Lactobacillus plantarum XZFMCC101.23155, probiotic agent and application of lactobacillus plantarum XZFMCC101.23155

Abstract

The invention provides lactobacillus plantarum XZFMCC101.23155, a probiotic agent and application of the lactobacillus plantarum XZFMCC101.23155 and belongs to the technical field of microorganisms. The invention provides lactobacillus plantarum XZFMCC101.23155, and the preservation number of the lactobacillus plantarum XZFMCC101.23155 is GDMCC No: 65783. The lactobacillus plantarum XZFMCC101.23155 can tolerate hydrogen peroxide, a fermentation product of the lactobacillus plantarum XZFMCC101.23155 has the capacity of removing DPPH, hydroxyl free radicals and superoxide anions, and the total antioxidant activity is high. The fermentation product can also significantly reduce the MDA content in mouse serum, significantly improve the SOD level in mouse serum, improve the GSH content in mouse serum and the like. The lactobacillus plantarum XZFMCC101.23155 and commercial zymophyte are jointly used as a leavening agent to prepare the cheese, and the antioxidant activity of the cheese can be remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Lactobacillus plantarum XZFMCC101.23155, a probiotic agent, and its application. Background Technology

[0002] Oxidative stress in cells can render their own antioxidant mechanisms ineffective in combating the damage caused by reactive oxygen species (ROS), leading to various diseases and aging. During normal metabolism, cells produce free radicals. Under the influence of external factors, such as ultraviolet radiation and smoking, the number of free radicals in the body increases, and their accumulation is one of the causes of aging and various chronic diseases. Research has found that probiotics can improve the host's metabolic level and enhance its antioxidant capacity, providing a new direction for the development of antioxidant products. However, most known probiotic strains currently exhibit low DPPH free radical scavenging rates, FRAP values, or SOD activities, making it difficult to meet the requirements for highly effective antioxidants. Currently, no *Lactobacillus plantarum* strains have been found to possess good antioxidant properties. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the purpose of this invention is to provide a plant lactobacillus with good antioxidant properties that can be applied to the body for antioxidant purposes.

[0004] The objective of this invention is achieved through the following technical solution:

[0005] This invention provides a Lactiplantibacillus plantarum XZFMCC101.23155, the preservation number of which is GDMCCNo: 65783.

[0006] This invention provides a probiotic agent comprising Lactobacillus plantarum XZFMCC101.23155 as described in the above technical solution.

[0007] Preferably, the dosage form of the probiotic agent includes granules, capsules, liquids, tablets, or powders.

[0008] This invention provides a method for preparing the probiotic agent described in the above technical solution, comprising:

[0009] Lactobacillus plantarum XZFMCC101.23155 was cultured in a culture medium to obtain a probiotic agent.

[0010] Preferably, the culture temperature is 30–37°C and the culture time is 12–48 h.

[0011] This invention provides the application of Lactobacillus plantarum XZFMCC101.23155 described in the above technical solution, the probiotic agent described in the above technical solution, or the probiotic agent prepared by the preparation method described in the above technical solution in the preparation of antioxidant products and / or the improvement of the antioxidant activity of products.

[0012] Preferably, the antioxidant includes one or more of the following (1) to (7): (1) improving DPPH scavenging ability; (2) improving ABTS cationic free radical scavenging ability; (3) improving hydroxyl free radical scavenging ability; (4) improving total antioxidant capacity; (5) reducing malondialdehyde content; (6) increasing superoxide dismutase content; (7) increasing glutathione content.

[0013] Preferably, the product includes pharmaceuticals, cosmetics, or food.

[0014] This invention provides a mozzarella cheese, the active ingredients of which include Lactobacillus plantarum XZFMCC101.23155 as described in the above technical solution, the probiotic agent as described in the above technical solution, or the probiotic agent prepared by the preparation method described in the above technical solution.

[0015] The present invention provides a method for preparing mozzarella cheese as described in the above technical solution, comprising: using the Lactobacillus plantarum XZFMCC101.23155 and / or the probiotic agent and lactic acid bacteria compound as a starter culture to ferment milk, thereby preparing cheese and obtaining mozzarella cheese.

