A strain of Lactobacillus plantarum YYY01 and its application in anti-inflammation and production of γ-aminobutyric acid

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a strain of Lactobacillus plantarum YYY01 and its application in anti-inflammatory and production of γ-aminobutyric acid. The Lactobacillus plantarum YYY01 provided by the present invention is deposited with GDMCC NO: 65147. The Lactobacillus plantarum YYY01 has good biosafety, survives well in the gastrointestinal environment and has a certain intestinal adhesion ability; Lactobacillus plantarum YYY01 can produce high γ-aminobutyric acid, has anti-inflammatory, intestinal flora regulation and immune barrier enhancement effects. It shows good application prospects in increasing the yield of γ-aminobutyric acid and preparing products containing γ-aminobutyric acid.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a strain of Lactobacillus plantarum YYY01 and application thereof in anti-inflammation and production of gamma-aminobutyric acid. Background Art

[0002] GABA is a compound with the chemical formula C 4 H 9 NO 2 , also known as 4-aminobutyric acid (γ-aminobutyricacid, GABA), is an amino acid that is widely found in vertebrates, plants and microorganisms. In animals, plants and microorganisms, γ-aminobutyric acid is an important intermediate and plays an important role in the response of organisms to environmental stress. In mammals, γ-aminobutyric acid is an important inhibitory neurotransmitter in the nervous system, and plays a very important role in brain development, anti-anxiety, lowering blood pressure and promoting growth. At the same time, γ-aminobutyric acid is also an important bioactive ingredient additive in food. It has been confirmed that γ-aminobutyric acid, as a small molecular weight non-protein amino acid, is safe for consumption and can be used in the production of beverages and other foods. Studies have shown that the intake of a certain amount of γ-aminobutyric acid has physiological effects such as improving the body's sleep quality and lowering blood pressure. In summary, γ-aminobutyric acid has a very wide range of applications in the fields of medicine, food and health products.

[0003] As a common probiotic, lactic acid bacteria have glutamate decarboxylase activity, which can decarboxylate glutamate to produce γ-aminobutyric acid. It is an important type of microorganism that ferments and synthesizes γ-aminobutyric acid. Lactobacillus plantarum is a widely distributed and widely used lactic acid bacterium that can survive in the gastrointestinal tract of fermented foods, meat, plants and mammals. As one of the important components of the normal intestinal microbial system and accompanying the host throughout life, it is of great significance for maintaining the balance of intestinal microecology. In recent years, Lactobacillus plantarum has become a hot topic of research in recent years due to its huge characteristic advantages, and has been valued in the food and health care product industry. Patent CN118497086B isolates and purifies a strain of Lactobacillus plantarum from a kimchi sample, which has multiple functions such as high production of γ-aminobutyric acid, lowering cholesterol, lowering blood sugar and antioxidant properties. Patent CN116496948A discloses a strain of Lactobacillus plantarum that produces γ-aminobutyric acid, and the γ-aminobutyric acid content in the fermented radish product produced by the Lactobacillus plantarum reaches 0.20 mg / mL.

[0004] Therefore, the development of a plant lactobacillus that can produce γ-aminobutyric acid and has anti-inflammatory effects is of great significance in the fields of medicine, food, and health care products, and provides certain reference and guidance for the development of products rich in γ-aminobutyric acid. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a strain of Lactobacillus plantarum YYY01 and its application in anti-inflammatory and production of γ-aminobutyric acid. The Lactobacillus plantarum YYY01 provided by the present invention is deposited with GDMCC NO: 65147. The Lactobacillus plantarum YYY01 has good biosafety, survives well in the gastrointestinal environment and has a certain intestinal adhesion ability; Lactobacillus plantarum YYY01 can produce high γ-aminobutyric acid, has anti-inflammatory, regulates intestinal flora and enhances immune barrier effects. It shows good application prospects in increasing the yield of γ-aminobutyric acid and preparing products containing γ-aminobutyric acid.

[0006] The technical solution of the present invention includes:

[0007] In a first aspect, the present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum ), the plant lactobacillus is plant lactobacillus YYY01, and its deposit number is GDMCC NO: 65147.

[0008] In a second aspect, the present invention provides a Lactobacillus plantarum preparation, comprising one or more of the Lactobacillus plantarum cells, fermentation broth, fermentation supernatant, fermentation broth precipitate, and freeze-dried powder of claim 1.

[0009] Specifically, the lyophilized powder also includes a lyophilization protectant.

[0010] Preferably, the lyoprotectant comprises: one or more of fucose, trehalose, sucrose, lactose, stachyose, sorbitol, glycerol, erythritol, fructooligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides, inulin, isomaltooligosaccharides, resistant dextrin, whey protein, collagen, skim milk, xylose, arabinose, ribose, rhamnose, galactose, mannose, fructose, sorbose, galactitol, xylitol, mannitol, maltitol, lactitol, raffinose, manno-oligosaccharides, maltodextrin and polydextrose.

[0011] Further preferably, the lyoprotectant consists of xylooligosaccharides and sucrose.

[0012] In a further preferred embodiment, the weight ratio of xylooligosaccharide to sucrose is 1:1.

[0013] Specifically, it is characterized in that the preparation process of the freeze-dried powder comprises: evenly mixing the Lactobacillus plantarum YYY01 mud with a freeze-drying protective agent and then freeze-drying.

[0014] More specifically, the freeze-drying process is as follows:

[0015] S1, keep the temperature of the control plate at -40 ~ -60℃ for 3-5h;

[0016] S2, control the plate at -20 ~ -40℃, 0.1-0.3mbar for 20-40h;

[0017] S3, control the layer plate at 20-30℃ and 0mbar for 15-20h;

[0018] S4, repeat steps S1-S3 to perform secondary freeze-drying.

[0019] Preferably, the freeze-drying process is as follows:

[0020] S1, keep the control layer at -50℃ for 4h;

[0021] S2, control the shelf at -30℃, 0.1-0.3mbar for 30h;

[0022] S3, control the shelf at 25°C and 0 mbar for 18 hours;

[0023] S4, repeat steps S1-S3 to perform secondary freeze-drying.

