Lactobacillus brevis and application thereof
By using Lactobacillus brevis Myh YC-01 to prepare fermented feed for crabs, the problem of microbial colonization in the crab intestines was solved, improving the hepato-intestinal health and immunity of crabs, and realizing the efficient utilization of fermented feed and the improvement of crab growth performance.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YANGZHOU UNIV
- Filing Date
- 2025-03-04
- Publication Date
- 2026-07-31
AI Technical Summary
The microbial strains used in existing fermented feed for crabs are difficult to colonize efficiently in the crab's intestines, resulting in the inability of probiotics to function continuously, and serious problems of feed resource shortage and pollution.
Lactobacillus brevis Myh YC-01, selected from healthy river crabs raised in the ecological environment of Gaoyou Lake, was used to prepare fermented feed. It has antioxidant and antibacterial capabilities. The fermented feed for river crabs was prepared by anaerobic fermentation at 37℃ for 7 days, which improved liver and intestinal health and immunity.
It significantly improves the liver and intestinal health of crabs, enhances their immunity, increases feed utilization efficiency, reduces the feed conversion ratio, and strengthens their growth performance and immunity.
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Figure CN120082472B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a type of Lactobacillus brevis and its application in the preparation of fermented feed for river crabs, belonging to the field of microbial technology. Background Technology
[0002] The Chinese mitten crab (Eriocheir sinensis), also known as the river crab, is widely distributed in freshwater lakes throughout my country. Its unique flavor and rich nutritional content make it highly popular among Chinese consumers. Therefore, river crab farming has developed rapidly in recent years, becoming an important aquaculture species in my country. Currently, many animal protein feeds for river crabs on the market use fresh or frozen juvenile fish. However, feeding large quantities of frozen small fish can easily lead to frequent crab diseases and pollute the water. In 2020, the Ministry of Agriculture and Rural Affairs issued a document proposing five major actions to promote green and healthy aquaculture, among which "replacing juvenile fish with formulated feed" requires the use of formulated feed throughout the entire river crab farming process.
[0003] In recent years, with the rapid development of crab farming, the output of crab feed has been continuously increasing. However, due to the shortage of feed resources and the high price of raw materials, cheap miscellaneous meals cannot be digested and absorbed by animals and are largely discarded. Faced with this problem, bio-fermented feed has gradually become a hot topic in the industry. Bio-fermented feed is a general term for a new type of feed that uses feed or feed raw materials as processing objects and biotechnology such as fermentation engineering and protein engineering as processing methods. It mainly includes: microbial fermented feed, fermented and enzymatically hydrolyzed feed, and feed enzyme preparations. Fermented feed uses a wide variety of probiotics, generally Bacillus, lactic acid bacteria, yeast, etc. Through single or compound strains, feed protein is decomposed into microbial cell protein, bioactive small peptide amino acids, and active probiotics, which can remove anti-nutritional factors in feed while also facilitating the absorption and utilization of nutrients by animals.
[0004] Most of the strains used in the fermented feed for crabs currently in use are not isolated from farmed animals, resulting in the inability of microorganisms to colonize the intestines efficiently and maintain the continuous function of probiotic strains. Summary of the Invention
[0005] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a strain of Lactobacillus brevis. This strain is screened from healthy ecologically farmed river crabs in Gaoyou Lake and has good antioxidant, pathogen inhibition and oxygen tolerance capabilities. It can efficiently colonize the intestines of river crabs and can be used to prepare fermented feed for river crabs, which can effectively improve the liver and intestinal health and immunity of river crabs.
[0006] To achieve the above objectives, the present invention adopts the following technical solution:
[0007] A Lactobacillus brevis strain, Myh YC-01, was deposited on January 16, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33441.
[0008] The Lactobacillus brevis MyhYC-01 strain of this invention was screened from healthy farmed river crabs in Gaoyou Lake. It has good antioxidant, pathogen inhibition and oxygen tolerance capabilities; it can improve the quality of fermented feed, significantly improve the liver and intestinal health of river crabs, and enhance their immunity.
[0009] The above-mentioned applications of Lactobacillus brevis in antioxidant activity.