[0016] The beneficial effects of this invention are:

[0017] This invention provides a *Lactiplantibacillus plantarum* XZFMCC101.23155, whose preservation number is GDMCCNo: 65783. This *Lactiplantibacillus plantarum* XZFMCC101.23155 is resistant to hydrogen peroxide, and its fermentation products have the ability to scavenge DPPH, hydroxyl radicals, and superoxide anions, exhibiting high total antioxidant activity. The fermentation products can also significantly reduce MDA levels in mouse serum, significantly increase SOD levels in mouse serum, and increase GSH levels in mouse serum. This invention uses *Lactiplantibacillus plantarum* XZFMCC101.23155 in combination with commercial fermentation bacteria as a starter culture in cheese preparation, which can significantly improve the antioxidant activity of cheese. Furthermore, the *Lactobacillus plantarum* XZFMCC101.23155 strain exhibits good tolerance to gastric juice, intestinal juice, and bile salts, moderate resistance to kanamycin and tetracycline, and resistance to minocycline and erythromycin. In summary, the *Lactobacillus plantarum* XZFMCC101.23155 strain provided by this invention can be used for antioxidant applications in the body.

[0018] Biological Preservation Instructions

[0019] Lactobacillus plantarum XZFMCC101.23155, classified as Lactiplantibacillusplantarum, was deposited on January 14, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCCNo: 65783. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The colony morphology of XZFMCC101.23155 is shown.

[0022] Figure 2 The growth curve and acid production curve of XZFMCC101.23155 are shown.

[0023] Figure 3 This is a graph showing the DPPH free radical scavenging rate detection results of the strains obtained in the initial screening of Example 3;

[0024] Figure 4 This is a graph showing the ABTS cationic free radical scavenging rate detection results of the strains obtained in the initial screening of Example 3;

[0025] Figure 5 This is a graph showing the detection results of the hydroxyl radical scavenging rate of the strains obtained in the initial screening of Example 3;

[0026] Figure 6 This is a graph showing the total antioxidant activity detection results of the strains obtained in the initial screening of Example 3;

[0027] Figure 7 The graph shows the results of detecting malondialdehyde (MDA), a lipid oxidation product, in the serum of mice in each experimental group (n=7).

[0028] Figure 8 The graph shows the results of SOD (superoxide dismutase) levels in the serum of mice in each experimental group (n=7).

[0029] Figure 9 The graph shows the results of detecting the level of glutathione (GSH), an antioxidant, in the serum of mice in each experimental group (n=7).

[0030] Figure 10 Image of the compound fermented cheese prepared for the experimental group;

[0031] Figure 11 Bubble diagram of the metabolic pathway of mozzarella cheese;

[0032] Figure 12 A diagram showing the metabolic pathways of alanine, aspartic acid, and glutamic acid in mozzarella cheese.

[0033] Figure 13 Diagram of the biosynthetic metabolic pathways of valine, leucine, and isoleucine in mozzarella cheese;

[0034] Figure 14 Diagram showing the biosynthetic pathways of phenylalanine, tyrosine, and tryptophan in mozzarella cheese;

[0035] Figure 15 A diagram showing the metabolic pathways of arginine and proline in mozzarella cheese.

[0036] Figure 16 This refers to the phenylalanine metabolic pathway in mozzarella cheese. Detailed Implementation

[0037] This invention provides a Lactiplantibacillus plantarum XZFMCC101.23155, the preservation number of which is GDMCCNo: 65783.

[0038] The *Lactobacillus plantarum* XZFMCC101.23155 provided by this invention is a purified strain isolated from local Tibetan yeast samples. *Lactobacillus plantarum* XZFMCC101.23155 grows on MRS plates, forming round colonies with neat edges, opaque surfaces, a raised center, and a smooth, milky-white color, classifying it as a Gram-positive bacterium. The 16S rDNA sequence of *Lactobacillus plantarum* XZFMCC101.23155 is shown in SEQ ID NO.1. *Lactobacillus plantarum* XZFMCC101.23155 provided by this invention is sensitive to polymyxins and penicillin, moderately resistant to kanamycin and tetracycline, and resistant to minocycline and erythromycin. *Lactobacillus plantarum* XZFMCC101.23155 provided by this invention is resistant to gastric juice, intestinal juice, and bile salts.