[0024] Still more preferably, the freeze-drying process is as follows:

[0025] S1, control the temperature of the plate to drop from room temperature to -50℃ within 1 hour and maintain it for 4 hours;

[0026] S2, control the temperature of the layer plate to -30℃ for 1.3h and maintain it at a vacuum of 0.2mbar for 30h;

[0027] S3, control the temperature of the layer plate to 25℃ for 1 hour and maintain it under 0mbar vacuum condition for 18 hours;

[0028] S4, repeat steps S1-S3 to perform secondary freeze-drying.

[0029] In a third aspect, the present invention provides a fermentation method of the above-mentioned Lactobacillus plantarum, the fermentation method comprising: activating Lactobacillus plantarum YYY01 and then inoculating it into a culture medium.

[0030] Specifically, the fermentation method includes: bacterial activation, primary fermentation, secondary fermentation and tertiary fermentation.

[0031] More specifically, the primary fermentation is to inoculate Lactobacillus plantarum YYY01 into a seed culture medium for primary fermentation to obtain a primary fermentation liquid.

[0032] Preferably, the seed culture medium includes but is not limited to: MRS seed culture medium.

[0033] Preferably, the primary fermentation culture condition is 35-39° C. for 8-12 h.

[0034] More specifically, the secondary fermentation is to inoculate the primary fermentation liquid into the fermentation medium for secondary fermentation to obtain the secondary fermentation liquid.

[0035] Preferably, the inoculation amount of the primary fermentation broth is 1%-3%.

[0036] Further preferably, the inoculation amount of the primary fermentation broth is 2%.

[0037] Preferably, the conditions of the secondary fermentation culture are temperature 35-39° C., pH 5.0-7.0, and fermentation culture time 8-10 h.

[0038] Further preferably, the conditions of the secondary fermentation culture are temperature 37° C. and pH 6.0.

[0039] Preferably, the fermentation medium contains the following substances per liter:

[0040] Peptone 10g, beef extract 10g, yeast extract 5.0g, glucose 20g, Tween 80 1mL, dipotassium hydrogen phosphate 2g, sodium acetate 5, diammonium citrate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, dipotassium hydrogen phosphate 2.0g, sodium L-glutamate 5g, add deionized water to 1L. Adjust pH to 6.2±0.2.

[0041] More specifically, the tertiary fermentation is to inoculate the secondary fermentation liquid into the fermentation medium for tertiary fermentation culture.

[0042] Specifically, the inoculation amount of the secondary fermentation broth is 1%-3%.

[0043] Further preferably, the inoculation amount of the secondary fermentation broth is 2%.

[0044] Preferably, the conditions of the tertiary fermentation culture are temperature 35-39° C. and pH 5.0-7.0.

[0045] Further preferably, the conditions of the tertiary fermentation culture are temperature 37° C. and pH 6.0.

[0046] Preferably, the fermentation medium contains the following substances per liter:

[0047] Peptone 10g, beef extract 10g, yeast extract 5.0g, glucose 20g, Tween 80 1mL, dipotassium hydrogen phosphate 2g, sodium acetate 5, diammonium citrate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, dipotassium hydrogen phosphate 2.0g, sodium L-glutamate 5g, add deionized water to 1L. Adjust pH to 6.2±0.2.

[0048] Preferably, the three-stage fermentation culture is carried out until OD 600nm No more increase, fermentation is over.

[0049] Preferably, during the tertiary fermentation culture, when the carbon source is lower than 2 g / L, feed medium is added.

[0050] Further preferably, the feed medium is deionized water containing 400 g / L glucose.

[0051] In a fourth aspect, the present invention provides the use of the above-mentioned Lactobacillus plantarum or Lactobacillus plantarum preparation in increasing the yield of γ-aminobutyric acid.

[0052] In a fifth aspect, the present invention provides the use of the above-mentioned Lactobacillus plantarum or Lactobacillus plantarum preparation in the preparation of a product containing γ-aminobutyric acid.

[0053] Specifically, the products include food, health products or medicines.

[0054] Preferably, the health care product helps to regulate intestinal flora or enhance immunity.

[0055] Preferably, the medicine is used for preventing, treating or assisting in treating colitis.

[0056] In a sixth aspect, the present invention provides a food containing the above-mentioned Lactobacillus plantarum or the Lactobacillus plantarum preparation.

[0057] Specifically, the food also includes nutritional additives that are nutritionally acceptable.

[0058] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0059] Preferably, the food includes candy flakes, soy milk, yogurt, canned food, biscuits, chocolate, cakes, cream, cheese, sour cream, milk powder, formula milk powder, ice cream, jam, puree, candied fruit, preserved fruit, bread, egg rolls, protein drinks, solid drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee or puffed food.

[0060] Preferably, the food comprises human food or animal food.

[0061] In a seventh aspect, the present invention provides a health product containing the above-mentioned Lactobacillus plantarum or Lactobacillus plantarum preparation.

[0062] Specifically, the health care product also includes nutritional additives that are nutritionally acceptable.

[0063] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0064] Preferably, the dosage form of the health care product includes tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

[0065] Preferably, the health care product helps to regulate intestinal flora or enhance immunity.

[0066] In an eighth aspect, the present invention provides a medicine containing the above-mentioned Lactobacillus plantarum or Lactobacillus plantarum preparation.

[0067] Specifically, the medicine further includes one or more physiologically acceptable auxiliary materials or pharmaceutically acceptable excipients.

[0068] Preferably, the physiologically acceptable excipients include, but are not limited to, erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate or dextrin.

[0069] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0070] Preferably, the medicine is used for preventing, treating or assisting in treating colitis.

[0071] The beneficial effects of the present invention include:

[0072] The Lactobacillus plantarum YYY01 provided by the present invention has a deposit number of GDMCC NO: 65147. The Lactobacillus plantarum YYY01 has good biosafety, survives well in the gastrointestinal environment and has a certain intestinal adhesion ability; the Lactobacillus plantarum YYY01 can produce high amounts of γ-aminobutyric acid, has anti-inflammatory, intestinal flora regulating and immune barrier enhancing effects, and shows good application prospects in increasing the yield of γ-aminobutyric acid and preparing products containing γ-aminobutyric acid.

[0073] Collection Instructions

[0074] Deposited strains: Lactiplantibacillus plantarum YYY01

[0075] Category naming: Lactiplantibacillus plantarum ;

[0076] Deposit number: GDMCC NO: 65147;

[0077] Preservation time: September 20, 2024;

[0078] Depository: Guangdong Microbiological Culture Collection Center;

[0079] Collection address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 This is the colony morphology of Lactobacillus plantarum YYY01.

