Lactobacillus plantarum strain LS2 and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum strain LS2 and application thereof.The lactobacillus plantarum strain LS2 is separated from cheese, has the high GABA production capacity and the nitrite degradation capacity, has the antibacterial effect on enteric pathogenic bacteria including escherichia coli, salmonella enteritidis and campylobacter jejuni, and has the advantages that the lactobacillus plantarum strain LS2 is high in GABA production capacity and nitrite degradation capacity; the strain has good antibacterial function, good acid resistance and cholate resistance, and can well survive in artificial intestinal juice and artificial gastric juice; in order to improve the yield of GABA, the accelerant capable of remarkably promoting the growth of the strain LS2 is obtained by researching the accelerant, 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 98% or above, the yield of GABA is improved to 3.3 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 LS2 and its application. BACKGROUND

[0002] GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter of the central nervous system, has many physiological functions, especially in regulating nerve excitability, improving sleep, relieving anxiety, etc. The preparation of gamma-aminobutyric acid is mainly by chemical synthesis, biological catalysis, microbial fermentation and other methods. Among them, the microbial fermentation method mainly uses the endogenous glutamate decarboxylase (GAD) in the microbial cell to catalyze L-glutamic acid decarboxylation to generate gamma-aminobutyric acid (GABA) and carbon dioxide (CO2). At present, the core microorganisms capable of producing GABA include lactic acid bacteria, yeast and aspergillus, etc. Among these microorganisms, lactic acid bacteria are often used for GABA production because of their high GABA yield and safety, and are often used as GABA fermentation strains.

[0003] In addition, in the prior art, lactic acid bacteria not only have the function of high-yield GABA, but also have the function of degrading nitrite. Nitrite as an additive, if taken in excess, can cause toxic effects, and even endanger life in severe cases. Under acidic and high-temperature conditions, it can 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 probability of breast cancer or prostate cancer.

[0004] Although the lactic acid bacteria reported in the prior art have the functions of producing GABA and degrading nitrite, usually due to the influence of the physiological environment, the strains with high GABA production are usually derived from dairy products, and the strains degrading nitrite are usually derived from traditional foods such as pickled vegetables. Due to the difference in habitat, strains capable of producing GABA may not necessarily be able to degrade nitrite, and strains capable of degrading nitrite may not necessarily be able to produce GABA. The strains capable of degrading nitrite and synthesizing GABA have also been screened out in the prior art, and the highest GABA yield is 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 capable of producing gamma-aminobutyric acid and degrading nitrite in the development process of functional fermented foods, which not only can improve the nutritional value and health function of the products, but also can enhance the market competitiveness and meet the growing demand of consumers for healthy foods. SUMMARY

[0005] In view of the above, it is necessary to provide a strain that can produce gamma-aminobutyric acid and has the ability to degrade nitrite, and at the same time, improve the activity of the strain freeze-dried powder by improving the freeze-drying process, improving the freeze-drying protectant, and optimizing the growth promoter.

[0006] To achieve the above purpose, the present application screens a new strain: Lactiplantibacillus plantarum LS2, which is classified and named as: Lactiplantibacillus plantarum, Chinese classification name: Lactiplantibacillus plantarum, preservation number: GDMCC NO: 66633; the strain is preserved in Guangdong Microbial Culture Collection Center, address: 59, 5th Floor, Institute of Microbiology, Guangdong Academy of Sciences, 100, Martyrs' Road, Guangzhou, preservation date: July 3, 2025.

[0007] The present application also includes a promoter for promoting the Lactiplantibacillus plantarum strain LS2, which consists of methionine, valine and / or riboflavin.

[0008] Further, the concentration of methionine is 0.4g / L-1.2g / L, the concentration of valine is 0.4g / L-0.8g / L, and the concentration of riboflavin is 0.8g / L-1.2g / L.

[0009] Further, the concentration of methionine is 0.4g / L, the concentration of valine is 0.8g / L, and the concentration of riboflavin is 0.8g / L.

[0010] The present application also includes a freeze-dried powder containing the Lactiplantibacillus plantarum strain LS2, and the protectant in the freeze-dried powder consists of the following components: 28-32g / 100mL of skim milk, 10-12g / 100mL of sucrose, 2-4g / 100mL of fructose and 0.02g / 100mL of zinc sulfate.

[0011] Further, the protectant consists of the following components: 32g / 100mL of skim milk, 12g / 100mL of sucrose, 4g / 100mL of fructose and 0.02g / 100mL of zinc sulfate.

