Lactobacillus plantarum strain LM2 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum strain LM2 and application of the lactobacillus plantarum strain LM2. The strain has relatively high GABA (gamma-aminobutyric acid) production capacity and nitrite degradation capacity, has a bacteriostatic effect on intestinal pathogens such as staphylococcus aureus, salmonella enteritidis, listeria monocytogenes and campylobacter jejuni, has a good bacteriostatic function, also has good acid resistance and cholate resistance, and can be used for preparing a bacterial strain for treating intestinal diseases. The strain can well survive in artificial intestinal juice and artificial gastric juice; in order to increase the yield of GABA, the accelerant capable of remarkably promoting the growth of the strain LM2 is obtained by researching the accelerant, and the highest survival rate of the freeze-dried powder prepared by optimizing the accelerant and the freeze-dried powder can reach 99% or above, the nitrite degradation rate can reach 99% or above, the yield of GABA is increased to 3.0 g / L or above, and the freeze-dried powder is bacterial powder capable of being widely applied.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to Lactobacillus plantarum strain LM2 and its application. BACKGROUND

[0002] Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid, which is an important inhibitory neurotransmitter in the central nervous system of the human body. A large number of studies have shown that GABA has multiple physiological functions, including mood regulation, blood pressure reduction, anti-diabetic, anti-inflammatory, anti-cancer and other effects, and has attracted much attention in the fields of medicine and functional foods.

[0003] Nitrite is a class of compounds containing nitrite root anion (NO2 - ), which usually exists in the form of salt, has antioxidant effect, is often used as food additive for meat curing products, and also has the effects of bacteriostasis, color protection, and taste improvement. If nitrite is used improperly or is ingested in excess, it can cause toxic effects, and even endanger life in severe cases; under acidic and high-temperature conditions, it is easy to react with protein decomposition products to generate carcinogenic nitrosamines. Epidemiological studies have confirmed that long-term excessive intake of nitrite not only causes hypoxia poisoning in the human body, but also is significantly positively correlated with the incidence of cardiovascular diseases such as hypertension; the higher the intake, the higher the incidence of breast cancer or prostate cancer.

[0004] Nitrite is a class of compounds containing nitrite root anion (NO2 - ), which usually exists in the form of salt, has antioxidant effect, is often used as food additive for meat curing products, and also has the effects of bacteriostasis, color protection, and taste improvement. If nitrite is used improperly or is ingested in excess, it can cause toxic effects, and even endanger life in severe cases; under acidic and high-temperature conditions, it is easy to react with protein decomposition products to generate carcinogenic nitrosamines. Epidemiological studies have confirmed that long-term excessive intake of nitrite not only causes hypoxia poisoning in the human body, but also is significantly positively correlated with the incidence of cardiovascular diseases such as hypertension; the higher the intake, the higher the incidence of breast cancer or prostate cancer.

[0005] Currently, the preparation of GABA is mainly carried out by chemical synthesis, biological catalysis, microbial fermentation and the like. The chemical synthesis method has high production rate, but the product has many impurities, and the raw materials or reagents used are toxic, so the synthesized GABA is not suitable for application in food, medicine and the like. The biological catalysis method has high synthesis efficiency and purity, but the catalyst is high in cost and easy to be inactivated, and the substrate is narrow in application range, so it is limited in large-scale production. The microbial fermentation method utilizes endogenous glutamate decarboxylase (GAD) in microbial cells to catalyze L-glutamic acid decarboxylation to generate GABA and carbon dioxide (CO2), and has low preparation cost, simple process and is easy to scale up. Yeast, lactic acid bacteria, fungi and many other microorganisms have the ability to synthesize GABA, which is mainly catalyzed by glutamate decarboxylase (GAD) pathway. Lactic acid bacteria are generally recognized as safe (GRAS) food microorganisms, which can produce GABA during fermentation, and are widely used in the development of fermented foods, health products and drugs. In addition to producing GABA, lactic acid bacteria can also effectively reduce and inhibit the production of nitrite. Due to the influence of the physiological environment, strains with high GABA production are usually derived from dairy products, and strains that degrade nitrite are usually derived from traditional foods such as pickled vegetables. Due to the different habitats, strains that can produce GABA may not necessarily be able to degrade nitrite, and strains that can degrade nitrite may not necessarily be able to produce GABA. The existing technology also screens out strains that can both degrade nitrite and synthesize GABA, with the highest GABA yield of 71.77±3.35 μg / mL, which is not very high. Therefore, it is of great importance to isolate and screen lactic acid bacteria strains that can both produce GABA and degrade nitrite in the development process of functional fermented foods, which can not only improve the nutritional value and health function of the products, but also enhance their market competitiveness and meet the growing demand of consumers for healthy foods. SUMMARY

[0006] In view of the above, it is necessary to provide a strain that can both produce GABA and degrade nitrite, and also improve the freeze-drying process and optimize the growth promoter to improve the activity of the freeze-dried powder of the strain.