[0081] Figure 2 This is the cell morphology of Lactobacillus plantarum YYY01.

[0082] Figure 3 This is a Gram staining microscopic examination image of Lactobacillus plantarum YYY01.

[0083] Figure 4 It is the result of hemolytic evaluation test.

[0084] Figure 5 This is the result of the digestive tract environment resistance test.

[0085] Figure 6 The effect of Lactobacillus plantarum YYY01 on body weight changes.

[0086] Figure 7 The effect of Lactobacillus plantarum YYY01 on DAI index; **** in the figure represents P <0.0001.

[0087] Figure 8 The effect of Lactobacillus plantarum YYY01 on colon length; ns in the figure means no significant difference; *** means P <0.001; **** represents P <0.0001.

[0088] Fig. 9 The effect of Lactobacillus plantarum YYY01 on tissue pathology.

[0089] Fig.10 The effect of Lactobacillus plantarum YYY01 on TNF-α and IL-1β; ** in the figure representsP <0.01; *** represents P <0.001; **** represents P <0.0001.

[0090] Fig.11 The effect of Lactobacillus plantarum YYY01 on IL-6, IFN-γ and IL-17; * in the figure represents P <0.05; ** represents P<0.01; *** represents P <0.001; **** represents P <0.0001.

[0091] Fig.12 The effect of Lactobacillus plantarum YYY01 on the mRNA expression of ZO-1 in colon tissue; ns in the figure means no significant difference; * means P <0.05.

[0092] Fig.13 Effects of Lactobacillus plantarum YYY01 on colonic macrophage infiltration and morphology in colitis mice; ns in the figure represent no significant difference; * in the figure represent P <0.05; ** represents P <0.01; *** represents P <0.001.

[0093] Fig.14 The effect of Lactobacillus plantarum YYY01 on the diversity of intestinal flora; ns in the figure means no significant difference; * in the figure means P <0.05; ** represents P <0.01; *** represents P <0.001.

[0094] Fig.15 This is the effect of the blank group and DSS group on the β-diversity of intestinal flora.

[0095] Fig.16 This is the effect of DSS group and Lactobacillus plantarum YYY01 group on the β-diversity of intestinal flora.

[0096] Fig.17 This is the freeze-dried survival rate of Lactobacillus plantarum YYY01 in different types of protective agents.

[0097] Fig.18 This is the freeze-drying survival rate of Lactobacillus plantarum YYY01 in composite protective agents with different molecular weights. DETAILED DESCRIPTION

[0098] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0099] Basic Experimental Example 1 Materials and Instruments Used in the Present Invention

[0100] 1. Culture medium

[0101] MRS seed medium (content per liter): peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, K 2 HPO 4 2g, NH 4 C 6 H 5 O 7 2g, MgSO 4 7H 2 O 0.2g, MnSO 4 ·5H 2 O 0.05g, add deionized water to 1L.

[0102] Fermentation medium 1 (content per liter): yeast extract FM803 40g, glucose 100g, MgSO 4 7H 2 O 0.3g, MnSO 4 ·5H 2 O 0.35g, add deionized water to 1L.

[0103] Fermentation medium 2 (content per liter): peptone 10g, beef extract 10g, yeast extract 5.0g, glucose 20g, Tween 80 1mL, dipotassium hydrogen phosphate 2g, sodium acetate 5, diammonium citrate 2g, magnesium sulfate 0.2g, manganese sulfate 0.05g, dipotassium hydrogen phosphate 2.0g, sodium L-glutamate 5g, add deionized water to 1L. Adjust pH to 6.2±0.2

[0104] Feed medium: deionized water containing 400 g / L glucose.

[0105] Bromocresol purple-MRS solid medium (content per liter): peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g, triammonium citrate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.58g, manganese sulfate monohydrate 0.20, TWEEN-80 1mL, agar powder 15g, bromocresol purple 0.04g, add deionized water to 1L. Adjust pH to 6.5 and sterilize at 121℃ for 15min.

[0106] 2. Main instruments and equipment

[0107] The main equipment information used in the present invention is shown in Table 1:

[0108] Table 1 Main instrument and equipment information

[0109]

[0110] 3. Main reagents and materials

[0111] Yeast powder and peptone were purchased from Sigma Aldrich; pig bile salts, antibiotics, glycine and glutamic acid were purchased from Sinopharm Reagent Company; pepsin and trypsin were purchased from Beijing Chembase Technology Co., Ltd.; genome extraction kits and gene fragment recovery were purchased from Sangon Biotech (Shanghai) Co., Ltd.; DNA polymerase and DNA Marker were purchased from Takara.

[0112] Example 1 Isolation, purification and identification of strains

[0113] 1.1 Isolation and purification

[0114] Weigh 1.0g healthy breast milk, dilute it 10 times with 9.0mL 0.85% sterile saline, then take 0.1mL of different breast milk dilutions and spread them on bromocresol purple-MRS solid medium, culture at 37℃ for 48h and observe the colony morphology. Select a colony with uniform round protrusions, very smooth, dense, and yellow surface on the plate for streaking, repeat 2-3 times, and obtain a single colony with consistent morphology.

[0115] 1.2 Morphological observation

[0116] The purified lactic acid bacteria were streaked onto MRS plates and cultured at 36°C for 48 h. The colony morphology was observed and recorded. The colonies were stained with Gram stain and examined under an optical microscope to observe the bacterial morphology.

[0117] The morphological characteristics of the strains Figure 1 and Figure 2 As shown, Gram staining microscopy is shown in Figure 3As shown. The results showed that the phenotypic characteristics of the target strain included: Gram-positive, rod-shaped, non-spore-forming, facultative anaerobic, and able to ferment a variety of carbohydrates to produce lactic acid under anaerobic conditions. It grew on an MRS plate supplemented with bromocresol purple, and a yellow color change zone was produced around the colony.