[0012] The application also includes an application of the Lactiplantibacillus plantarum strain LS2 in preparing a bacteriostatic agent, wherein the pathogenic bacteria inhibited by the bacteriostatic agent are Escherichia coli, Salmonella enterica subsp.enterica serovar Enteritidis and Campylobacter jejuni.

[0013] The application also includes an application of the Lactiplantibacillus plantarum strain LS2 or the freeze-dried powder in improving the production of gamma-aminobutyric acid, reducing the content of nitrite, improving the tolerance to cholic acid and / or improving the acid tolerance.

[0014] The application also includes an application of the freeze-dried powder in food processing.

[0015] The application also includes a method for preparing the freeze-dried powder containing the Lactiplantibacillus plantarum strain LS2, wherein the method comprises the following steps:

[0016] (1) strain activation and culture: inoculating the Lactiplantibacillus plantarum strain LS2 into MRS culture medium, performing seed culture by standing overnight, after one-time subculture activation, inoculating the Lactiplantibacillus plantarum strain LS2 into MRS culture medium containing the accelerator for scale-up culture;

[0017] (2) centrifugal collection of bacterial bodies and sub-packing: after sub-packing the fermentation liquor, centrifugation, removing the supernatant fermentation liquor and adding physiological saline for resuspension, obtaining bacterial slurry after repeated centrifugation; mixing and oscillating the bacterial slurry with 1 / 5 volume of the protective agent solution of the original fermentation liquor to make the mixture uniform, and preparing a bacterial suspension;

[0018] (3) pre-freezing: pouring the bacterial suspension into a sterile culture dish with a thickness of 0.5 cm, and pre-freezing at-80℃ for 12 h;

[0019] (4) vacuum freeze-drying: after pre-freezing the bacterial slurry, freeze-drying under a vacuum degree of 20-30 Pa for 24 h, so that the moisture content of the freeze-dried bacterial powder is 3%, and the freeze-dried bacterial powder is obtained.

[0020] The application has the following beneficial effects:

[0021] 1. The LS2 strain of the present application is isolated from cheese. The strain has high GABA (gamma-aminobutyric acid) production capacity, and also has the ability to degrade nitrite, inhibit intestinal pathogenic bacteria such as Escherichia coli, Salmonella enteritidis and Campylobacter jejuni, and has good antibacterial function. In addition, the strain has good acid and bile salt tolerance and can survive well in artificial intestinal fluid and artificial gastric juice. In addition, in order to improve the yield of GABA, the research group obtained the growth promoters that can significantly promote the growth of the strain LS2 through the study of the promoters: methionine, valine and / or riboflavin. Through orthogonal optimization experiment, the mass concentration of the above growth promoters is obtained, that is, the concentration of methionine is 0.4 g / L-1.2 g / L, the concentration of valine is 0.4 g / L-0.8 g / L, and the concentration of riboflavin is 0.8 g / L-1.2 g / L. In addition, through the optimization of the growth promoters and the freeze-dried powder, the highest survival rate of the prepared freeze-dried powder can reach more than 99%, the nitrite degradation rate can reach more than 98%, and the GABA yield can be increased to more than 3.3 g / L. It is a kind of bacteria powder that can be widely applied. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a plate growth chart of the LS2 strain of the present application.

[0023] Figure 2 It is a microscope examination chart of the LS2 strain of the present application under a microscope.

[0024] Figure 3 It is a growth curve chart of the LS2 strain of the present application.

[0025] Figure 4 It is a result chart of the acid production experiment of the LS2 strain of the present application.

[0026] BIOLOGICAL MATERIAL PRESERVATION INFORMATION

[0027] The strain information preserved in the present application is: Lactiplantibacillus plantarum LS2, which is classified and named as: Lactiplantibacillus plantarum, Chinese classification name: Lactiplantibacillus plantarum, preservation number: GDMCC NO: 66633; the strain is preserved in Guangdong Microbial Culture Collection Center, address: No. 59, Building 5, Institute of Microbiology, Guangdong Academy of Sciences, 100, Martyrs Road, Guangzhou, preservation date: July 3, 2025. DETAILED DESCRIPTION

[0028] All features disclosed in this specification, or all steps of any methods or processes disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0029] Any feature in the foregoing specification that is expressed as a particular combination of features, unless specifically stated otherwise, can be expressed or performed with reference to alternate combinations characterizing alternate embodiments.