[0010] The above-mentioned Lactobacillus brevis was used in the preparation of fermented feed for crabs to improve liver and intestinal health and enhance their immunity.
[0011] A method for preparing fermented feed for river crabs: First, sterilized water and basic feed are mixed evenly, then the above-mentioned Lactobacillus brevis liquid is added, and the mixture is anaerobic fermented at 37°C for 7 days to obtain fermented feed for river crabs.
[0012] The basic feed consists of the following components in the indicated weight percentages: fish meal 8-12%, chicken meal 6-10%, hemoglobin meal 2-3%, soybean meal 18-22%, peanut meal 14-16%, rapeseed meal 15-17%, yeast protein 1-3%, wheat flour 11-13%, lysine 0.3-1%, methionine 0.1-0.3%, calcium dihydrogen phosphate 1-2.5%, soybean oil 2-4%, shrimp paste 1-3%, multivitamin and mineral premix 0.5-1.5%, antioxidant 0.1-0.2%, sodium humate 1-3%, molting agent 0.1-0.2%, and bentonite 4-4.5%.
[0013] Furthermore, the mass ratio of the basic feed to sterilized water is 1:0.4.
[0014] Furthermore, the amount of *Lactobacillus brevis* added is 10. 6 CFU / g.
[0015] Furthermore, the method for preparing the Lactobacillus brevis culture is as follows: a single colony of Lactobacillus brevis is picked from the isolated and purified solid culture medium and placed in MRS liquid culture medium. It is then anaerobically cultured at 37°C for 24 hours. The colony is then inoculated into MRS culture medium at an inoculation rate of 1% (volume percentage) and anaerobically cultured at 37°C for 6-8 hours to obtain the Lactobacillus brevis culture.
[0016] The beneficial effects of this invention are as follows: This invention provides a *Lactobacillus brevis* strain, Myh YC-01, which is the first strain isolated and screened from the intestines of river crabs. This strain possesses strong anti-inflammatory and antibacterial properties. It can be used to effectively ferment feed, pre-decompose nutrients in the feed, and improve feed utilization efficiency. Aquaculture experiments have shown that this strain has a good effect on improving the hepatointestinal health and immunity of river crabs. The *Lactobacillus brevis* strain Myh YC-01 of this invention has the potential to be used as a probiotic in aquaculture and has a very broad application prospect. Attached Figure Description
[0017] Figure 1 Phylogenetic tree of Lactobacillus brevis MyhYC-01 based on 16S rDNA sequence;
[0018] Figure 2 The growth curve of Lactobacillus brevis Myh YC-01;
[0019] Figure 3 Morphological characteristics of Lactobacillus brevis MyhYC-01;
[0020] Figure 4 The results of the antibacterial activity test of Lactobacillus brevis MyhYC-01 against three pathogens;
[0021] Figure 5 The effect of Lactobacillus brevis fermented feed on antioxidant enzymes in the hepatopancreas of crabs;
[0022] Figure 6 The effect of Lactobacillus brevis fermented feed on the activity of blood immune enzymes in crabs;
[0023] Figure 7 The effect of Lactobacillus brevis fermented feed on the expression of immune-related genes in the hepatopancreas of crabs;
[0024] Figure 8 To investigate the effects of Lactobacillus brevis fermented feed on the expression of genes related to intestinal health in crabs. Detailed Implementation
[0025] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments.
[0026] Example 1
[0027] 1. Isolation and Identification of Lactobacillus brevis
[0028] The inventors collected the intestines of wild river crabs from Gaoyou Lake for the isolation and screening of *Lactobacillus brevis*. The specific method was as follows: Intact intestines of the river crabs were picked up with sterile forceps. The foregut and midgut were placed in 1 mL of pre-cooled sterile PBS buffer and homogenized thoroughly using a high-throughput tissue homogenizer. The homogenate was serially diluted, and 0.1 mL was spread onto MRS solid medium and incubated at 37°C for 48 h. Colonies that were white, with smooth edges and a raised center were picked and inoculated into MRS liquid medium for expansion and streaking. This was repeated at least three times until the colony morphology was completely uniform. Single colonies were transferred to liquid medium for enrichment and then stored in 20% glycerol at -80°C.