[0039] This invention provides a probiotic agent comprising *Lactobacillus plantarum* XZFMCC101.23155 as described in the above-mentioned technical solution. As an optional embodiment of this invention, the viability of the probiotic agent can be ≥1×10⁻⁶. 8 CFU / mL or ≥1×10 8 CFU / g can also be 1×10 9 CFU / mL or 1×109 CFU / g. As an optional embodiment of the present invention, the dosage form of the probiotic agent includes granules, capsules, liquids, tablets, or powders. The present invention does not specifically limit the preparation method of different dosage forms of probiotic agents; any conventional probiotic agent preparation method in the art can be used.

[0040] This invention provides a method for preparing the probiotic agent described in the above-mentioned technical solution, comprising: culturing *Lactobacillus plantarum* XZFMCC101.23155 in a culture medium to obtain the probiotic agent. As an optional embodiment of this invention, the culture medium includes MRS medium; the culture temperature can be 30–37°C, or 30, 37, 32, 33, 34, 35, 36, or 37°C; the culture time can be 12–48 hours, or 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours. After culture, a fermentation broth is obtained. As an optional embodiment of the present invention, the fermentation broth can be directly used as a probiotic agent. Alternatively, the supernatant and bacterial cells of the obtained fermentation broth can be separated; the supernatant can be directly used as a probiotic agent, and the bacterial cells can be resuspended to obtain a bacterial suspension, which can also be used as a probiotic agent.

[0041] This invention provides the application of *Lactobacillus plantarum* XZFMCC101.23155 described in the above-mentioned technical solution, the probiotic agent described in the above-mentioned technical solution, or the probiotic agent prepared by the preparation method described in the above-mentioned technical solution in the preparation of antioxidant products and / or the improvement of the antioxidant activity of products. As an optional embodiment of this invention, the antioxidant effect includes one or more of the following (1) to (7): (1) improving DPPH scavenging ability; (2) improving ABTS cationic free radical scavenging ability; (3) improving hydroxyl free radical scavenging ability; (4) improving total antioxidant capacity; (5) reducing malondialdehyde content; (6) increasing superoxide dismutase content; (7) increasing glutathione content. As an optional embodiment of this invention, the product includes pharmaceuticals, cosmetics, or food.

[0042] The *Lactobacillus plantarum* XZFMCC101.23155 or the probiotic agent provided by this invention can improve DPPH scavenging ability. The fermentation product or fermentation supernatant of the *Lactobacillus plantarum* XZFMCC101.23155 provided by this invention has a high DPPH scavenging ability, which is beneficial for improving the DPPH scavenging ability of the organism and / or the product.

[0043] The *Lactobacillus plantarum* XZFMCC101.23155 or the probiotic agent provided by this invention can enhance the ABTS cationic free radical scavenging ability. The fermentation product or fermentation supernatant of the *Lactobacillus plantarum* XZFMCC101.23155 provided by this invention has a high ABTS cationic free radical scavenging ability, which is beneficial for improving the ABTS cationic free radical scavenging ability of the organism and / or the product.

[0044] The *Lactobacillus plantarum* XZFMCC101.23155 or the probiotic agent provided by this invention can enhance the hydroxyl radical scavenging ability. The fermentation product or fermentation supernatant of the *Lactobacillus plantarum* XZFMCC101.23155 provided by this invention has a high hydroxyl radical scavenging ability, which is beneficial for improving the hydroxyl radical scavenging ability of the body and / or products.

[0045] The *Lactobacillus plantarum* XZFMCC101.23155 or the probiotic agent provided by this invention can improve total antioxidant capacity. The fermentation product or fermentation supernatant of the *Lactobacillus plantarum* XZFMCC101.23155 provided by this invention has high iron ion reducing capacity and high total antioxidant capacity, which is beneficial for improving the total antioxidant capacity of the organism and / or the product.

[0046] The *Lactobacillus plantarum* XZFMCC101.23155 or the probiotic agent provided by this invention can reduce malondialdehyde (MDA) content. The results of the embodiments of this invention show that *Lactobacillus plantarum* XZFMCC101.23155 can significantly reduce MDA content in mouse serum.