[0030] Example 1

[0031] This embodiment is a screening method of Lactiplantibacillus plantarum strain LS2:

[0032] 1. Isolation and purification of lactic acid bacteria: papaya pickles, pickled mustard, pickled cowpea, pickled bamboo shoots, cheese and other samples were diluted with physiological saline in gradient, 100 μL of the diluted solution was spread on MRS agar medium containing 2% calcium carbonate, and incubated at 37°C for 48h. Different single colonies were selected according to the calcium ring, colony size, shape, color and other characteristics, and purified by plate streaking. The purified strains were subjected to gram staining and hydrogen peroxide enzyme test, and the gram-positive and hydrogen peroxide enzyme-positive strains were selected and stored in 30% glycerol tubes at-80°C.

[0033] 2. The colony morphology of strain LS2 is shown in Figure 1 The microscopic results are shown in Figure 2 It can be seen from Figure 1 that the colony is round or oval, the surface is smooth and wet, the edge is neat, and the color is milky white; from Figure 2 It can be seen that after gram staining, the strain is gram-positive and rod-shaped, which is consistent with the morphological characteristics of Lactiplantibacillus.

[0034] 3. Molecular identification method of strain LS2: the purified strain was entrusted to Shengong Bioengineering (Shanghai) Co., Ltd. for 16S rDNA sequencing identification. The bacterial genomic DNA was extracted by kit, and the sequence obtained by 16S rDNA sequence amplification with 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') is shown in SEQ ID NO. 1. After 1% agarose gel electrophoresis, the sequencing sequence was submitted to the National Center for Biotechnology Information (NCBI) database for BLAS homology comparison. After comparison, the homology of the strain with Lactiplantibacillus plantarum reached 100%, and combined with the morphological characteristics, the strain LS2 was identified as Lactiplantibacillus plantarum.

[0035] Example 2

[0036] This embodiment is the screening of nitrite-degrading strains, which is as follows:

[0037] 1. Screening of nitrite-degrading strains: 60 strains screened by the lactic acid bacteria isolation and purification method according to Example 1 were subjected to preliminary screening of nitrite-degrading ability. The color change of the solution after colorimetric reaction was used to preliminarily judge the ability of lactic acid bacteria to degrade nitrite. If the color is close to or darker than that of the control tube, the higher the residual amount of nitrite, the lower the strain degradation ability or no degradation ability; the lighter the color, the lower the residual amount, the higher the degradation ability.

[0038] 2. Rescreening: the activated bacterial liquid of the strains screened by colorimetry was inoculated into MRS broth medium containing 200 mg / L nitrite at an inoculation amount of 2% (V / V), and incubated at 37°C for 24 h. The nitrite content in the MRS broth medium at 12 h and 24 h was determined by using the nitrite reagent kit of Nanjing Jiancheng, and the nitrite degradation rate was calculated according to the following formula.

[0039]

[0040] In the formula: V 对 is the mass of NaNO2 in the un-inoculated medium, mg; V 样 is the mass of NaNO2 in the fermentation bacterial liquid, mg.

[0041] Finally, 5 strains with the best nitrite degradation ability were screened out, as shown in Table 1:

[0042] Table 1 Nitrite degradation rates of different strains

[0043]

[0044] Note: Different lowercase letters in the table represent significant differences (p<0.05) in the same column, and the same letter represents no significant difference (p>0.05) in the same column. In addition, LM in the table represents the strain source of papaya pickle, LS represents the strain source of cheese, LJ represents the strain source of sour mustard, and LG represents the strain source of sour jar beans, and the following table is the same.

[0045] As can be seen from Table 1, when cultured in a medium containing 200 mg / L NaNO2 for 12 h, the nitrite degradation rate of strain LM2 was the highest at 93.12%, which was significantly higher than that of the other 4 strains (p<0.05). When cultured for 24 h, the nitrite degradation rate of strain LM2 was still the highest, and the degradation rate of strain LS2 reached 95.55%, which was not the highest but not significantly different from that of strain LM2 (p>0.05), indicating that the nitrite degradation rate would increase with time. In addition, from the top 5 strains in terms of degradation rate, except for LS2 from cheese, the other 4 strains were all from plant pickles, indicating that it was easier to screen strains capable of degrading nitrite in plant pickles.

[0046] Example 3

[0047] This example is the screening of the screening strain of the lactic acid bacteria producing γ-aminobutyric acid, which is specifically as follows:

[0048] 1. Screening of GABA-producing strains: 60 strains screened according to the lactic acid bacteria isolation and purification method of Example 1 were screened, and the specific method was as follows: the purified strains were inoculated into MRS broth medium for activation, and the activated strains were inoculated into TYG liquid medium at an inoculation amount of 4% (v / v), and cultured at 37°C and 150 r / min for 72 h. The bacterial liquid was centrifuged at 12000 r / min for 10 min, and the GABA content of the supernatant was determined; wherein, the TYG liquid medium was prepared as follows: tryptone 10 g / L, yeast extract 5 g / L, glucose 20 g / L, sodium succinate 5 g / L, L-glutamic acid 10 g / L, heated and dissolved, cooled to room temperature, adjusted to pH 6.5, and sterilized at 115°C for 25 min.