[0029] To further identify the strain, genomic DNA was extracted and amplified using universal primers for 16S rDNA. The 16S rDNA gene sequence was then obtained through sequencing. BLAST analysis was performed on the NCBI website, and a phylogenetic tree was constructed, as shown below. Figure 1 The results showed that the strain belonged to the Lactobacillus brevis species and was named Lactobacillus brevis MyhYC-01. It was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with the accession number CGMCC No. 33441.
[0030] 2. Growth performance and morphology of Lactobacillus brevis Myh YC-01
[0031] Take three anaerobic culture tubes containing sterilized MRS liquid culture medium, inoculate them with 1% activated Lactobacillus brevis culture solution, and incubate them at 37℃. Take a small amount of liquid at 0, 1, 2, 3, 4, 6, 8, 10, 12, 14 and 16 h to measure the OD value.
[0032] Figure 2 The growth curve of Lactobacillus brevis Myh YC-01 is shown. Figure 2 It can be seen that the strain enters the logarithmic growth phase 1-8 hours after inoculation, with the fastest growth rate occurring around 5 hours.
[0033] Figure 3 The image shows the morphological characteristics of Lactobacillus brevis MyhYC-01. It can be seen that the colonies of Lactobacillus brevis are raised, slightly transparent milky white, round and smooth, with neat edges.
[0034] 3. Drug susceptibility and antibacterial assays of Lactobacillus brevis Myh YC-01
[0035] (1) The drug susceptibility test was performed using the KB disc diffusion method. Specifically, drug susceptibility discs were dispersed and applied to plates coated with *Lactobacillus brevis*. After incubation at 37°C for 24 hours, the diameter of the inhibition zone was measured. R represents resistance (inhibition zone less than 10 mm); I represents moderate sensitivity (inhibition zone 10–20 mm); and S represents sensitivity (inhibition zone greater than 20 mm). The results are shown in Table 1.
[0036] Table 1 Results of drug susceptibility testing
[0037]
[0038]
[0039] Table 1 shows that this *Lactobacillus brevis* is resistant to 14 drugs, including penicillin, oxacillin, cephalexin, cefazolin, cefuroxime sodium, ceftazidime, ceftriaxone, streptomycin, norfloxacin, ciprofloxacin, vancomycin, trimethoprim-sulfamethoxazole, clindamycin, and levofloxacin; moderately sensitive to 6 drugs, including ampicillin, cefoperazone, amikacin, kanamycin, erythromycin, and lincomycin; and sensitive to 10 drugs, including piperacillin, gentamicin, tetracycline, minocycline, azithromycin, polymyxin B, chloramphenicol, imipenem, doxycycline, and florfenicol.
[0040] (2) The antibacterial experiment used the perforation method. Specifically, sterile medical cotton swabs were used to collect bacterial suspensions of three common aquatic pathogens: Staphylococcus aureus, Aeromonas vera, and Aeromonas hydrophila, and evenly spread them on nutrient agar solid medium. A 1 mL pipette tip (outer diameter: 9 mm) was used to make perforations in the nutrient agar medium. Lactobacillus brevis fermentation broth was added to the corresponding perforations, ensuring the perforations were full but not overflowing. The culture dishes containing the samples were placed in a 4℃ refrigerator for 3 hours, then transferred to a 37℃ incubator for anaerobic incubation for 12 hours. The diameter of the inhibition zone in the plates was measured and recorded to determine the antibacterial effect of Lactobacillus brevis. The test results are shown in Table 2 and... Figure 4 :
[0041] Table 2. Diameter of inhibition zones for three pathogens
[0042]
[0043] From Table 2 and Figure 4 It was found that the inhibition zone diameter of *Lactobacillus brevis* against *Aeromonas hydrophila* and *Aeromonas vesiculosus* was >15 mm, therefore, *Lactobacillus brevis* has a high inhibitory effect on both of these pathogens. The inhibition zone diameter of 15 mm is greater than that of *Staphylococcus aureus* (>10 mm), therefore, *Lactobacillus brevis* has a moderate inhibitory effect on *Staphylococcus aureus*.
