bacteria are Escherichia coli ATCC 25922, Salmonella enteritidis CCTCC AB 94018, Klebsiella pneumoniae ATCC 10031, Enterobacter sakazakii ATCC 29544, Vibrio parahaemolyticus ATCC 10031, Salmonella typhimurium ATCC 14028, Shewanella putrefaciens ATCC 8071, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa PAO1.

6. An intestinal antibacterial drug containing the Lactobacillus brevis according to claim 1, wherein the drug inhibits Staphylococcus aureus RN4220, Listeria monocytogenes ATCC 19115, Escherichia coli ATCC 25922, Salmonella enteritidis CCTCC AB94018, Klebsiella pneumoniae ATCC 10031, Enterobacter sakazakii ATCC 29544, Vibrio parahaemolyticus ATCC 10031, Salmonella typhimurium ATCC 14028, Shewanella putrefaciens ATCC 8071, Pseudomonas fluorescens ATCC 13525, and Pseudomonas aeruginosa PAO1.

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