[0049] 2. GABA determination method: Berthelot colorimetric method was used for determination. Specifically, the Berthelot colorimetric method was used for determination: 200 ul of supernatant was taken after centrifugation (10000 x g, 15 min), 6% redistilled phenol 1.0 mL, borate buffer solution (pH = 9.5) 0.5 mL were added, mixed well, 5.2% hydrogen peroxide solution 1 mL was added, stood for 5 min, heated in boiling water bath for 10 min, then ice bath for 10 min, then 60% ethanol solution 2 mL was added, mixed well, and the absorbance value was measured at 645 nm, and the reagent blank was zeroed.

[0050] The top 5 GABA-producing strains obtained are shown in Table 2:

[0051] Table 2 GABA production of different strains

[0052]

[0053] Note: LM in the table represents the strain source of papaya pickle, and LS represents the strain source of cheese.

[0054] As can be seen from Table 2, the strain LS2 has the highest yield of 1.71 g / L, followed by the strain LM2, with a yield of 1.58 g / L, and the difference between the two strains is not significant (p>0.05), which is significantly higher than that of strains LS3, LS6 and LS9 (p<0.05), and the GABA production of strains LS3, LS6 and LS9 is less than 1.0 g / L. In addition, from the top 5 GABA-producing strains, except that LM2 is derived from plant pickling products, the other 4 strains are derived from cheese, indicating that it is easier to screen GABA-producing strains in dairy products.

[0055] In summary, in this screening experiment, strains LM2 and LS2 have the functions of degrading nitrite sodium and producing GABA, and strain LS2 has higher GABA production. The two strains are derived from plant pickles and dairy products, respectively, and have high research value.

[0056] Example 4

[0057] In this example, the acid tolerance of the strains was identified.

[0058] Eight strains (LM2, LS2, LJ1, LJ3, LG1, LS3, LS6, and LS9) selected from Example 2 and Example 3 were subjected to acid tolerance identification. The method was as follows: the optical density value of the bacterial solution was adjusted to OD 600 nm=1.0(±0.1), and the bacterial solution was inoculated into MRS liquid medium with pH values of 2.0, 3.0, 4.0, and 5.0 at an inoculation amount of 2%(v / v). After 24h of culture at 37°C, the OD 600 nm value of the bacterial solution was measured. The OD 600 nm value of the bacterial solution in MRS liquid medium with pH 6.5 was taken as the control. The survival rate was calculated according to the following formula.

[0059]

[0060] In the formula: OD 600 value of the sample group is the OD 600 value of the bacterial solution in different pH MRS liquid medium, the OD 600 value of the control group is the OD 600 value of the bacterial solution in MRS liquid medium with pH 6.5.

[0061] Table 3 Acid tolerance of eight strains of lactic acid bacteria

[0062]

[0063] As shown in Table 3, as the pH of the culture environment increased, the survival rate of the strains also increased, indicating that the acidic environment had a great impact on the growth of lactobacillus. At pH 2.0 and pH 3.0, the survival rate of the strains was low, and the growth activity was inhibited. At pH 4.0 and pH 5.0, the survival rate of the strains increased significantly. At pH 4.0, the survival rates of strains LM2 and LS2 were both higher than 80%, significantly higher than those of other strains (p<0.05). At pH 5.0, the survival rate of the strains reached more than 88%, of which 7 strains reached more than 97%. In summary, strains LM2 and LS2 performed best in terms of acid tolerance and had good application potential.

[0064] Example 5

[0065] In this example, the bile salt tolerance of the strains was identified.

[0066] Adjust the optical density of the bacterial solution to OD 600 nm = 1.0 (±0.1), inoculate in MRS liquid medium containing 0.1, 0.2, 0.3% bovine bile salt at an inoculum of 2% (v / v), incubate at 37°C for 24h, and measure the OD at 24h 600 nm value, using MRS liquid medium without bovine bile salt as a control, and calculate the survival rate of the strain to bile salt according to the following formula.

[0067]

[0068] In the formula: OD 600 value of the MRS liquid medium containing bile salt, OD 600 value of the control group, OD 600 value of the MRS liquid medium without bile salt, and OD 600 value.