[0118] 1.3 Molecular biological identification

[0119] Extract the genomic DNA of the strain according to the instructions of the bacterial genome extraction kit. Use the genomic DNA of the strain as a template and use universal primers for PCR amplification. Detect the PCR amplification product by agarose gel electrophoresis to observe whether there is a clear target band. Sequence the PCR amplification product, and its 16s rDNA sequence result is shown in SEQ ID NO.1. The sequencing results were subjected to BLAST analysis on the NCBI website, and the sequence information was compared with the known sequences in GenBank, and then the phylogenetic tree was constructed using the neighbor-joining method using MEGAX software.

[0120] SEQ ID NO.1:

[0121]

[0122] Lactobacillus plantarum Lactiplantibacillus plantarum GCF 014131735.1 is on a branch and has the closest relationship. Combined with the morphological results, strain YYY01 was finally identified as a plant lactobacillus ( Lactiplantibacillus plantarum ).

[0123] 1.4 Strain preservation

[0124] The above strain was deposited in Guangdong Microbial Culture Collection Center on September 20, 2024, with the deposit number GDMCC NO: 65147, and was named Lactobacillus plantarum ( Lactiplantibacillus plantarum )YYY01.

[0125] Example 2 Safety Evaluation

[0126] 2.1 Drug resistance evaluation

[0127] The drug sensitivity of Lactobacillus plantarum YYY01 was tested, and the MIC values ​​were compared according to the (NCCLS) guidelines provided by the World Health Organization (WHO). The results are shown in Table 2 below.

[0128] Table 2 Drug resistance evaluation

[0129]

[0130] Note: In the table, "R" stands for drug resistance; "I" stands for intermediate sensitivity; "S" stands for sensitive; and "resistant" stands for drug resistance.

[0131] The analysis results show that lactobacillus plantarum derived from breast milk is resistant to multiple antimicrobial drugs such as aminoglycosides and glycopeptides; it is moderately sensitive to the antimicrobial effects of tetracyclines; and it shows a certain degree of drug sensitivity to antimicrobial drugs such as penicillins, macrolides, cephalexin, and chloramphenicol.

[0132] 2.2 Hemolytic evaluation

[0133] Lactobacillus may become potential hemolytic bacteria in some cases, and in severe cases, it may lead to the occurrence of sepsis. Therefore, hemolytic assay is one of the indicators that must be measured for lactic acid bacteria. In this example, the plate detection method (GB 4789.11) was used to find that Lactobacillus plantarum YYY01 had no hemolytic phenomenon on the blood plate, indicating that the strain had no hemolytic ability ( Figure 4 ).

[0134] Example 3 Resistance to digestive tract environment

[0135] Artificial gastric juice preparation: KH 2 PO 4 0.24g / L, Na 2HPO 4 1.44 g / L, NaCl 8.00 g / L, KCl 0.20 g / L, pepsin 3.00 g / L, adjust the pH to 2.5 with 0.1 mol / L HCl, and filter through a 0.22 μm filter membrane.

[0136] Artificial intestinal fluid configuration: KH 2 PO 4 0.24g / L, Na 2 HPO 4 1.44g / L, NaCl 8.00g / L, KCl 0.20g / L, trypsin 1.00g / L, porcine bile salt 18.00g / L, 0.1mol / L NaOH adjusted to pH 8.0, and filtered through a 0.22μm filter membrane.

[0137] Preparation of strain fermentation broth: The activated Lactobacillus plantarum YYY01 was inoculated into fermentation medium 1 at a 5% inoculum amount and cultured at a pH of 6.5 and a temperature of 37° C. for 24 h.

[0138] The fermentation liquid of Lactobacillus plantarum YYY01 cultured for 24 hours was inoculated into artificial gastric fluid at a volume ratio of 1:10, and cultured at a constant temperature of 37°C. Samples were taken at 0, 1, 2, and 3 hours, and the live bacteria in the artificial gastric fluid were diluted and spread for counting. The suspension inoculated in artificial gastric fluid for 3 hours was further added to artificial intestinal fluid at a volume ratio of 1:10, and samples were taken and counted at 4, 5, 6, 7, and 8 hours to obtain the number of live bacteria CFU / mL, and lg (CFU) was taken for graphical analysis.

[0139] The results of the test are as follows Figure 5 As shown, when the fermentation liquid of Lactobacillus plantarum YYY01 was introduced into the artificial simulated gastrointestinal environment, the number of live bacteria decreased slightly, and the inhibitory effect of the strong acid and anaerobic environment was not significant. Lactobacillus plantarum YYY01 showed good resistance to artificial gastric juice and intestinal juice, indicating that it can survive well in the gastrointestinal environment.

[0140] Example 4 Evaluation of hydrophobicity and self-aggregation

[0141] 4.1 Hydrophobicity evaluation

[0142] The fermentation liquid of Lactobacillus plantarum YYY01 cultured for 24 hours was centrifuged at 3000r / min for 10 minutes, the supernatant was discarded, and the liquid was resuspended in distilled water. The concentration of the liquid was adjusted to make the OD 600 0.8-1, 3 mL of the adjusted bacterial suspension was mixed with 1 mL of n-butanol, n-hexane, and ethyl acetate for 2 min, and allowed to stand at 37 °C for 3 h. The non-organic phase was taken to measure its OD 600 , the surface hydrophobicity is calculated according to the following formula:

[0143] Surface hydrophobicity%=(1-A i / A 0 )×100%;

[0144] Where: A i is the OD of the aqueous phase after 3 hours 600 ; A 0 Initial OD 600 .

[0145] 4.2 Self-aggregation evaluation

[0146] The fermentation liquid of Lactobacillus plantarum YYY01 cultured for 24 hours was centrifuged at 3000r / min for 10 minutes, the supernatant was discarded, and the liquid was resuspended in distilled water. The concentration of the liquid was adjusted to make the OD 600 0 is 0.8-1, and after adjusting the concentration, place at 37°C for 24h and measure the OD of the supernatant 600 , find the self-aggregation rate:

[0147] Self-aggregation rate % = (1-A i / A 0 )×100%;

[0148] Where: A i OD for 24 h treatment 600 ; A0 is the initial OD 600 .

[0149] The test results showed that the surface hydrophobicity of Lactobacillus plantarum YYY01 was 74.03% and the 24h surface aggregation was 84.65%. The intestinal adhesion ability of lactic acid bacteria plays an important role in its probiotic function. The self-aggregation rate and hydrophobicity reflect the adhesion of the strain to intestinal epithelial cells. The higher the self-aggregation rate and hydrophobicity, the stronger the adhesion ability. The surface characteristics of Lactobacillus plantarum YYY01 have high adhesion. The results show that they have a certain intestinal adhesion ability, which has an important impact on maintaining the balance and health of the host's intestinal flora.