[0047] The *Lactobacillus plantarum* XZFMCC101.23155 or the probiotic agent provided by this invention can increase the content of superoxide dismutase (SOD). The results of the embodiments of this invention show that *Lactobacillus plantarum* XZFMCC101.23155 can significantly increase the SOD level in mouse serum.

[0048] The *Lactobacillus plantarum* XZFMCC101.23155 or the probiotic agent provided by this invention can increase glutathione content. The results of the embodiments of this invention show that *Lactobacillus plantarum* XZFMCC101.23155 can significantly increase GSH levels in mouse serum.

[0049] This invention provides a mozzarella cheese, the active ingredients of which include Lactobacillus plantarum XZFMCC101.23155 as described in the above technical solution, the probiotic agent as described in the above technical solution, or the probiotic agent prepared by the preparation method described in the above technical solution.

[0050] This invention provides a method for preparing the mozzarella cheese described in the above-mentioned technical solution, comprising: using *Lactobacillus plantarum* XZFMCC101.23155 and / or a probiotic agent combined with lactic acid bacteria as a starter culture to ferment milk, thereby preparing cheese and obtaining mozzarella cheese. As an optional embodiment of this invention, the lactic acid bacteria include the commercial strain DOM1 lactic acid bacteria. This invention does not impose any particular limitation on the proportion of the compounding; any conventional compounding proportion in the art can be used. This invention does not impose any particular limitation on the preparation method of the mozzarella cheese; any conventional preparation method in the art can be used.

[0051] This invention uses Lactobacillus plantarum XZFMCC101.23155 and commercial lactic acid bacteria to ferment milk to prepare cheese, which can enhance the antioxidant activity of mozzarella cheese.

[0052] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1: Isolation and Purification of Strains

[0054] Take samples of local Tibetan yeast and perform serial dilutions of 10-fold, gradually reducing each dilution to 10-fold. -7 Four suitable dilutions were selected, and 100 μL of each was spread onto MRS agar plates. After incubation at 37°C for 48 h, single colonies with different morphologies were selected and isolated using the streak plate method to obtain multiple purified strains. One of these strains was XZFMCC101.23155, abbreviated as XZFMCC101.23155. The colony morphology of XZFMCC101.23155 is shown below. Figure 1 As shown, the colonies are round, with neat edges, opaque, raised in the middle, smooth, and milky white, belonging to Gram-positive bacteria.

[0055]

[0056] The 16S rDNA sequence of strain XZFMCC101.23155 has 100% similarity to the 16S rDNA sequence of Lactiplantibacillus plantarumstrain LRCC5195, further confirming that strain XZFMCC101.23155 is Lactobacillus plantarum. Strain XZFMCC101.23155 is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 65783.

[0057] Example 2: Growth curves and acid production curves of the strain

[0058] The bacterial concentration of strain XZFMCC101.23155 isolated in Example 1 was adjusted to 1×10⁻⁶. 9 The bacterial suspension was inoculated into MRS liquid medium at a rate of 1% (V / V) and incubated at 30°C for 48 hours.

[0059] The sampling time for the growth curve was as follows: samples were taken every 2 hours during the first 16 hours, and every 4 hours from 16 to 48 hours, to measure the OD of the bacterial culture. 600nm The sampling time for the acid production curve was as follows: samples were taken every 2 hours within the first 8 hours, and every 4 hours from 8 to 48 hours to measure the pH value. Results are shown below. Figure 2 .

[0060] Example 3: Determination of the strain's tolerance to hydrogen peroxide

[0061] Oxidants can stimulate the body's oxidative defense system to produce various enzymes. When microorganisms are treated with low concentrations of H2O2, the cells develop adaptive abilities to protect themselves from the harm of higher concentrations of oxidants. Therefore, H2O2 tolerance can be used as an indicator to measure the antioxidant activity of microorganisms.

[0062] The cell concentration of the purified strain obtained in Example 1 was adjusted to 1×10⁻⁶. 9 CFU / mL bacterial suspensions were inoculated at 3% (V / V) into liquid culture media with different H2O2 concentrations (1 mmol / L and 2 mmol / L, respectively), and incubated at 37℃ for 8 h. The OD values ​​of the bacterial suspensions at different H2O2 concentrations were then measured using a multi-functional microplate reader. 600nm value.