[0069] Table 4: Bile salt tolerance of 8 strains of lactic acid bacteria

[0070]

[0071] As shown in Table 4, as the concentration of bile salt increases, the survival rate decreases. After 24h of incubation in medium containing 0.1-0.3% bile salt, the survival rate of strain LM2 varied less, followed by strain LS2, and the survival rates of these two strains were significantly higher than those of the other strains (p<0.05). When the concentration of bile salt was 0.1%, the survival rates of 6 strains reached more than 90%; when the concentration of bile salt was 0.2%, the survival rates of 4 strains reached more than 80%; and when the concentration of bile salt was 0.3%, the survival rates of 2 strains reached more than 80%. Therefore, strains LM2 and LS2 showed higher bile salt tolerance and good adaptability in complex environments, and had great application potential.

[0072] Example 6

[0073] This example is for the identification of the artificial gastrointestinal fluid tolerance of the strain.

[0074] The specific method is as follows: take 1 mL of bacterial suspension and add it to 9 mL of simulated gastric juice, mix thoroughly, and digest at 37°C at 150 r / min for 3h. Plate count the bacteria solution at 0h and 3h of digestion, using the number of viable bacteria at 0h of digestion as a control, and calculate the survival rate. Take 1 mL of simulated gastric juice and add it to 9 mL of simulated intestinal juice, incubate at 37°C for 3h, plate count the bacteria solution at 0h and 3h of digestion, using the number of viable bacteria at 0h of digestion as a control, and calculate the tolerance of the strain to simulated gastric juice according to the following formula.

[0075]

[0076] In the formula: N 0hViable count at 0h of inoculation, lg CFU / mL; N 3h Viable count at 3h of inoculation, lg CFU / mL.

[0077] Table 5 Artificial gastric and intestinal fluid tolerance of 8 strains of lactic acid bacteria

[0078]

[0079]

[0080] From Table 5, the simulated artificial gastric and intestinal fluid tolerance experiment reflects the potential of the strains to resist the erosion of the digestive fluid and maintain activity in the actual human gastrointestinal environment. Due to the unique cell structure and physiological characteristics, lactic acid bacteria can effectively resist the strong acid environment of artificial gastric juice and the degradation of pepsin, and maintain 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 immune regulation. If the viable count is too low, it is difficult to achieve the expected probiotic effect. From the simulation experiment, in artificial gastric juice, strains LM2 and LS2 can reach more than 90%, which is significantly higher than other strains (p<0.05), among which strain LS2 has the highest survival rate of 96.46%, followed by strain LM2 with a survival rate of 91.43%, and the survival rates of LM2 and LS2 are significantly different (p<0.05); in simulated artificial intestinal fluid, strains LM2 and LS2 can reach more than 80%, which is significantly higher than other strains (p<0.05), among which strain LM2 has the highest survival rate of 89.58%, followed by strain LS2 with a survival rate of 85.31%, and the survival rates of LM2 and LS2 are significantly different (p<0.05), and LS2 is more suitable for survival in artificial gastric juice than LM2 in terms of tolerance.

[0081] In summary of the above experiments, we found that in this screening, strains LM2 and LS2 showed good survival rate and tolerance in nitrite degradation rate, GABA production, acid tolerance, bile salt tolerance, artificial intestinal fluid and artificial gastric juice. Therefore, we considered these two strains as the focus of further research in the later stage, and this application mainly carried out related research on strain LS2.

[0082] Example 7

[0083] In this example, the growth and acid production ability of strain LS2 were identified.

[0084] 1. Strain growth curve: inoculate the activated bacterial solution into MRS liquid medium at an inoculation amount of 2% (V / V), and cultivate at 37℃ for 24h. Take samples every 2h to measure OD 600 nm absorbance value, and draw the growth curve of the strain.

[0085] For example,Figure 3 As shown: strain LS2 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 LS2 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 LS2 produced the highest amount of acid, reaching 21.08 g / L.

[0088] Example 8

[0089] Antibacterial experiment of strain LS2 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 LS2 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 LS2 against common pathogens.

[0092]

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

[0094] From Table 6, the strain GX-7 has bacteriostatic effect on Escherichia coli, Salmonella enterica subsp. enterica serovar Enteritidis and Campylobacter jejuni, and has no bacteriostatic effect on Staphylococcus aureus, Listeria monocytogenes and Shigella flexneri; from the bacteriostatic circle, the bacteriostatic circle of Escherichia coli is the highest, followed by Campylobacter jejuni, and then Salmonella enterica subsp. enterica serovar Enteritidis; the difference between Escherichia coli and Campylobacter jejuni is not significant (p>0.05), and the difference between Campylobacter jejuni and Salmonella enterica subsp. enterica serovar Enteritidis is not significant (p>0.05); but the bacteriostatic circle of Escherichia coli is significantly higher than that of Salmonella enterica subsp. enterica serovar Enteritidis (p<0.05).