[0007] To achieve the above purpose, the present application screens a new strain: Lactiplantibacillus plantarum LM2, which is classified and named as Lactiplantibacillus plantarum, Chinese classification name is Lactiplantibacillus plantarum, preservation number is GDMCC NO: 66632; the strain is preserved in Guangdong Microbial Culture Collection Center, address: No. 59, Building 5, Institute of Microbiology, Guangdong Academy of Sciences, 100, Xianlie Middle Road, Guangzhou, and the preservation date is July 3, 2025.

[0008] The application also comprises a promoter of the Lactiplantibacillus plantarum strain LM2, which is composed of histidine, arginine and / or ornithine.

[0009] Further, the concentration of histidine is 0.4g / L-0.8g / L, the concentration of arginine is 0.4g / L-1.2g / L, and the concentration of ornithine is 0.4g / L-1.2g / L.

[0010] Further, the concentration of histidine is 0.4g / L, the concentration of arginine is 0.4g / L, and the concentration of ornithine is 0.4g / L.

[0011] The application also comprises a freeze-dried powder containing the Lactiplantibacillus plantarum strain LM2, and the protective agent in the freeze-dried powder is composed of 30-35g / 100mL of skimmed milk, 8-9g / 100mL of glucose, 1-3g / 100mL of fucoidan, and 0.8-1.0g / 1000mL of folic acid.

[0012] Further, the protective agent is composed of 33g / 100mL of skimmed milk, 7g / 100mL of glucose, 2g / 100mL of fucoidan, and 0.9g / 1000mL of folic acid.

[0013] The application also comprises an application of the Lactiplantibacillus plantarum strain LM2 in preparing an antibacterial agent, and the pathogenic bacteria inhibited by the antibacterial agent are Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis, Listeria monocytogenes, and Campylobacter jejuni.

[0014] The application also comprises an application of the Lactiplantibacillus plantarum strain LM2 or the freeze-dried powder in increasing the production of gamma-aminobutyric acid, reducing the content of nitrite, improving the ability to resist bile salts, and / or improving the acid resistance.

[0015] The application also comprises an application of the freeze-dried powder in food processing.

[0016] The present invention also includes a method for preparing the lyophilized powder containing the Lactobacillus plantarum strain LM2, characterized in that the method comprises the following steps:

[0017] (1) Activation and culture of strain: The Lactobacillus plantarum strain LM2 was inoculated into MRS medium and allowed to stand overnight for seed culture. After one subculture activation, the Lactobacillus plantarum strain LM2 was inoculated into MRS medium containing the promoter for expansion culture.

[0018] (2) Centrifugation to collect bacterial cells and aliquoting: After aliquoting the fermentation broth, centrifuge, remove the supernatant fermentation broth, add physiological saline to resuspend, and repeat centrifugation to obtain bacterial sludge; mix the bacterial sludge with 1 / 5 of the original fermentation broth volume of the protective agent solution and shake to make it uniform, and prepare a bacterial suspension.

[0019] (3) Pre-freezing: Pour the bacterial suspension into a sterile petri dish to a thickness of 0.5 cm and pre-freeze at -80℃ for 12 h;

[0020] (4) Vacuum freeze drying: After the fungal mud is pre-frozen, it is freeze-dried for 24 hours under a vacuum of 20-30 Pa to make the moisture content of the freeze-dried fungal powder 3%.

[0021] The present invention has the following beneficial effects:

[0022] 1. The LM2 strain of this invention was isolated from sour papaya. This strain has a high GABA (γ-aminobutyric acid) production capacity and the ability to degrade nitrite. It also exhibits antibacterial effects against intestinal pathogens such as Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Campylobacter jejuni, demonstrating good antibacterial function. Furthermore, it has good acid and bile salt tolerance and can survive well in artificial intestinal and gastric fluids. In addition, to improve GABA production, the research group studied growth promoters and found that a growth promoter can significantly promote the growth of strain LM2. The growth promoters are histidine, arginine, and / or ornithine. Orthogonal optimization experiments were conducted to determine the optimal concentrations of these promoters: histidine at 0.4 g / L-0.8 g / L, arginine at 0.4 g / L-1.2 g / L, and ornithine at 0.4 g / L-1.2 g / L. Furthermore, through optimization of the promoters and lyophilized powder, the prepared lyophilized powder achieved a survival rate exceeding 99%, a nitrite degradation rate exceeding 99%, and a GABA yield exceeding 3.0 g / L, making it a widely applicable microbial powder. Attached Figure Description

[0023] Figure 1 Growth of LM2 strain of this application on a petri dish

[0024] Figure 2 Microscopic examination of LM2 strain of this application

[0025] Figure 3 Growth curve of LM2 strain of this application

[0026] Figure 4 Results of acid production experiment of LM2 strain of this application

[0027] Biological material preservation information

[0028] The preserved strain information of this application is: Lactiplantibacillus plantarum LM2, its taxonomic name is: Lactiplantibacillus plantarum, the Chinese taxonomic name is: Lactobacillus plantarum, and the preservation number is GDMCC NO: 66632; this strain is preserved in the Guangdong Provincial Center for Microbiological Culture Collection, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the preservation date is July 3, 2025. Specific implementation manners

[0029] All features disclosed in this specification, or steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0030] Any feature disclosed in this specification (including any additional claims, abstract), unless specifically stated, each feature is only an example of a series of equivalent or similar features.