[0044] 4. Oxygen tolerance experiment, physiological and biochemical reaction experiment and DPPH free radical scavenging rate experiment of Lactobacillus brevis Myh YC-01.
[0045] (1) The fermentation supernatant of Lactobacillus brevis Myh YC-01 cultured for 24 h was diluted and spread on plates. After being exposed to air for 0 min, 15 min, and 30 min respectively, the plates were sequentially placed in an anaerobic incubator at 37℃ for 24 h. The growth status of the strains on each plate was observed and counted. Each sample was repeated three times to ensure the accuracy of the experimental results. The survival rate of Lactobacillus brevis was calculated as 100% based on the number of viable bacteria after 0 min of exposure. The test results are shown in Table 3:
[0046] Table 3 Results of the oxygen tolerance test
[0047]
[0048] As shown in Table 3, Lactobacillus brevis has a high tolerance to oxygen.
[0049] (2) Physiological and biochemical experiments were conducted on Lactobacillus brevis Myh YC-01 using Lactobacillus biochemical reaction tubes (Qingdao Haibo Biotechnology Co., Ltd.).
[0050] Test results: Lactobacillus brevis can utilize cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin and lactose, but cannot utilize aescin.
[0051] (3) DPPH free radical scavenging rate represents the antioxidant capacity of the strain. 1 mL of fermentation supernatant from *Lactobacillus brevis* Myh YC-01 was mixed thoroughly with 1 mL of 1.50 mmol / L DPPH anhydrous ethanol solution. The mixture was incubated at room temperature in the dark for 30 min, and the absorbance was measured at 517 nm. The blank group used an equal volume of ethanol instead of DPPH solution, and the control group used an equal volume of sterile water instead of the *Lactobacillus brevis* fermentation supernatant. 0.125 g / L vitamin C was used as a positive control. The scavenging rate was calculated using the following formula:
[0052] Clearance rate / % = [1 - (A1 - A) / A0] × 100%
[0053] In the formula, A is the absorbance value of the blank group, A0 is the absorbance value of the control group, and A1 is the absorbance value of the sample group.
[0054] The above experimental steps showed that Lactobacillus brevis Myh YC-01 had a good DPPH free radical scavenging rate of 62.53%.
[0055] Example 2
[0056] The effects of Lactobacillus brevis on the growth performance, immunity, and hepatointestinal health of crabs:
[0057] One hundred and twenty river crabs were selected for the experiment and divided into three groups: a control group, a blank fermented feed group, and a Lactobacillus brevis fermented feed group, with four replicates per group and ten crabs per replicate. The crabs were raised for ten weeks and fed twice daily (08:00 and 17:00) until full. The control group was fed a basal diet, the blank fermented feed group was fed a blank fermented feed, and the Lactobacillus brevis fermented feed group was fed the Lactobacillus brevis fermented feed. Uneaten feed and feces were removed one hour after feeding.
[0058] Preparation method of fermented feed with Lactobacillus brevis: Lactobacillus brevis Myh was picked from isolated and purified solid culture medium. Single colonies of YC-01 were cultured in MRS liquid medium at 37°C for 24 hours under static anaerobic conditions. Then, 1% (volume percentage) of the culture was inoculated into MRS medium and cultured at 37°C under static anaerobic conditions until the end of the logarithmic phase to obtain *Lactobacillus brevis* bacterial suspension. Sterile water was mixed with a basic feed (basic feed formula: fish meal 10%, chicken meal 8%, hemoglobin powder 2.5%, soybean meal 20%, peanut meal 15%, rapeseed meal 16%, yeast protein 2%, wheat flour 12%, lysine 0.5%, methionine 0.1%, calcium dihydrogen phosphate 1.5%, soybean oil 3%, shrimp paste 2%, multivitamin and mineral premix 1%, antioxidant 0.1%, sodium humate 2%, molting agent 0.1%, bentonite 4.2%) at a mass ratio of 1:0.4. The *Lactobacillus brevis* bacterial suspension was then added to the sterilized MRS liquid medium and mixed into the feed (the initial addition of *Lactobacillus brevis* to the feed was 10... 6 The sample (CFU / g) was immediately vacuum-sealed and placed in a 37°C incubator for 7 days to obtain fermented Lactobacillus brevis feed. After removal, it was stored at 4°C.