[0150] Example 5 GABA production function

[0151] Activation of strains: Streak Lactobacillus plantarum YYY01 stored in a -80°C glycerol tube onto an MRS plate using an inoculation loop and culture at 37°C for 12 h to activate the strain.

[0152] Primary fermentation: Pick the bacterial moss into 5 mL of MRS seed medium and culture at 37°C for 10 h.

[0153] Secondary fermentation: The primary fermentation liquid is inoculated into the seed tank (inoculation amount 2%). The seed tank uses fermentation medium 2, cultured at 37°C and pH 6.0. After fermentation culture for 8-10 hours, it is inoculated into the fermentation tank.

[0154] The third stage fermentation was inoculated with 2% of the second stage fermentation broth, pH 6.0, temperature 37°C. When the carbon source in fermentation medium 2 was lower than 2 g / L, feed medium (8 mL / (L·h)) was added until OD 600nm No more increase, fermentation ends (about 11-13h).

[0155] Determination method: Use GABA standard to accurately prepare 0.25, 0.5, 0.75, 1.00, 1.25, 1.50 mg / mL standard solutions, take 0.5 mL of each and place in an ice bath, add 0.2 mL of pH 9.0 boric acid-borax buffer solution, 1 mL of 6% phenol solution, 0.4 mL of sodium hypochlorite solution, shake thoroughly, react in a water bath for 7 minutes, cool in an ice bath for 9 minutes, add 2 mL of distilled water, and measure the absorbance at 630 nm. Draw a standard curve with concentration as the horizontal axis and absorbance as the vertical axis (the standard curve is y = 1.6752x - 0.4945). Take 0.5 mL of fermentation broth and place in an ice bath, add 0.2 mL of buffer solution, 1 mL of phenol solution, and 0.4 mL of sodium hypochlorite solution, shake thoroughly, react in a water bath for 7 minutes, cool in an ice bath for 9 minutes, add 2 mL of distilled water, measure the absorbance at 630 nm, and calculate the GABA content in the sample. The measurement results showed that the concentration of γ-aminobutyric acid was 0.43g / L.

[0156] Example 6 Evaluation of anti-inflammatory efficacy

[0157] C57BL / 6J mice are an ideal model strain for dextran sodium sulfate (DSS)-induced colitis, so C57BL / 6J mice were selected in this experiment. C57BL / 6J mice were 6-8 weeks old male mice, and there were 10 mice in each group.

[0158] 6.1 Experimental Animal Grouping

[0159] The specific groups, treatment methods for each group and modeling methods are described in Table 3:

[0160] Table 3 Experimental animal groups

[0161] Group Name 1-7 days 8-14 days 15 days Blank control group Free drinking of distilled water + intragastric administration of normal saline as control Free drinking of distilled water + intragastric administration of normal saline as control Sacrifice and take samples and test various indicators DSS Group Free drinking of distilled water + intragastric administration of normal saline as control Free drinking of DSS water (2.5%) + intragastric administration of normal saline as control Sacrifice and take samples and test various indicators Positive drug group Free drinking of distilled water + oral administration of 10 mg / mL mesalazine 200 μL Free drinking of DSS water (2.5%) + intragastric administration of 10 mg / mL mesalazine 200 μL Sacrifice and take samples and test various indicators Lactobacillus plantarum YYY01 group <![CDATA[Free drinking of distilled water + intragastric administration of 5 × 10 9 CFU / mL Lactiplantibacillus plantarum YYY01, 200 μL]]> <![CDATA[Free drinking of DSS water (2.5%) + intragastric administration of 5 × 10 9 CFU / mL Lactiplantibacillus plantarum YYY01, 200 μL]]> Sacrifice and take samples and test various indicators

[0162] The specific processing is as follows:

[0163] Week 1: Adaptation period

[0164] Week 2-3 (experimental period: 0-14 days, modeling is 8-14 days):

[0165] Normal control group (Con group): During the entire experimental period (day 0-day 14), mice had normal diet and water, and were gavaged with 200 μL of normal saline as a control;

[0166] Modeling group (DSS group): Drinking 2.5% DSS solution from the 8th day to the end of modeling for 7 consecutive days, and gavage with 200 μL of normal saline as a control during the whole experiment;

[0167] Positive drug group (5-ASA group): 7 days before modeling, mice were gavaged with 200 μL of mesalazine at a concentration of 10 mg / mL, and drank 2.5% DSS solution from the 8th day;

[0168] Lactobacillus plantarum YYY01 group: 7 days before modeling, mice were gavaged with 200 μL of Lactobacillus plantarum YYY01, with a viable count of 5×10 9 CFU / mL, drinking 2.5% DSS solution from the 8th day.

[0169] After the experiment, mice were anesthetized with 1% sodium pentobarbital solution and then killed by cervical dislocation. After blood was collected, the blood was allowed to stand for 30 minutes and then centrifuged at 2500g to obtain serum. The ileum, cecum, liver, spleen, etc. of the mice were immediately placed in liquid nitrogen; 1 cm of the distal colon was removed and immediately placed in 4% paraformaldehyde solution, the contents of the remaining colon were scraped out, and the contents and intestinal tube were placed in 1.5 mL EP tubes, respectively, and immediately placed in liquid nitrogen.

[0170] 6.2 Experimental Methods

[0171] 6.2.1 Disease Activity Index

[0172] During the modeling period, the mice were weighed, feces were collected, blood in the stool was measured, and the DAI index was calculated. The disease activity index (DAI) is an indicator of the degree of colitis inflammation. The score refers to Murthy's scoring system, which includes three aspects: weight change, blood in the stool, and stool characteristics. DAI = weight change score + blood in the stool score + stool characteristics score. The specific scores are shown in Table 4.

[0173] Table 4 Disease Activity Index Scoring Criteria

[0174]

[0175] 6.2.2 Colon length measurement

[0176] The length of the colon reflects the severity of colitis in mice to a certain extent. After the mice were killed, the entire colon (from the end of the cecum to the anus) was taken and the length was measured.