[0031] Example 1:

[0032] The screening method of Lactiplantibacillus plantarum strain LM2 in this example is as follows:

[0033] 1. Isolation and purification of lactic acid bacteria: Samples such as papaya pickles, pickled mustard, pickled cowpeas, pickled bamboo shoots, and cheese are diluted step by step with physiological saline. Take 100 μL of the diluted solution and spread it on an MRS agar medium plate containing 2% calcium carbonate, and incubate it in an inverted manner at 37°C for 48 h. Select different single colonies according to the calcium circle, colony size, shape, color, etc. of the colonies, and use the plate for streaking to purify to single colonies. Perform Gram staining microscopy and catalase test on the purified strains, and select Gram-positive and catalase-positive bacteria and store them in 30% glycerol tubes at -80°C.

[0034] 2. The colony morphology of strain LM2 is asFigure 1 As shown, the microscopic examination results are as follows: Figure 2 As shown, from Figure 1 As can be seen, the colonies are round or oval, with a smooth, moist surface, neat edges, and a milky white color; from Figure 2 It can be seen that, after Gram staining, the strain is a Gram-positive bacterium and is rod-shaped, which is consistent with the morphological characteristics of Lactobacillus plantarum.

[0035] 3. Molecular identification method of strain LM2: The purified strain was sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequencing identification. Bacterial genomic DNA was extracted using a kit, and 16S rDNA sequences were amplified using 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The resulting sequence is shown in SEQ ID NO.1 of the sequence listing. After 1% agarose gel electrophoresis, the sequencing sequence was submitted to the National Center for Biotechnology Information (NCBI) database for BLAS homology comparison. The comparison showed that the homology between this strain and *Lactiplantibacillus plantarum* reached 100%. Combined with morphological characteristics, strain LM2 was identified as *Lactiplantibacillus plantarum*.

[0036] Example 2:

[0037] This example describes the screening of strains with nitrite-degrading capabilities, as detailed below:

[0038] 1. Screening of nitrite-degrading strains: Sixty strains obtained according to the lactic acid bacteria isolation and purification method in Example 1 were preliminarily screened for their nitrite-degrading ability. Specifically, the color change of the solution after the reaction was used to preliminarily determine the lactic acid bacteria's ability to degrade nitrite. If the color was close to or darker than the control tube, the higher the nitrite residue, indicating a lower or no degradation ability; the lighter the color, the lower the residue, indicating a higher nitrite-degrading ability.

[0039] 2. Secondary screening: The activated bacterial suspension of the strains initially screened by colorimetric method was inoculated at a rate of 2% (V / V) into MRS broth medium containing 200 mg / L nitrite and incubated statically at 37°C for 24 h. The nitrite content in the MRS broth medium was determined at 12 and 24 h using the Nanjing Jiancheng nitrite reagent kit, and the nitrite degradation rate was calculated according to the following formula.

[0040]

[0041] In the formula: V 对 The mass of NaNO2 in the uninoculated culture medium is expressed in mg; V 样The value represents the mass of NaNO2 in the fermentation broth, expressed in mg.

[0042] Five strains with the best ability to degrade nitrite were finally selected, as shown in Table 1:

[0043] Table 1. Nitrite degradation rate of different strains

[0044]

[0045] Note: Different lowercase letters in the table indicate significant differences in the same column of data (p<0.05), and the same letters indicate no significant differences in the same column (p>0.05). In addition, LM represents the source of the strain: papaya kimchi, LS represents the source of the strain: cheese, LJ represents the source of the strain: pickled mustard greens, and LG represents the source of the strain: pickled peas. The same applies to the following tables.

[0046] As shown in Table 1, when cultured in a medium containing 200 mg / L NaNO2 for 12 h, strain LM2 exhibited the highest nitrite degradation rate at 93.12%, significantly higher than the other four strains (p<0.05). After 24 h of culture, strain LM2 still had the highest nitrite degradation rate, while strain LS2 achieved a degradation rate of 95.55%. Although not reaching the highest rate, the difference between LS2 and strain LM2 was not significant (p>0.05). This indicates that the nitrite degradation rate increases with time. Furthermore, among the top five strains in terms of degradation rate, except for LS2 which originated from cheese, the other four strains were derived from plant-based pickled products. This suggests that it is easier to screen for strains capable of degrading nitrite from plant-based pickled products.