[0063] When the initial H2O2 concentration was 2 mM, the OD after 8 h of growth 600nmStrains with H2O2 tolerance values ​​above 1.00 were considered to have strong H2O2 tolerance. A total of seven strains were identified: XZFMCC101.2329, XZFMCC101.2366, XZFMCC107.2341, XZFMCC107.2303, XZFMCC101.23155, XZFMCC101.23157, and XZFMCC101.23186. Further screening tests were conducted.

[0064] Example 4

[0065] The seven strains screened in Example 3 were used at a concentration of 1.01 ± 0.05 × 10⁻⁶. 9 After culturing the seed culture with CFU / mL for 16 h, the supernatant was collected after centrifugation at 4000 r / min for 10 min and then subjected to the following measurements:

[0066] 1) The DPPH scavenging capacity of the supernatant was determined using the BC4750 kit from Beijing Solarbio Science & Technology Co., Ltd.

[0067] DPPH free radical scavenging rate = [[A blank - (A determination - A control)] ÷ A blank] × 100%; where, A blank: absorbance of the blank group (containing only DPPH solution + solvent, without the test sample); A determination: absorbance of the test sample group (DPPH solution + test antioxidant); A control: absorbance of the control group (test sample + solvent, without DPPH).

[0068] 2) The ABTS cation radical scavenging ability of the supernatant was determined using the BC4770 kit from Beijing Solarbio Science & Technology Co., Ltd.

[0069] ABTS free radical scavenging rate = [A blank - (A assay - A control)] ÷ A blank × 100%.

[0070] 3) The hydroxyl radical scavenging capacity of the supernatant was determined using the BC1320 kit from Beijing Solarbio Science & Technology Co., Ltd.

[0071] Hydroxyl radical scavenging rate = (Atest - Apair) ÷ (Ablank - Apair) × 100%.

[0072] 4) The total antioxidant capacity (FRAP) of the supernatant was determined using the BC1315 kit from Beijing Solarbio Science & Technology Co., Ltd.

[0073] Standard curve plotting: based on Fe 2+ A standard curve was established based on the final concentration and absorbance. The δA measurement was then substituted into the standard curve equation to calculate the equivalent Fe. 2+ Concentration (x).

[0074] Total antioxidant capacity (μmol / mL) = x × V 反总 ÷V 样 = 34 × x; where x represents the concentration of antioxidants (μmol / L) measured by the standard curve; V 反 Always the total volume of the reaction system (mL); V 样 This is the actual amount of sample used (mL); V 反 The total / V sample is 34.

[0075] The results are shown in Table 1 and Figures 3-6 .

[0076] Table 1. Results of antioxidant activity detection of strains obtained from the initial screening in Example 3.

[0077]

[0078] From Table 1 and Figures 3-6 The DPPH scavenging rates of the strains ranged from 55.41% to 78.49%, with significant differences in DPPH scavenging ability among different strains, and XZFMCC101.2366 exhibiting the strongest DPPH scavenging capacity. The ABTS scavenging capacity also showed significant differences among different strains, with XZFMCC101.23155 demonstrating a scavenging capacity of 92.24%, indicating good ABTS cationic free radical scavenging ability. The hydroxyl radical scavenging rates of the strains ranged from 14.3% to 26.28%, with significant differences in hydroxyl radical scavenging capacity among different strains, and XZFMCC101.23157 exhibiting the strongest hydroxyl radical scavenging capacity. The total antioxidant activity of the strains ranged from 13.07 to 23.36 μmol / mL, with significant differences in total antioxidant capacity among different strains. Lactobacillus plantarum XZFMCC101.23155 had a total antioxidant capacity of 21.33 μmol / mL, indicating that Lactobacillus plantarum XZFMCC101.23155 possesses good total antioxidant capacity.

[0079] Example 5: Determination of the survival rate of the strain in artificial gastric fluid

[0080] In the real human gut environment, pepsin and trypsin hydrolyze the proteins of microorganisms, and may even inhibit or kill them. Therefore, the tolerance of lactic acid bacteria to artificially simulated gastrointestinal fluids was analyzed.