[0095] Example 9

[0096] Activity improvement experiment of strain LS2:

[0097] Amino acids are the basic premise for microorganisms to synthesize proteins, cell proliferation and division, which can be used as nitrogen source and carbon source for microbial growth, but the types and concentrations of amino acids required by different microorganisms and different strains are not the same, and improper use of amino acid types and concentrations will inhibit the growth of strains, and some amino acids may be essential amino acids for strains, which can promote the growth of microorganisms. Therefore, in combination, amino acids have different regulatory effects on different strains, therefore, we consider selecting some amino acids for research in order to find the types and concentrations of amino acids that can promote the growth of strain LS2. The addition of amino acids will also lack any essential amino acid, which will significantly inhibit or even completely stop the growth.

[0098] In addition, vitamins have a significant impact on the growth, metabolism and activity of lactic acid bacteria. Most vitamins act as coenzymes or cofactors, directly participating in various key biochemical reactions of lactic acid bacteria, especially B vitamins. However, different bacterial genera and even different strains have great differences in the demand and synthesis capacity for specific vitamins. Therefore, we consider selecting amino acids and B vitamins for optimization, and selecting growth promoters suitable for strain LS2

[0099] The optical density of the bacterial solution was adjusted to OD 600 nm = 1.0 (± 0.1), inoculated in MRS liquid medium containing 1 g / L promoter (promoter as shown in Table 7) at an inoculation amount of 2% (v / v), incubated at 37°C for 24 h, and the OD at 24 h was measured 600 nm value; calculate the nitrite degradation rate at 24 h according to the method of Example 2; calculate the GABA yield according to the method of Example 3. The results are shown in Table 7.

[0100] Table 7 Effect of different promoters on strain LS2

[0101]

[0102] As can be seen from Table 7, histidine, arginine, ornithine and valine can significantly increase the OD value of strain LS2, promote the growth of strain LS2 (p<0.05), and methionine can significantly inhibit the growth of strain LS2 (p<0.05). From the nitrite degradation rate and GABA yield, the promotion effect of valine is not obvious, histidine, arginine and ornithine significantly increase the degradation rate of nitrite, and from the GABA yield, histidine and ornithine are significantly higher than the control group, arginine is increased, but the difference is not significant (p>0.05); from the nitrite degradation rate, the promotion of a single promoter at 24 h has reached 99%,

[0103] According to the above research results, histidine, arginine and ornithine are selected for ratio experiment to find the nitrite degradation rate and GABA yield of strain LS2, as follows:

[0104] Orthogonal experiments were designed by adding histidine, arginine and ornithine to the basic medium, and after 12 h of culture, the nitrite degradation rate and the GABA yield after 24 h of culture were calculated, and the 24 h nitrite degradation rate and the 24 h GABA yield were used as screening indicators, as follows:

[0105] Table 8 Factor levels of orthogonal experiment of strain LS2

[0106]

[0107] The above ratio orthogonal analysis and its results are shown in Table 9, respectively:

[0108] Table 9 orthogonal experiment and results

[0109]

[0110] From Table 9, the nitrite degradation rate of the 2nd, 5th and 7th test groups was still above 98% after 24 hours, maintaining a high degradation rate, which was not much different from the highest 98.23% in Table 7; the nitrite degradation rate of the 1st, 3rd-4th test groups was below 95%, which was not much different from the CK group in Table 7, indicating that the ratio of the test groups did not promote the survival rate of strain LS2, thereby affecting the degradation rate of nitrite; the nitrite degradation rate of the 8th-9th test groups was below 90%, which was significantly lower than the CK group in Table 7; from the range, the range of riboflavin > the range of valine > the range of methionine, which indicated that riboflavin had the greatest impact on the 24h degradation rate of nitrite, followed by valine, and methionine had the least impact; from the GABA content, the GABA yield of the 2nd, 5th and 7th test groups was above 2.90g / L, which was higher than the highest 2.09g / L in Table 7, the GABA content of the 1st, 3rd and 9th groups was not significantly different, which was not much different from the highest 2.09g / L in Table 7, and the GABA content of the 8th group was significantly lower than the CK group in Table 7; from the range, the range of riboflavin > the range of valine > the range of methionine, which indicated that riboflavin had the greatest impact on the yield of GABA, followed by valine, and methionine had the least impact. According to the above experimental results, therefore, we prefer the concentration of the 2nd, 5th and 7th test groups of the promoting agent, i.e. the concentration of methionine is 0.4g / L-1.2g / L, the concentration of valine is 0.4g / L-0.8g / L, and the concentration of riboflavin is 0.8g / L-1.2g / L. The optimal test group is considered to be the case where the difference in nitrite degradation rate is not significant, and the GABA yield of test 2 is significantly improved, therefore, we prefer test 2, i.e. the concentration of methionine is 0.4g / L, the concentration of valine is 0.8g / L, and the concentration of riboflavin is 0.8g / L.