[0047] Example 3:

[0048] This example describes the screening of γ-aminobutyric acid-producing lactic acid bacteria strains, as detailed below:

[0049] 1. Screening of γ-aminobutyric acid (GABA)-producing strains: Sixty strains obtained according to the lactic acid bacteria isolation and purification method in Example 1 were screened. Specifically, the purified strains were inoculated into MRS broth medium for activation. The activated strains were then inoculated into TYG liquid medium at a 4% (v / v) inoculation rate and cultured at 37℃ and 150 rpm for 72 h. The bacterial culture was centrifuged at 12000 rpm for 10 min, and the GABA content of the supernatant was determined. The TYG liquid medium was prepared as follows: 10 g / L tryptone, 5 g / L yeast extract, 20 g / L glucose, 5 g / L sodium succinate, and 10 g / L L-glutamic acid were dissolved by heating, cooled to room temperature, and the pH was adjusted to 6.5. The medium was then sterilized at 115℃ for 25 min.

[0050] 2. GABA determination method: The Berthelot colorimetric method was used. Specifically, the Berthelot colorimetric method was used as follows: the fermentation broth was centrifuged (10000×g, 15min), and 200ul of the supernatant was collected. 1.0mL of 6% redistilled phenol and 0.5mL of borate buffer solution (pH=9.5) were added, mixed well, and then 1mL of 5.2% hydrogen peroxide solution was added. The mixture was allowed to stand for 5min, heated in a boiling water bath for 10min, and then placed in an ice bath for 10min. 2mL of 60% ethanol solution was added, mixed well, and the absorbance was measured at a wavelength of 645nm. The reagent blank was used to zero the instrument.

[0051] The yields of the top 5 GABA producers are shown in Table 2:

[0052] Table 2. GABA production of different strains

[0053]

[0054] Note: In the table, LM represents the strain source of papaya kimchi, and LS represents the strain source of cheese.

[0055] As shown in Table 2, strain LS2 had the highest GABA yield at 1.71 g / L, followed by strain LM2 at 1.58 g / L. The difference between the two strains was not significant (p>0.05), but LM2 was significantly higher than strains LS3, LS6, and LS9 (p<0.05). The GABA yields of strains LS3, LS6, and LS9 were all below 1.0 g / L. Furthermore, among the top 5 GABA-producing strains, except for LM2 which originated from plant-based preserved products, the other 4 strains all originated from cheese. This indicates that it is easier to screen for GABA-producing strains in dairy products.

[0056] In summary, strains LM2 and LS2 demonstrated the ability to both degrade sodium nitrite and produce GABA in this screening experiment. Furthermore, since the two strains originated from plant-based pickled products and dairy products, respectively, they possess significant research value.

[0057] Example 4:

[0058] This example demonstrates the acid resistance assessment of the strain:

[0059] Eight bacterial strains (LM2, LS2, LJ1, LJ3, LG1, LS3, LS6, and LS9) from Examples 2 and 3 were selected for acid resistance identification. The specific method was as follows: the absorbance of the bacterial suspension was adjusted to OD... 600 nm = 1.0 (±0.1), and inoculated into MRS liquid medium at pH 2.0, 3.0, 4.0, and 5.0 at an inoculum rate of 2% (v / v). After incubation at 37℃ for 24 h, the OD of the bacterial culture was measured. 600 nm value, in pH 6.5 MRS liquid medium OD 600The nm value is the control, and the survival rate is calculated according to the following formula.

[0060]

[0061] Where: OD of sample group 600 The values ​​are different pH values ​​for MRS liquid culture medium OD. 600 Value, control group OD 600 For pH 6.5 MRS liquid culture medium OD 600 value.

[0062] Table 3. Acid resistance of 8 lactic acid bacteria strains

[0063]

[0064] As shown in Table 3, the survival rate of the strains increased with increasing pH, indicating that an acidic environment has a significant impact on the growth of Lactobacillus. At pH 2.0 and pH 3.0, the survival rate of the strains was low, and their growth activity was inhibited; at pH 4.0 and pH 5.0, the survival rate increased significantly. At pH 4.0, the survival rates of strains LM2 and LS2 were both above 80%, significantly higher than other strains (p<0.05). At pH 5.0, the survival rate reached over 88%, with 7 strains reaching over 97%. In summary, strains LM2 and LS2 exhibited the best performance in acid resistance and have good application potential.

[0065] Example 5:

[0066] This example demonstrates the identification of bile salt tolerance in a bacterial strain.