[0081] Artificial gastric juice: 0.35g pepsin was dissolved in 100mL of 0.2% sterile physiological saline, and the pH was adjusted to 3.0 with hydrochloric acid before filtration for sterilization. The XZFMCC101.23155 strain obtained in Example 1 was cultured in MRS medium for 18h, and the bacterial cells were collected by centrifugation at 4000r / min for 10min. After washing twice with sterile physiological saline, the cells were resuspended in an equal volume of sterile physiological saline to prepare 1×10⁻⁶ cells / mL. 9CFU / mL bacterial suspension. 1 mL of bacterial suspension was added to 9 mL of artificial gastric juice, mixed thoroughly, and incubated at 37°C for 3 h. Samples were taken at 0 h and 3 h for plate counting to calculate the tolerance of each strain to artificial gastric juice.

[0082] Survival rate = (lgN / lgN0) × 100%;

[0083] In the formula: N represents the number of viable bacteria after treatment with artificial simulated gastric juice, in CFU / mL; N0 represents the number of viable bacteria without treatment with artificial simulated gastric juice, in CFU / mL.

[0084] The survival rate of Lactobacillus plantarum XZFMCC101.23155 after 3 hours of culture in artificial gastric juice was 46.2%, indicating that Lactobacillus plantarum XZFMCC101.23155 has a good survival rate in gastric juice and good gastric juice tolerance.

[0085] Example 6: Determination of the survival rate of the strain in artificial intestinal fluid

[0086] Artificial intestinal fluid: According to the method provided in the 2015 edition of the Chinese Pharmacopoeia, accurately weigh 6.8 g of potassium dihydrogen phosphate and add 500 mL of water. Adjust the pH to 6.8 with 0.4% NaOH solution; separately dissolve 10 g of trypsin in an appropriate amount of water. Mix the two solutions and add water to a final volume of 1000 mL. Adjust the pH to 8.0 with 0.1 mol / L NaOH, then thoroughly dissolve the contents and filter through a 0.22 μm microporous membrane for sterilization. Store at 4°C.

[0087] 1×10⁻⁶ of Lactobacillus plantarum XZFMCC101.23155 9 CFU / mL bacterial suspension was inoculated into artificial intestinal fluid and cultured at 37°C for 3 h. The survival rate of XZFMCC101.23155 in artificial intestinal fluid was then detected.

[0088] Survival rate = (lgN / lgN0) × 100%;

[0089] In the formula: N represents the number of viable bacteria after treatment with artificial intestinal fluid, in CFU / mL; N0 represents the number of viable bacteria without treatment with artificial intestinal fluid, in CFU / mL.

[0090] The survival rate of Lactobacillus plantarum XZFMCC101.23155 in artificial intestinal fluid was 88.8%, significantly higher than 60%, indicating that Lactobacillus plantarum has a good survival rate in intestinal fluid and has good intestinal fluid tolerance.

[0091] Example 7: Determination of the strain's tolerance to bile salts

[0092] The small intestine is an important site for lactic acid bacteria to exert their probiotic effects. The bile salt content in the normal human small intestine fluctuates within the range of 0.03% to 0.3%. Bile salts can alter the permeability of the bacterial outer membrane and damage the bacterial deoxyribonucleic acid, thereby affecting the colonization of lactic acid bacteria in the human intestine. Therefore, bile salt tolerance is an important indicator for evaluating whether lactic acid bacteria can exert their probiotic effects in the human gastrointestinal tract.

[0093] 1×10⁻⁶ bacterial suspension of strain XZFMCC101.23155 9 CFU / mL was inoculated at a 3% (v / v) inoculum into MRS liquid medium containing 0.2% ox bile salt and without ox bile salt, respectively. OD was measured after incubation at 37°C for 24 h. 600nm Value. Calculate the bile salt tolerance of lactic acid bacteria according to the formula.

[0094] Lactic acid bacteria bile salt tolerance (%) = (A1 / A0) × 100%

[0095] In the formula: A1 represents the OD after 24 h of culture in MRS medium containing 0.3% ox bile salts. 600nm Value; A0 represents the OD value after 24 hours of culture in MRS medium without ox bile salts. 600nm value.