[0059] Preparation method of blank fermented feed: without adding Lactobacillus brevis liquid, only add sterile water and sterile MRS liquid culture medium for fermentation (the remaining steps are the same as above) to obtain blank fermented feed, which is then stored at 4℃.
[0060] At the end of the breeding experiment, each crab was weighed and counted to calculate its growth performance. Muscle, hepatopancreas, and intestinal samples were collected from each group of Chinese mitten crabs to determine hepatopancreatic enzyme activity and hepatopancreatic and tract gene expression. Hemolymph was collected from the joint membrane at the base of the third walking leg of the crab, anticoagulant was added, and the mixture was centrifuged at 12000 rpm for 10 min at 4℃. The supernatant was collected for the determination of immunoenzyme activity.
[0061] Enzyme activity assay method:
[0062] Intestinal and hepatopancreatic samples were homogenized using a high-throughput tissue homogenizer. Intestinal digestive enzymes and hepatopancreatic antioxidant enzymes were measured according to kits from Nanjing Jiancheng Technology Co., Ltd. Alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM), and peroxidase (POD) in blood samples were measured according to the kit instructions from Nanjing Jiancheng Technology Co., Ltd.
[0063] Methods for measuring gene expression levels:
[0064] Gene expression levels were measured using cDNA synthesized using TransScript All-in-One First-strand cDNA Synthesis SuperMix for qPCR reagent (Transgen Biotech, Beijing, China). Gene primers were synthesized by TransGen Biotech (Beijing, China). qRT-PCR reactions were performed on a real-time quantitative PCR instrument using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reaction system and conditions were set according to the kit instructions. The total reaction volume was 20 μL, with β-actin as the internal control gene, and a 2... -△△Ct The relative expression level of gene mRNA can be calculated using this method.
[0065] Experimental results:
[0066] The results of the test on the effects of Lactobacillus brevis fermented feed on the survival rate, growth performance and feed conversion ratio of crabs are shown in Table 4:
[0067] Table 4. Effects of Lactobacillus brevis fermented feed on survival rate, growth performance, and feed conversion ratio of crabs.
[0068]
[0069] As shown in Table 4, the weight gain rate of the Lactobacillus brevis fermented feed group was significantly higher than that of the other two groups (P<0.05), and the feed conversion ratio was significantly lower (P<0.05), indicating that the Lactobacillus brevis fermented feed had better utilization efficiency.
[0070] The effects of Lactobacillus brevis fermented feed on antioxidant enzymes in the hepatopancreas of crabs are shown in [reference needed]. Figure 5 ,in, Figure 5 A represents the effect on T-AOC. Figure 5 B represents the impact on GSH-PX. Figure 5C represents the effect on MDA. GSH-PX and T-AOC are important indicators of the antioxidant capacity of crabs, while the level of MDA indicates the degree of damage to hepatopancreatic cells. The results showed that feeding with Lactobacillus brevis fermented feed significantly promoted the activity of GSH-PX and T-AOC (P<0.05), and enhanced the antioxidant capacity of the crab's hepatopancreas; at the same time, the MDA level was significantly reduced (P<0.05), and the hepatopancreas was healthy.
[0071] Figure 6 The effect of Lactobacillus brevis fermented feed on the activity of blood immune enzymes in crabs, among which, Figure 6 A represents the effect on lysozyme LZM. Figure 6 B represents the effect on peroxidase POD. Figure 6 C represents the effect on acid phosphatase ACP. Figure 6 D represents the effect on alkaline phosphatase (AKP). The results showed that the activities of POD, LZM, and AKP in the blood of the Lactobacillus brevis fermented feed group were significantly increased (P < 0.05), indicating that feeding enhanced the non-specific immunity of crabs.