[0067] Adjust the absorbance of the bacterial solution to OD. 600 nm = 1.0 (±0.1), inoculated at a rate of 2% (v / v) into MRS liquid medium containing 0.1%, 0.2%, and 0.3% ox bile salts, and incubated at 37℃ for 24 h. OD was measured after 24 h. 600 The nm value was used as a control in MRS liquid medium without ox bile salts, and the survival rate of the strain to bile salts was calculated according to the following formula.

[0068]

[0069] Where: OD of sample group 600 The value was OD of the MRS liquid culture medium containing bile salts. 600 Value, control group OD 600 MRS liquid culture medium OD for bile salt-free group 600 value.

[0070] Table 4. Bile salt tolerance of 8 lactic acid bacteria strains

[0071]

[0072] As shown in Table 4, the survival rate decreased with increasing bile salt concentration. After culturing for 24 hours in a medium containing 0.1–0.3% bile salt, strain LM2 showed the smallest variation in survival rate, followed by strain LS2. The survival rates of these two strains were significantly higher than other strains (p<0.05). At a bile salt concentration of 0.1%, six strains achieved a survival rate of over 90%; at 0.2%, four strains achieved a survival rate of over 80%; and at 0.3%, two strains achieved a survival rate of over 80%. This demonstrates that strains LM2 and LS2 exhibit high bile salt tolerance and good adaptability in complex environments, showing great application potential.

[0073] Example 6:

[0074] This example demonstrates the identification of the strain's tolerance to artificial gastrointestinal fluid.

[0075] The specific method is as follows: 1 mL of bacterial suspension is added to 9 mL of simulated gastric fluid, thoroughly mixed, and digested at 37℃ and 150 r / min for 3 h. Plate counts are performed on the 0-hour and 3-hour digestion solutions, using the viable bacteria count from the 0-hour digestion as a control, and the survival rate is calculated. 1 mL of simulated gastric fluid is added to 9 mL of simulated intestinal fluid and incubated at 37℃ for 3 h. Plate counts are performed on the 0-hour and 3-hour digestion solutions, using the viable bacteria count from the 0-hour digestion as a control, and the strain's tolerance to the simulated gastrointestinal fluid is calculated using the following formula.

[0076]

[0077] Where: N 0h The viable bacterial count at 0 h after inoculation is 1g CFU / mL; N 3h The viable bacterial count at 3 hours after inoculation is 1g CFU / mL.

[0078] Table 5. Tolerance of 8 strains of lactic acid bacteria to artificial gastrointestinal fluid

[0079]

[0080] Table 5 shows that the simulated gastrointestinal fluid tolerance experiment reflects the potential of the strains to resist digestive fluid erosion and maintain activity in the actual human gastrointestinal environment. Due to their unique cell structure and physiological characteristics, lactic acid bacteria can effectively resist the highly acidic environment of simulated gastric fluid and the degradation effect of pepsin, maintaining a high viable count in the gastrointestinal tract. The viable count is closely related to the probiotic functions of lactic acid bacteria, such as regulating intestinal flora balance, strengthening intestinal barrier function, and achieving immunomodulatory effects. If the viable count is too low, the expected probiotic effects are difficult to achieve. The simulation experiments showed that in simulated gastric fluid, strains LM2 and LS2 achieved a survival rate of over 90%, significantly higher than other strains (p<0.05). Among them, strain LS2 had the highest survival rate at 96.46%, followed by strain LM2 at 91.43%, and the survival rates of LM2 and LS2 were significantly different (p<0.05). In simulated intestinal fluid, strains LM2 and LS2 achieved a survival rate of over 80%, significantly higher than other strains (p<0.05). Among them, strain LM2 had the highest survival rate at 89.58%, followed by strain LS2 at 85.31%, and the survival rates of LM2 and LS2 were significantly different (p<0.05).

[0081] Based on the above experiments, we found that strains LM2 and LS2 showed good survival rates and tolerance in nitrite degradation rate, GABA production, acid resistance, bile salt resistance, artificial intestinal fluid, and artificial gastric fluid during this screening. Therefore, we plan to focus on these two strains for further research. This application mainly focuses on the relevant research of strain LM2.

[0082] Example 7:

[0083] The growth and acid production capacity of strain LM2 in this embodiment were identified as follows:

[0084] 1. Growth curve of the strain: The activated bacterial culture was inoculated into MRS liquid medium at an inoculum of 2% (V / V) and incubated at 37°C for 24 hours. OD was measured every 2 hours. 600 The growth curve of the strain was plotted based on the absorbance value in nm.

[0085] like Figure 3 As shown: strain LM2 exhibits a typical S-shaped growth curve, with OD values ​​in the early stages of culture (0–6 h) being [missing data]. 600 The relatively small increase in nm value indicates that the lactic acid bacteria are in the adaptation phase, with slow metabolic activity and minimal proliferation. After 6 hours, they enter the logarithmic growth phase, and the OD value rises rapidly, indicating enhanced metabolic activity, vigorous cell division, and increased cell density. After 18 hours, the increase in OD value slows down and tends to stabilize, indicating that the lactic acid bacteria have entered the stationary phase, at which point the cell proliferation and death rates are roughly equal, and the bacterial concentration reaches a relatively stable state.