[0096] The survival rate of Lactobacillus plantarum XZFMCC101.23155 in bovine bile salts was 25.73%, significantly higher than 10%, indicating that XZFMCC101.23155 has good bile salt tolerance.

[0097] Example 8: Determination of drug susceptibility of strains

[0098] Antibiotic susceptibility testing was performed using the disk agar diffusion method (KB method). 100 μL of activated solution at a concentration of 10... 7 ~10 8 A CFU / mL suspension of XZFMCC101.23155 was evenly spread onto an MRS agar plate using a spreader. Antimicrobial tablets were then placed on top, and the plates were incubated anaerobically at 37°C for 48 hours. The diameter of the inhibition zone was measured, and three replicates were performed for each antibiotic. The results are shown in Table 2.

[0099] Table 2. Results of antibiotic susceptibility of Lactobacillus plantarum XZFMCC101.23155.

[0100] antibiotic XZFMCC101.23155 Minocycline R tetracycline I Kanamycin I Erythromycin R penicillin S Polymyxin S

[0101] Note: In the table, S: sensitive; I: moderately resistant; R: resistant.

[0102] Table 2 shows that Lactobacillus plantarum XZFMCC101.23155 is sensitive to polymyxin and penicillin, moderately resistant to kanamycin and tetracycline, and resistant to minocycline and erythromycin.

[0103] Example 9: Establishing a mouse model

[0104] XZFMCC101.23155 Sample Preparation: Take OD 600nm Seed culture of Lactobacillus plantarum XZFMCC101.23155 with a growth rate between 0.8 and 1.0 was inoculated into MRS liquid medium and cultured on a shaker at 37°C and 200 rpm for 24 h. The bacterial culture was then transferred to sterile EP tubes, centrifuged at 5000×g and 4°C for 10 min, and filtered through a 0.22 μm microporous membrane. The supernatant was used as the XZFMCC101.23155 sample for the following experiments.

[0105] Male C57BL / 6J mice aged 8-10 weeks were purchased from SpeyBio (Suzhou) Co., Ltd. and housed in a standardized laboratory with a room temperature of 25±2℃, relative humidity of 50±5%, and 12h light / 12h darkness. The experiment began after one week of acclimatization feeding.

[0106] After 7 days of acclimatization, the mice were weighed and randomly divided into five groups: blank control group, model group, positive control group, experimental group 1 (low dose of sample XZFMCC101.23155), and experimental group 2 (high dose of sample XZFMCC101.23155), with 7 mice in each group.

[0107] The positive control group received daily oral administration of 0.20 mL of vitamin C solution, at a dose of 150 mg / kg per mouse. Experimental group 1 received daily oral administration of 0.2 mL of XZFMCC101.23155 sample. Experimental group 2 received daily oral administration of 0.4 mL of XZFMCC101.23155 sample. The blank control group and model group received daily oral administration of 0.2 mL of physiological saline. Except for the blank control group, oxidative damage was induced 30 minutes after oral administration using D-galactose (D810319-100g, Shanghai Maclean Biochemical Technology Co., Ltd.) at a dose of 600 mg / kg. D-galactose was administered intraperitoneally at a dose of 0.1 mL / 10g. The oral administration and modeling process continued for 35 days. During the 35-day continuous gavage and modeling process, the modeling agent was injected after each daily gavage administration.

[0108] Example 10: Determination of malondialdehyde (MDA) in mouse serum

[0109] Malondialdehyde (MDA) is an important metabolite produced by oxygen free radicals attacking unsaturated fatty acids in lipids within living organisms. MDA is cytotoxic, causing cross-linking and polymerization of biomolecules such as proteins, leading to alterations in cell membrane structure and function, and causing severe damage to cells.

[0110] In each of the five groups of experiments in Example 9, the same number of mice were randomly selected, and serum was collected from the mice. MDA levels in the mouse serum were measured using a kit from Nanjing Jiancheng Bioengineering Institute (catalog number A003-1-2), as shown in Table 3. Figure 7 As shown. Compared with the model group, ***p<0.001.

[0111] Table 3. Detection results of malondialdehyde (MDA) levels in serum of mice in each experimental group (n=7)

[0112]

[0113] From Table 3 and Figure 7 It was found that ingestion of Lactobacillus plantarum XZFMCC101.23155 in mice reduced the MDA content in their serum.