[0177] 6.2.3 Colon paraffin sections, H&E staining, and tissue damage scores

[0178] Tissue sections and tissue scores are the most important indicators for determining the severity of colitis. Therefore, we measured this. After the mice were killed, 1 cm of the distal colon (1 cm from the anus) was taken for subsequent fixation, embedding, sectioning, staining and other pathological studies. The Dieleman scoring system was used to score the tissue damage of the colon tissue sections of each group. The tissue damage score includes four aspects: degree of inflammation, lesion depth, crypt destruction and lesion range. The specific standards are shown in Table 5.

[0179] Table 5 Tissue damage scoring criteria

[0180]

[0181] 6.2.4 ELISA determination of inflammatory factors in colon tissue

[0182] The determination was performed according to the instructions of the ELISA kits for TNF-α, IL-1β, IL-6, IL-10 and IL-5. The ELISA kits were purchased from Shanghai ELISA Co., Ltd.

[0183] 6.2.5 Number of total macrophages and M1 macrophages in colon tissues of mice in different groups

[0184] Colon tissue was placed in 4% paraformaldehyde fixative, dehydrated, embedded, and sliced. The slices were then placed in sodium citrate buffer (pH 6.0, 0.01 mol / L) and blocked with goat serum for 1 h. The samples were incubated with primary monoclonal antibodies (iNOS, F4 / 80), incubated with secondary antibodies, and DAPI was added to the samples. The slices were sealed with 50% glycerol. iNOS was detected using confocal laser scanning microscopy + and F4 / 80 + ImageJ was used to calculate F4 / 80 + Cells and iNOS + The number of cells.

[0185] 6.2.6 Alcian blue staining and PAS goblet cell staining

[0186] The intestine is covered with a layer of mucus, which protects the intestine as a physical barrier. The main component of the mucus layer is the mucin MUC2 secreted by goblet cells, and Alcian blue can dye mucin blue, so the present invention uses Alcian blue staining to detect the integrity of the colon mucus layer. After the mouse was killed, 1 cm of the distal colon was taken, paraffin sectioned, and then Alcian blue staining and PAS goblet cell staining were performed.

[0187] 6.2.6 Immunohistochemistry of colonic tight junction proteins

[0188] In order to understand the distribution of tight junction proteins in the colon, the present invention uses immunohistochemistry to measure the distribution of tight junction proteins.

[0189] 6.2.7 Determination of intestinal flora

[0190] During the modeling period, 2-3 feces were collected from each mouse and immediately placed in a -80°C refrigerator for subsequent intestinal flora measurement.

[0191] 6.3 Experimental Results

[0192] 6.3.1 Effects of Lactobacillus plantarum YYY01 on pathological parameters of colitis mice

[0193] During the DSS modeling period, mice will experience a sharp decrease in weight, shortened colon length, diarrhea, and bloody stools. Therefore, measuring the weight, DAI index, and colon length after dissection during the modeling period can well reveal the effect of Lactobacillus plantarum YYY01 intervention in colitis mice. The results are shown in Figure 6 . First, the effect of Lactobacillus plantarum on the body weight of mice during DSS modeling was analyzed. On the seventh day of modeling, the body weight of mice in the DSS group decreased by 11.0%, while the intervention of mesalazine and Lactobacillus plantarum YYY01 significantly inhibited the decrease in body weight. Therefore, inhibiting the weight loss of colitis mice is an important manifestation of lactic acid bacteria in alleviating colitis.

[0194] The DAI index is a comprehensive score of weight change, blood in stool and stool characteristics, which can better reflect the success of DSS modeling. The results of DAI index determination are shown in Figure 7 The DAI index of mice in the DSS group reached 9.8 points on the seventh day of modeling. The mice had visible blood in their stools and loose stools, and their body weight dropped by 11.0%, indicating that the colitis modeling was successful. Compared with the modeling group, the DAI index of mice in the mesalazine and Lactobacillus plantarum YYY01 groups decreased by 48.9% and 20.4%, respectively ( p <0.01).

[0195] The length of the mouse colon is closely related to inflammation and is one of the key indicators to characterize the degree of inflammation. Therefore, after the mouse is dissected, the length of the complete colon is measured. Figure 8 The colon length of mice in the DSS group was 2.97 cm, which was significantly shorter than that of the blank group (6.21 cm). The colon length of mice in the mesalazine and Lactobacillus plantarum YYY01 groups increased to 4.86 cm and 4.57 cm, respectively. p <0.05). Therefore, Lactobacillus plantarum YYY01 has the effect of reducing the disease activity index score of colitis in mice.

[0196] Colon section Fig. 9As shown in the figure, the blank group mice had normal crypts, evenly distributed goblet cells, and no inflammatory cell infiltration. The DSS group mice had lost crypts, partially lost goblet cells, partially depleted mucin, and severe inflammatory cell infiltration. Compared with the DSS group, the colon crypts of the mice in the Lactobacillus plantarum YYY01 group were restored, the number of goblet cells was restored, mucin increased, and there was almost no inflammatory cell infiltration, which significantly alleviated the colon pathology of the mice. Therefore, Lactobacillus plantarum YYY01 can effectively slow down the severity of histopathology in mice with colitis.

[0197] 6.3.2 Regulation mechanism of Lactobacillus plantarum YYY01 on the immune barrier of colitis mice

[0198] (1) Regulatory effect of Lactobacillus plantarum YYY01 on cytokines

[0199] IBD patients are often accompanied by severe immune response disorders. Epithelial cells can respond to bacterial invasion by secreting IL-8, TNF-α, and IL-6, and then recruit neutrophils, macrophages, and T cells to enhance protective immunity. Cytokines play an important role in regulating the immune system, and contain anti-inflammatory cytokines and pro-inflammatory cytokines in inflammatory responses, which are related to Th1 or Th2 cells. Therefore, the pro-inflammatory and anti-inflammatory cytokines in the colon of mice after intervention with Lactobacillus plantarum YYY01 were measured.