[0111] Example 10

[0112] The strain LS2 was prepared into a freeze-dried powder, and the specific preparation method was as follows:

[0113] (1) Strain activation and culture: inoculate strain LS2 into MRS medium, and stand overnight for seed culture. After one generation of activation, inoculate strain LS2 into MRS medium added with the promoting agent of Test 1 (the concentration of methionine is 0.4 g / L, the concentration of valine is 0.8 g / L, and the concentration of riboflavin is 0.8 g / L) at an inoculation amount of 2% (v / v), and expand culture at 30°C under the condition of 40 r / min of a shaker for 24 h.

[0114] (2) Centrifugal collection of bacterial cells and sub-packing: after sub-packing the fermentation liquor, centrifuge at 7500 rpm for 10 min under the condition of 4°C, remove the supernatant fermentation liquor, resuspend by adding physiological saline, and obtain bacterial slurry after repeated centrifugation; mix the bacterial slurry with a solution of freeze-drying protective agent in a volume of 1 / 5 of the original fermentation liquor, and oscillate to make it uniform, to prepare a bacterial suspension.

[0115] (3) Pre-freezing: pour the bacterial suspension into a sterile culture dish with a thickness of about 0.5 cm, and pre-freeze at -80°C for 12 h.

[0116] (4) Vacuum freeze-drying: after pre-freezing the bacterial slurry, freeze-dry under the condition of a vacuum degree of 30 Pa (only one test condition is disclosed in this embodiment, and actually, the effect of freeze-drying can be achieved at 20-30 Pa) for 24 h, so that the moisture content of the freeze-dried bacterial powder is about 3%.

[0117] After the orthogonal optimization of the freeze-drying protective agent by the applicant, it is considered that the components that have the greatest impact on the protective agent are skim milk, glucose, fructose, and vitamin B, which are positively correlated with the survival rate of strain LS2. The components in the protective agent are: 28-32 g / 100 mL of skim milk, 10-12 g / 100 mL of sucrose, 2-4 g / 100 mL of fructose, and 0.02 g / 100 mL of zinc sulfate. After the orthogonal experiment, some formulations with significant differences are selected for experiments, and the specific formulations are shown in Table 10:

[0118] Table 10 Selection of freeze-dried powder protective agent

[0119]

[0120] The freeze-dried powder prepared from the freeze-dried powder protective agent prepared according to Table 10 is subjected to survival rate determination, nitrite degradation capacity determination, and salt tolerance determination, and the determination methods are as follows:

[0121] (1) Survival rate determination: at the 24th hour, the viable cell count of Lactobacillus plantarum before freeze-drying and the viable cell count of the same volume of bacterial slurry after freeze-drying are determined, respectively.

[0122] Freeze-drying survival rate / % = (viable cell count of 1 mL of bacterial slurry after freeze-drying) / (viable cell count of 1 mL of bacterial slurry before freeze-drying)

[0123] (2) The freeze-dried powder was inoculated into MRS medium at an inoculation amount of 5% for activation for 24 h, and then the activated bacterial liquid was inoculated into the culture medium at an inoculation amount of 2%. The GABA yield was calculated according to the method of Example 3.

[0124] (3) The freeze-dried powder was inoculated into MRS medium at an inoculation amount of 5% for activation for 24 h, and then the activated bacterial liquid was inoculated into the culture medium at an inoculation amount of 2%. The GABA yield was calculated according to the method of Example 2.