[0086] 2. Acid production curve of the strain: The activated bacterial solution was inoculated into MRS liquid medium at an inoculum size of 2% (V / V) and cultured at 37℃ for 24 h. The total acid content was measured every 2 h, and the acid production curve of the strain was plotted.

[0087] like Figure 4 As shown, the acid production of strain LM2 showed an increasing trend with increasing culture time. This is because the strain produces acidic metabolites during its metabolism, causing the acidity of the culture medium to rise continuously. At 24 hours of culture, strain LM2 produced the highest amount of acid, reaching 22.5 g / L.

[0088] Example 8:

[0089] Antibacterial experiment of strain LM2 against common intestinal pathogens:

[0090] Six indicator bacteria—Escherichia coli, Staphylococcus aureus, Salmonella enterica subsp. enterericaserovar Enteritidis, Listeria monocytogenes, Campylobacter jejuni, and Shigella flexneri—were activated and cultured for later use. The antibacterial activity of strain LM2 against these six indicator bacteria was determined using the perforated agar diffusion method. The diameter of the clear zone was measured (inhibition zone diameter = measurement diameter - hole diameter), and the results are shown in Table 6.

[0091] Table 6. Antibacterial effects of strain LM2 against common pathogens.

[0092]

[0093] Note: “—” in the table indicates no inhibitory effect.

[0094] Table 6 shows that strain GX-7 exhibits antibacterial effects against Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis, Listeria monocytogenes, and Campylobacter jejuni, but shows no antibacterial effect against Escherichia coli and Shigella flexneri. In terms of inhibition zones, the inhibition zone against Staphylococcus aureus is significantly higher than that against other pathogens (p<0.05), followed by Salmonella enterica subsp. enterica serovar Enteritidis, and then Listeria monocytogenes. The strain LM2 showed no significant difference in efficacy against Listeria monocytogenes and Campylobacter jejuni (p>0.05).

[0095] Example 9:

[0096] Experiment to enhance the activity of strain LM2:

[0097] Amino acids are essential for microbial protein synthesis, cell proliferation, and division. They serve as nitrogen and carbon sources for microbial growth. However, different microorganisms and strains require different types and concentrations of amino acids. Improper use of amino acid types and concentrations can inhibit strain growth, while certain amino acids may be essential for strains and promote microbial growth. Therefore, amino acids have different regulatory effects on different strains. Thus, we considered selecting a subset of amino acids for research in order to find the types and concentrations of amino acids that can promote the growth of strain LM2. The addition of amino acids and the lack of any essential amino acid can significantly inhibit or even completely stop growth.

[0098] Adjust the absorbance of the bacterial solution to OD. 600 nm = 1.0 (±0.1), inoculated at a rate of 2% (v / v) into MRS liquid medium containing 1 g / L promoter (promoters are shown in Table 7), and incubated at 37℃ for 24 h. OD was measured after 24 h. 600nm value; nitrite degradation rate over 12 hours was calculated according to the method in Example 2; GABA yield was calculated according to the method in Example 3. The results are shown in Table 7.

[0099] Table 7 Effects of different promoters on strain LM2

[0100]

[0101] As shown in Table 7, histidine, arginine, ornithine, and valine significantly increased the OD value of strain LM2, promoting its growth (p<0.05), while methionine significantly inhibited its growth (p<0.05). Regarding nitrite degradation rate and GABA production, valine's effect was not significant. Histidine, arginine, and ornithine significantly increased the nitrite degradation rate. In terms of GABA production, histidine and ornithine showed significant increases compared to the control group, while arginine showed an increase, but the difference was not significant (p>0.05). Regarding nitrite degradation rate, the increase achieved by a single promoter reached 99% within 24 hours.

[0102] Based on the above research results, histidine, arginine, and ornithine were selected for a ratio experiment to determine the nitrite degradation rate and GABA production of strain LM2, as follows:

[0103] An orthogonal experiment was designed by adding histidine, arginine, and ornithine to the basal medium at specific concentrations. After 12 hours of culture, GABA production was calculated based on nitrite degradation rate and after 24 hours of culture. The nitrite degradation rate at 12 hours and the GABA production at 24 hours were used as screening indicators, as detailed below:

[0104] Table 8. Orthogonal experimental factor levels for strain LM2

[0105]

[0106] The orthogonal analysis of the above proportions and the results are shown in Table 9:

[0107] Table 9. Orthogonal Experiments and Results

[0108]