[0200] Depend on Fig.10 The quantitative results showed that the TNF-α concentration in the blank group was 5.98±0.30pg / mL, while that in the DSS group was 37.41±3.76pg / mL. Under the intervention of Lactobacillus plantarum YYY01, the TNF-α concentration was significantly reduced to 25.78±3.64g / mL, a decrease of 31.15% compared with the DSS group. The IL-1β concentration in the blank group was 172.86±76.78pg / mL, while that in the DSS group was 3199.24±281.13pg / mL. Under the intervention of Lactobacillus plantarum YYY01, the IL-1β concentration was significantly reduced to 2119.01±232.01pg / mL, a decrease of 33.77% compared with the DSS group.

[0201] Depend on Fig.11The quantitative results showed that the IL-6 concentration in the blank group was 11.88±3.11pg / mL, while the IL-6 concentration in the DSS group was 410.35±61.86pg / mL. Under the intervention of Lactobacillus plantarum YYY01, the IL-6 concentration was significantly reduced to 110.66±20.87pg / mL, which was 73.03% lower than that in the DSS group. The IFN-γ concentration in the blank group was 9.9±0.92pg / mL, while the IFN-γ concentration in the DSS group was 24.09±4.06pg / mL. Under the intervention of Lactobacillus plantarum YYY01, the IFN-γ concentration was significantly reduced to 13.7±3.19pg / mL, which was 43.1% lower than that in the DSS group. The IL-17 concentration in the blank group was 11.86±1.82pg / mL, while the IL-17 concentration in the DSS group was 62.83±3.73pg / mL. The IL-17 concentration was significantly reduced to 29.95±3.29pg / mL under the intervention of Lactobacillus plantarum YYY01, which was reduced by 52.33% compared with the DSS group.

[0202] (2) Effect of Lactobacillus plantarum YYY01 on the mRNA expression of ZO-1 in colon tissue

[0203] Depend on Fig.12 The quantitative results showed that the mRNA expression of ZO-1 in the blank group was 1.42±0.14, the mRNA expression of ZO-1 in the DSS group was 0.77±0.22, and the mRNA expression of ZO-1 under the intervention of Lactobacillus plantarum YYY01 was significantly increased to 1.20±0.16, which was 61.04% higher than that in the DSS group, and there was no significant difference with the blank group. Therefore, Lactobacillus plantarum YYY01 can effectively promote the mRNA expression of tight junction protein ZO-1 in colon tissue.

[0204] (3) Effects of Lactobacillus plantarum YYY01 on colonic macrophage infiltration and morphology in colitis mice

[0205] Infiltration of lamina propria macrophages and M1 macrophages aggravates colitis in mice. F4 / 80+ cells were used to replace total lamina propria macrophages, and iNOS+ cells were used to replace M1 macrophages. Fig.13The quantitative results showed that the total number of macrophages in the blank group was 23.33±1.53, while the total number of macrophages in the DSS group was 29.33±1.53. The total number of macrophages under the intervention of Lactobacillus plantarum YYY01 was 25.67±2.52, which was 3.66 less than that in the DSS group. The quantitative results showed that the number of M1 macrophages in the blank group was 8.67±0.58, while the number of M1 macrophages in the DSS group was 12.33±0.58. The number of M1 macrophages under the intervention of Lactobacillus plantarum YYY01 was 9.67±0.58, which was 1.13 less than that in the DSS group, and there was no significant difference with the blank group. Therefore, Lactobacillus plantarum YYY01 can effectively reduce the number of total macrophages and M1 macrophages in the colon.

[0206] 6.3.3 Effects of Lactobacillus plantarum YYY01 on intestinal flora

[0207] (1) Effect of Lactobacillus plantarum YYY01 on intestinal flora diversity

[0208] Dysbiosis of the intestinal microbiota is an important factor in the pathogenesis of colitis. Mannosyl-exopolysaccharide is a promising prebiotic candidate for alleviating colitis and has the potential to modulate the intestinal microbiota. Fig.14 It can be seen that the Shannon index of the blank group was 6.33±0.35, and that of the DSS group was 5.07±0.25. The Shannon index of mice intervened by Lactobacillus plantarum YYY01 was 5.87±0.15, which was 15.78% higher than that of the DSS group, and there was no significant difference with the blank group. The number of species observed in the blank group was 406.7±20.82, and that in the DSS group was 256.67±30.55. The number of species observed in mice intervened by Lactobacillus plantarum YYY01 was 411.33±8.08, which was 60.25% higher than that of the DSS group, and there was no significant difference with the blank group. Both the Shannon index and the number of species observed indicate that Lactobacillus plantarum YYY01 significantly improved the species diversity of the microbiota of DSS mice.

[0209] (2) Effect of Lactobacillus plantarum YYY01 on the β-diversity of intestinal flora

[0210] β-diversity shows the differences in microbial communities between sample groups. Figure 15-16 As shown, there was a significant difference in β-diversity between the blank group mice and the DSS group mice ( P <0.05, indicating that DSS-induced colitis would change the distribution of intestinal flora in mice. There was a significant difference in the intestinal flora between mice treated with Lactobacillus plantarum YYY01 and mice in the DSS group ( P <0.05), indicating that Lactobacillus plantarum YYY01 can change the species composition of the intestinal flora of mice.

[0211] Example 7 Lyophilized Agent of Lactobacillus plantarum YYY01

[0212] 7.1 Screening of Lyoprotectants

[0213] Lyoprotectants: blank, fucose, trehalose, sucrose, lactose, stachyose, sorbitol, glycerol, erythritol, fructooligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides, inulin, isomaltooligosaccharides, resistant dextrin, whey protein, collagen, skim milk, xylose, arabinose, ribose, rhamnose, galactose, mannose, fructose, sorbose, galactitol, xylitol, mannitol, maltitol, lactitol, raffinose, manno-oligosaccharides, maltodextrin, polydextrose, etc. Lyoprotectants and Lactobacillus plantarum YYY01 mud are compounded in a 1:1 ratio.

[0214] Freeze-drying process: control the temperature of the plate to drop from room temperature to -50℃ within 1 hour and maintain it for 4 hours; control the temperature of the plate to -30℃ within 1.3 hours and maintain it at a vacuum of 0.2mbar for 30 hours; control the temperature of the plate to rise to 25℃ within 1 hour and maintain it at a vacuum of 0mbar for 18 hours for secondary drying. Use the colony plate counting method to count the live bacteria of the sample before freeze-drying and record the data. After freeze-drying, take out and rehydrate the sample to the volume before freeze-drying, and repeat the above operation to determine the number of live bacteria contained in the sample.