[0072] Figure 7 The effects of Lactobacillus brevis fermented feed on the expression of immune-related genes in the hepatopancreas of crabs were investigated. Figure 7 A represents the effect on alf. Figure 7 B represents the effect on crustin1. Figure 7 C represents the effect on hsp70. Figure 7 D represents the effect on hsp90. Figure 7 E represents the effect on toll1. Figure 7 F represents the effect on pelle. Figure 7 G represents the effect on the tube. Figure 7 H represents the effect on myd88. The toll1, pelle, myd88, and tube genes are important components of the Toll immune pathway. Myd88 activates the production of inflammatory factors. Antimicrobial peptides alf and crustin1 are key genes for immune defense. Hsp70 and Hsp90 are both heat shock proteins, enabling animals to rapidly adapt to environmental changes and protecting cells from damage. They can induce and activate various immune factors, participating in immune responses. The results showed that feeding a fermented diet containing *Lactobacillus brevis* significantly upregulated the gene expression levels of toll1, toll2, pelle, tube, alf1, alf2, crustin, Hsp70, and Hsp90 (P < 0.05), and significantly downregulated the expression level of myd88 (P < 0.05). This indicates that the hepatopancreatic immune capacity of crabs was enhanced.
[0073] Figure 8 The effects of Lactobacillus brevis fermented feed on the expression of genes related to intestinal health in crabs were investigated. Figure 8A represents the effect on ilf2. Figure 8 B represents the effect on litaf. Figure 8 C represents the impact on p38MAPK. Figure 8 D represents the impact on Relish. Figure 8 E represents the impact on PM. Figure 8 F represents the effect on PT. Figure 8 G represents the effect on ZO-1. The expression levels of PT and PM can represent the health of the perifecal membrane barrier in the intestine of Chinese mitten crabs, and ZO-1 is related to intestinal permeability. ilf2 and litaf are two important pro-inflammatory factors in Chinese mitten crabs and are considered to be marker cytokines of inflammatory responses. p38MAPK initiates the release of litaf. The nuclear transcription factor relish is a member of the NF-κBs superfamily and is an important effector of immune signal transduction. The results showed that the expression levels of PT, PM, and ZO-1 were significantly upregulated in the Lactobacillus brevis fermented diet group (P < 0.05), and intestinal permeability was increased; the expression levels of pro-inflammatory factors were significantly downregulated (P < 0.05), indicating that Lactobacillus brevis has a good anti-inflammatory effect.
[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A Lactobacillus brevis Myh YC-01, characterized in that, The Lactobacillus brevis was deposited at the China General Microbiological Culture Collection Center on January 16, 2025, with accession number CGMCC No. 33441.
2. The application of the Lactobacillus brevis described in claim 1 in the preparation of fermented feed for crabs that improves liver and intestinal health and enhances the immune function of crabs.
3. A method for preparing fermented feed for river crabs, characterized in that, First, sterilized water is mixed evenly with basic feed, then the Lactobacillus brevis liquid described in claim 1 is added, and the mixture is anaerobic fermented at 37°C for 7 days to obtain fermented feed for river crabs. The basic feed consists of the following components in the indicated weight percentages: fish meal 8-12%, chicken meal 6-10%, hemoglobin meal 2-3%, soybean meal 18-22%, peanut meal 14-16%, rapeseed meal 15-17%, yeast protein 1-3%, wheat flour 11-13%, lysine 0.3-1%, methionine 0.1-0.3%, calcium dihydrogen phosphate 1-2.5%, soybean oil 2-4%, shrimp paste 1-3%, multivitamin and mineral premix 0.5-1.5%, antioxidant 0.1-0.2%, sodium humate 1-3%, molting agent 0.1-0.2%, and bentonite 4-4.5%.
4. The method for preparing fermented feed for river crabs as described in claim 3, characterized in that, The mass ratio of the basic feed to sterilized water is 1:0.
4.
5. The method for preparing fermented feed for river crabs as described in claim 3, characterized in that, The Lactobacillus brevis is added in an amount of 10 6 CFU / g.
6. The method for preparing fermented feed for river crabs as described in claim 3, characterized in that, The method for preparing the Lactobacillus brevis culture is as follows: a single colony of Lactobacillus brevis is picked from the isolated and purified solid culture medium and placed in MRS liquid culture medium. It is then anaerobically cultured at 37°C for 24 hours. The colony is then inoculated into MRS culture medium at an inoculation rate of 1% and anaerobically cultured at 37°C for 6-8 hours to obtain the Lactobacillus brevis culture.