[0109] Table 9 shows that the nitrite degradation rate of experimental groups 1-4 and 6-7 remained above 97% after 24 hours, maintaining a high degradation rate, not significantly different from the highest 99.02% in Table 7. The nitrite degradation rate of experimental groups 5 and 8-9 was only below 90%, indicating that the ratios in these experimental groups reduced the survival rate of strain LM2 and did not promote it, thus affecting the nitrite degradation rate. The ranges show that the range of arginine > histidine > ornithine, indicating that arginine has the greatest impact on the nitrite degradation rate after 12 hours, followed by histidine, with ornithine having the least impact. Regarding GABA content, experimental groups 1-4... The GABA yield of experimental groups 6 and 7 both reached above 2.2 g / L, higher than the highest 2.19 g / L in Table 7, but the differences between 2.2 g / L and 2.32 g / L and 2.19 g / L were not significant. The nitrite degradation rate of experimental groups 5 and 8-9 was only below 1.5 g / L, higher than the CK group in Table 7 (1.61 g / L), indicating that the ratio of these experimental groups reduced the survival rate of strain LM2 and did not promote it, thus affecting the GABA yield. The range shows that the range of histidine > the range of arginine > the range of ornithine, indicating that histidine has the greatest impact on GABA yield, followed by arginine, and ornithine has the least impact. Based on the above experimental results, considering that the differences between 2.2 g / L, 2.32 g / L and 2.19 g / L are not significant, we optimized the amino acid concentrations for experimental groups 1-4, namely: histidine concentration of 0.4 g / L-0.8 g / L, arginine concentration of 0.4 g / L-1.2 g / L, and ornithine concentration of 0.4 g / L-1.2 g / L. We considered Experiment 1 to be the optimal experimental group, where the difference in nitrite degradation rate was not significant, as it significantly increased GABA production. Therefore, we optimized Experiment 1, with histidine, arginine, and ornithine concentrations of 0.4 g / L.

[0110] Example 10:

[0111] The strain LM2 was prepared into a lyophilized powder, and the specific preparation method is as follows:

[0112] (1) Activation and culture of strain: Strain LM2 was inoculated into MRS medium and allowed to stand overnight for seed culture. After one subculture activation, strain LM2 was inoculated into MRS medium supplemented with the promoters of Experiment 1 (0.4 g / L histidine, 0.4 g / L arginine, and 0.4 g / L ornithine) at an inoculation rate of 2% (v / v) for expansion culture. The culture was carried out at 30°C and 40 r / min for 24 h.

[0113] (2) Centrifugation to collect bacterial cells and aliquot: After aliquoting the fermentation broth, centrifuge at 7500 rpm for 10 min at 4℃, remove the supernatant fermentation broth, add physiological saline to resuspend, and repeat centrifugation to obtain bacterial sludge; mix the bacterial sludge with 1 / 5 volume of the original fermentation broth freeze-drying protectant solution and shake to make it uniform, and prepare bacterial suspension.

[0114] (3) Pre-freezing: Pour the bacterial suspension into a sterile petri dish to a thickness of about 0.5 cm and pre-freeze at -80℃ for 12 hours.

[0115] (4) Vacuum freeze drying: After the fungal mud is pre-frozen, it is freeze-dried for 24 hours under a vacuum of 30 Pa (this embodiment only discloses one test condition, but in fact, the freeze-drying effect can be achieved at 20-30 Pa) so that the moisture content of the freeze-dried fungal powder is about 3%.

[0116] After orthogonal optimization by the applicant, the freeze-drying protectant was found to have the greatest impact on the survival rate of strain LM2, consisting of skim milk, glucose, fucoidan, and folic acid. The protectant's components were: 30-35 g / 100 mL skim milk, 8-9 g / 100 mL glucose, 1-3 g / 100 mL fucoidan, and 0.8-1.0 g / 1000 mL folic acid. Following orthogonal experiments, some formulations showing significant differences were selected for further testing. Specific formulations are shown in Table 10.

[0117] Table 10 Selection and Formulation of Lyophilized Powder Protectants

[0118]

[0119] Based on the lyophilization protectants prepared according to Table 10, the lyophilized powders prepared using the lyophilization protectants were subjected to survival rate determination, nitrite degradation ability determination, and salt tolerance determination. The determination methods are as follows:

[0120] (1) Survival rate determination: At 24h, the number of viable Lactobacillus plantarum before freeze-drying and the number of viable bacteria after freeze-drying the same volume of bacterial mud were determined respectively.

[0121] Freeze-dried survival rate (%) = (Number of viable bacteria in 1 mL of sludge after freeze-drying) / (Number of viable bacteria in 1 mL of sludge before freeze-drying)

[0122] (2) The freeze-dried powder was inoculated into MRS medium at a rate of 5% and cultured for 24 hours to activate it. Then the activated bacterial solution was inoculated into the culture medium at a rate of 2%, and the GABA yield was calculated according to the method in Example 3.