[0215] At present, the most commonly used protective agents are sugars (alcohols), proteins and polymers. Sugar (alcohol) protective agents have multiple hydroxyl groups, which can replace water molecules to form hydrogen bonds with phosphate groups in the bacterial cell membrane phospholipids or with polar groups in bacterial proteins during the freeze-drying process, thereby avoiding damage to the integrity of the cell membrane and protein structure and function; protein protective agents can form a protein film on the surface of bacteria, stabilize the bacterial cell membrane, provide a protective coat for the bacteria, and protect the bacteria from damage due to exposure to the external environment. In order to systematically explore the freeze-drying protective effects of these sugar (alcohol), protein and polymer protective agents on Lactobacillus, single-factor experiments were conducted on the sugar (alcohol), protein and polymer protective agents reported in the literature. Fig.17 It can be seen that the protection effects of most of the 34 different protective agents are significantly higher than those of the control sample of sterile water, among which the freeze-dried protection effect of oligosaccharides is the best, and the survival rate of Lactobacillus plantarum YYY01 after freeze-drying reaches about 60%.

[0216] 7.2 Effects of Lyoprotectants of Different Molecular Weights on Bacteria

[0217] By analyzing the freeze-drying protective effects of different types of freeze-drying protectants on Lactobacillus plantarum, it was confirmed that the protection methods of large and small molecules are different ( Fig.17). According to previous studies, a single protective agent cannot meet the requirements of freeze drying and bacterial resistance to adverse external conditions, so it is generally used in a mixed formula. Therefore, protective agents with different molecular weights, large molecular sugars (fructose, xylo-oligosaccharides, manno-oligosaccharides) and small molecular sugars (sucrose, stachyose) are selected and mixed in pairs (weight ratio 1:1) for freeze drying of Lactobacillus plantarum YYY01.

[0218] The results of the test are as follows Fig.18 As shown, the results showed that among the combined groups, only the oligoxylose + sucrose group was significantly improved compared with the single oligosaccharide group, while the other groups did not show significant improvement.

[0219] 7.3 Freeze-drying process of Lactobacillus plantarum YYY01

[0220] In summary, the freeze-drying process of Lactobacillus plantarum YYY01 is as follows:

[0221] Formula of freeze-dried protective agent: oligoxylose and sucrose protective agent. Preparation: Weigh and dissolve each component according to a weight ratio of 1:1, sterilize at 115°C for 20 minutes, and after the temperature drops to room temperature, refrigerate in a -4°C refrigerator for use.

[0222] After the fermentation of Lactobacillus plantarum YYY01 is completed, the bacterial sludge is collected by centrifugation. After the bacterial sludge is collected, a lyophilization protective agent is added according to a mass / volume ratio of bacterial sludge (kg): lyophilization protective agent (L) = 1:1, and the mixture is stirred to obtain a uniformly mixed bacterial powder.

[0223] Freeze drying: The evenly mixed bacterial powder was divided into freeze dryer trays, and then the freeze dryer trays were placed in the freeze dryer for freeze drying of the bacterial powder; the freeze drying process parameters were set as follows: the temperature of the control layer plate was cooled from room temperature to -50°C within 1 hour and maintained for 4 hours; the temperature of the control layer plate was heated to -30°C within 1.3 hours and maintained at a vacuum degree of 0.2 mbar for 30 hours; the temperature of the control layer plate was heated to 25°C within 1 hour and maintained at a vacuum condition of 0 mbar for 18 hours for secondary drying.

[0224] The freeze-dried powder was collected and the viable count was 4.1×10 11 CFU / g.

[0225] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized in that, The plant lactobacillus is plant lactobacillus YYY01, and its deposit number is GDMCC NO: 65147.

2. A plant lactobacillus preparation, characterized in that The plant lactobacillus preparation comprises one or more of the plant lactobacillus cells, fermentation liquid and freeze-dried powder of claim 1.

3. The plant lactobacillus preparation according to claim 2, characterized in that The freeze-dried powder also includes a freeze-dried protective agent; the freeze-dried protective agent includes: one or more of fucose, trehalose, sucrose, lactose, stachyose, sorbitol, glycerol, erythritol, fructooligosaccharides, xylooligosaccharides, galacto-oligosaccharides, inulin, isomaltooligosaccharides, resistant dextrin, whey protein, collagen, skim milk, xylose, arabinose, ribose, rhamnose, galactose, mannose, fructose, sorbose, galactitol, xylitol, mannitol, maltitol, lactitol, raffinose, manno-oligosaccharides, maltodextrin, and polydextrose.

4. The plant lactobacillus preparation according to claim 3, characterized in that The freeze-drying protective agent consists of oligoxylose and sucrose; the weight ratio of the oligoxylose to the sucrose is 1:

1.

5. The plant lactobacillus preparation according to claim 2, characterized in that The preparation process of the freeze-dried powder comprises: mixing the Lactobacillus plantarum YYY01 bacterial sludge and the freeze-drying protective agent uniformly and then freeze-drying; The freeze-drying process is as follows: S1, keep the temperature of the control plate at -40 ~ -60℃ for 3-5h; S2, control the plate at -20 ~ -40℃, 0.1-0.3mbar for 20-40h; S3, control the layer plate at 20-30℃ and 0mbar for 15-20h; S4, repeat steps S1-S3 to perform secondary freeze-drying.

6. The fermentation method of plant lactobacillus according to claim 1, characterized in that: The fermentation method comprises: activating plant lactobacillus YYY01 and then inoculating it into a culture medium.

7. Use of the Lactobacillus plantarum according to claim 1 or the Lactobacillus plantarum preparation according to any one of claims 2 to 5 in increasing the yield of γ-aminobutyric acid.

8. Use of the Lactobacillus plantarum according to claim 1 or the Lactobacillus plantarum preparation according to any one of claims 2 to 5 in the preparation of products containing γ-aminobutyric acid; the products include foods, health products or medicines.

9. The use according to claim 8, characterized in that: The health care product is helpful to regulate intestinal flora or enhance immunity; the medicine is used for preventing, treating or assisting in the treatment of colitis.

10. A product containing the Lactobacillus plantarum according to claim 1 or the Lactobacillus plantarum preparation according to any one of claims 2 to 4, characterized in that: The products include food, health products or medicines.

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