[0125] Table 11 Strain survival rate, nitrite degradation rate and GABA yield of different freeze-drying protectants

[0126]

[0127] As shown in Table 11, the survival rates of Groups 1-3 all reached more than 96%, which were significantly higher than those of Groups 4-6 (p<0.05), and the GABA yields were significantly higher than those of Groups 4-7. It is indicated that the survival rate of the freeze-dried bacterial powder can affect the nitrite degradation rate and GABA yield of the lactic acid bacteria and is positively correlated, and the components of the protectant are indispensable. The absence of any component can affect the survival rate of the strain, and further affect the nitrite degradation rate and GABA yield. Therefore, for the LS2 strain, the components of the protectant selected as 28-32 g / 100 mL of skim milk, 10-12 g / 100 mL of sucrose, 2-4 g / 100 mL of fructose and 0.02 g / 100 mL of zinc sulfate can well protect the strain LS2, and can keep the cell sap of the strain in balance. The optimal components of the protectant are selected as 32 g / 100 mL of skim milk, 12 g / 100 mL of sucrose, 4 g / 100 mL of fructose and 0.02 g / 100 mL of zinc sulfate.

[0128] In summary, the Lactobacillus plantarum LS2 of the present application has high GABA production capacity and nitrite degradation capacity, and has good inhibitory capacity on intestinal pathogenic bacteria compared with other strains, and also has good acid tolerance and bile salt tolerance, and can well survive in artificial intestinal fluid and artificial gastric juice. The Lactobacillus plantarum LS2 can be used for processing bacteriostatic agents, health foods and preparing pickled foods. After being prepared into freeze-dried powder by optimizing the promoting agent and the protectant, the Lactobacillus plantarum LS2 can still maintain high activity, and is a strain that can be widely applied.

[0129] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the present application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. A Lactiplantibacillus plantarum strain LS2, having a preservation number of GDMCC NO: 66633.

2. A promoter for promoting the Lactiplantibacillus plantarum strain LS2 of claim 1, characterized in that, The promoting agent consists of methionine, valine and / or riboflavin.

3. The accelerator according to claim 2, characterized in that, The concentration of methionine is 0.4 g / L-1.2 g / L, the concentration of valine is 0.4 g / L-0.8 g / L, and the concentration of riboflavin is 0.8 g / L-1.2 g / L.

4. The accelerator according to claim 2, characterized in that, The concentration of methionine is 0.4 g / L, the concentration of valine is 0.8 g / L, and the concentration of riboflavin is 0.8 g / L.

5. A lyophilized powder comprising the Lactiplantibacillus plantarum strain LS2 as claimed in claim 1, characterized in that, The protective agent in the freeze-dried powder consists of 28-32 g / 100 mL of skim milk, 10-12 g / 100 mL of sucrose, 2-4 g / 100 mL of fructose, and 0.02 g / 100 mL of zinc sulfate.

6. The lyophilized powder according to claim 5, characterized in that, The protective agent consists of 32 g / 100 mL of skim milk, 12 g / 100 mL of sucrose, 4 g / 100 mL of fructose, and 0.02 g / 100 mL of zinc sulfate.

7. Use of Lactiplantibacillus plantarum strain LS2 according to claim 1 for the preparation of an antibacterial agent, characterized in that, The pathogenic bacteria inhibited by the bacteriostatic agent are Escherichia coli, Salmonella enterica subsp. enterica serovar Enteritidis, and Campylobacter jejuni.

8. Use of the Lactiplantibacillus plantarum strain LS2 of claim 1 or the freeze-dried powder of claim 5 in improving the production of gamma-aminobutyric acid, reducing the content of nitrite, improving the tolerance to bile salts, and / or improving the acid tolerance.

9. Use of the freeze-dried powder of claim 5 in food processing.

10. A method of preparing the lyophilized powder comprising Lactiplantibacillus plantarum strain LS2 according to claim 5, characterized in that, The method comprises the following steps: (1) Strain activation and culture: inoculate the Lactiplantibacillus plantarum strain LS2 into MRS medium, and perform seed culture by standing overnight. After one passage of activation, inoculate the Lactiplantibacillus plantarum strain LS2 into MRS medium containing the promoting agent of claim 2 for expansion culture; (2) Centrifugal collection of bacterial cells, and sub-packing: after sub-packing the fermentation broth, centrifugation, and removal of the supernatant fermentation broth, resuspend with physiological saline, and repeat centrifugation to obtain bacterial slurry; Mix the bacterial slurry with 1 / 5 volume of the protective agent solution of claim 5, and oscillate to make it uniform to prepare a bacterial suspension; (3) Pre-freezing: pour the bacterial suspension into a sterile culture dish with a thickness of 0.5 cm, and pre-freeze at -80°C for 12 h; (4) Vacuum freeze-drying: after pre-freezing the bacterial slurry, freeze-dry under a vacuum degree of 20-30 Pa for 24 h, so that the moisture content of the freeze-dried bacterial powder is 3%, and the freeze-dried bacterial powder is obtained.

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