[0123] (3) The freeze-dried powder was inoculated into MRS medium at a rate of 5% and cultured for 24 hours to activate it. Then the activated bacterial solution was inoculated into the medium at a rate of 2%, and the GABA yield was calculated according to the method in Example 2.

[0124] Table 11. Strains' survival rate, nitrite degradation rate, and GABA production under different freeze-drying protectants.

[0125]

[0126] Table 11 shows that the survival rates of groups 1-3 all reached over 95%, significantly higher than those of groups 4-6 (p<0.05), and the GABA production was significantly higher than that of groups 4-7. This indicates that in freeze-dried bacterial powder, the survival rate affects the nitrite degradation rate and GABA production of lactic acid bacteria in a positive correlation, and the components of the protectant are indispensable. The absence of any component will affect the survival rate of the strain, and thus affect the nitrite degradation rate and GABA production. Therefore, for the LM2 strain, the components of the protectant are crucial. Selecting 30-35g / 100mL of skim milk, 8-9g / 100mL of glucose, 1-3g / 100mL of fucoidan, and 0.8-1.0g / 1000mL of folic acid can effectively protect strain LM2 and maintain the balance of cell sap within the strain. The optimal protective agent composition is 33g / 100mL of skim milk, 7g / 100mL of glucose, 2g / 100mL of fucoidan, and 0.9g / 1000mL of folic acid.

[0127] In summary, the *Lactobacillus plantarum* LM2 strain of this application possesses high GABA production and nitrite degradation capabilities, exhibits superior inhibitory activity against intestinal pathogens compared to other strains, and demonstrates excellent acid and bile salt tolerance. It can survive well in artificial intestinal and gastric fluids and can be used to process antibacterial agents, health foods, and pickled foods. Even after optimization of promoters and protectants to prepare freeze-dried powder, it retains high viability, making it a widely applicable strain.

[0128] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A strain of Lactiplantibacillus plantarum, LM2, with accession number GDMCCNO:66632.

2. A promoter for promoting the growth of Lactobacillus plantarum strain LM2 as described in claim 1, characterized in that, The promoter is composed of histidine, arginine and / or ornithine.

3. The accelerator according to claim 2, characterized in that, The concentrations of histidine, arginine, and ornithine are 0.4 g / L to 0.8 g / L, arginine, and ornithine, respectively.

4. The accelerator according to claim 2, characterized in that, The concentrations of histidine, arginine, and ornithine are 0.4 g / L.

5. A freeze-dried powder comprising the Lactobacillus plantarum strain LM2 as described in claim 1, characterized in that, The preservative in the freeze-dried powder consists of the following components: 30-35g / 100mL skim milk, 8-9g / 100mL glucose, 1-3g / 100mL fucoidan, and 0.8-1.0g / 1000mL folic acid.

6. The freeze-dried powder according to claim 5, characterized in that, The protective agent consists of the following components: 33g / 100mL skim milk, 7g / 100mL glucose, 2g / 100mL fucoidan and 0.9g / 1000mL folic acid.

7. The application of the Lactobacillus plantarum strain LM2 as described in claim 1 in the preparation of an antibacterial agent, characterized in that, The pathogens inhibited by the antibacterial agent are: Staphylococcus aureus, Salmonella enterica subsp. entericaserovar Enteritidis, Listeria monocytogenes, and Campylobacter jejuni.

8. The use of Lactiplantibacillus plantarum strain LM2 as described in claim 1 or the freeze-dried powder as described in claim 5 in increasing γ-aminobutyric acid production, reducing nitrite content, improving bile salt tolerance, and / or improving acid tolerance.

9. The application of the freeze-dried powder as described in claim 5 in food processing.

10. A method for preparing the lyophilized powder containing Lactobacillus plantarum strain LM2 as described in claim 5, characterized in that, The method includes the following steps: (1) Activation and culture of strain: The Lactobacillus plantarum strain LM2 was inoculated into MRS medium and allowed to stand overnight for seed culture. After one subculture activation, the Lactobacillus plantarum strain LM2 was inoculated into MRS medium containing the promoter as described in claim 2 for expansion culture. (2) Centrifugation to collect bacterial cells and aliquoting: After aliquoting the fermentation broth, centrifuge, remove the supernatant fermentation broth, add physiological saline to resuspend, and repeat centrifugation to obtain bacterial sludge; Mix the bacterial sludge with 1 / 5 of the original fermentation liquid volume of the protective agent solution as described in claim 5 and shake to make it uniform, thus preparing a bacterial suspension. (3) Pre-freezing: Pour the bacterial suspension into a sterile petri dish to a thickness of 0.5 cm and pre-freeze at -80℃ for 12 h; (4) Vacuum freeze drying: After the fungal mud is pre-frozen, it is freeze-dried for 24 hours under a vacuum of 20-30 Pa to make the moisture content of the freeze-dried fungal powder 3%.

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