[0114] Example 11: Determination of SOD / GSH levels in mouse serum

[0115] Superoxide dismutase (SOD) has antioxidant and anti-aging effects. Its mechanism of action is mainly to scavenge harmful superoxide anion free radicals (O2). - Superoxide dismutase (SOD) is an enzyme that catalyzes the conversion of superoxide into oxygen and hydrogen peroxide through a dismutation reaction. It is widely found in various animals, plants, and microorganisms and is an important antioxidant that protects cells exposed to oxygen. Glutathione (GSH) helps maintain normal immune system function and has antioxidant and detoxification effects. SOD and GSH have extremely wide applications, primarily in delaying aging, preventing disease, improving immunity, and as food and cosmetic additives.

[0116] In each of the five groups of experiments in Example 9, the same number of mice were randomly selected, and serum was collected from the mice. SOD levels in the mouse serum were measured using a kit from Jianglai Biotechnology (product number JL12237), and GSH levels were measured using a kit from Nanjing Jiancheng Bioengineering Institute (product number A006-2-1). See Table 4 and... Figures 8-9 As shown. Compared with the model group, *p<0.05, **p<0.01.

[0117] Table 4. Results of SOD and GSH levels in the serum of mice in each experimental group

[0118]

[0119] From Table 4 and Figures 8-9 It was found that ingestion of Lactobacillus plantarum XZFMCC101.23155 in mice increased the levels of SOD and GSH in their serum.

[0120] Example 12: Determination of the antioxidant capacity of the strain in cheese production

[0121] 1. Preparation of lactic acid bacteria starter: *Lactobacillus plantarum* XZFMCC101.23155 was inoculated into MRS liquid medium and cultured at 37℃ for 12 h. The supernatant was discarded after centrifugation at 5000 r / min for 10 min. The bacterial cells were washed twice with sterile water under the same conditions, and the bacterial count was adjusted to approximately 10⁻⁶ cells / mL with sterile water. 8 CFU / mL.

[0122] 2. Cheese preparation process: Raw milk (cow's milk) → pasteurization (61℃, 30min) → cooling to 30~32℃ → inoculation with starter culture and fermentation at 30~32℃ until the final pH value is 5.4 → curdling (adding Clerici rennet 0.03g / L) → cutting (2cm×2cm) → heating (slowly heating for 30min, then heating to 41℃) → whey discharge (curd blocks are cut into 2~3cm square pieces, and slowly shaken to discharge whey at 45℃ for 30min) → heat scalding and stretching (heat scalding temperature is 70℃, stretching is repeated 20 times until the surface is smooth, then formed into cheese balls and cooled) → shaping → cooling → packaging → post-ripening (vacuum packaged and placed at 4℃ for 21 days of post-ripening).

[0123] Samples of mozzarella cheese were taken during the post-ripening process for observation and measurement.

[0124] 3. Divide into two experimental groups: a blank control group and an experimental group.

[0125] In the blank control group, when preparing cheese according to the above cheese preparation process, 0.2% (w / v) of commercial DOM1 lactic acid bacteria powder (originating from Italy, model DOM1, distributor: Beijing Youduo Aite Biotechnology Co., Ltd.) was added to the milk during the fermentation process. The resulting mozzarella cheese was denoted as commercially fermented cheese.

[0126] When the experimental group prepared cheese according to the above cheese preparation process, during fermentation with the starter culture, 0.2% (w / v) commercial DOM1 lactic acid bacteria powder and 2% (v / v) of a 10% concentration of [unspecified ingredient] were added to the milk. 8 A CFU / mL suspension of XZFMCC101.23155 bacteria was used as the starter culture for fermentation. The mozzarella cheese prepared by the experimental group is denoted as compound fermented cheese. The compound fermented cheese prepared by the experimental group is shown below. Figure 10 As shown.

[0127] The scavenging rates of ABTS, hydroxyl, and DPPH free radicals in commercially fermented cheese and compound fermented cheese were measured on days 1, 7, 14, and 21 of post-ripening to compare the differences in antioxidant activity indicators of mozzarella cheese products fermented with different starter cultures. The specific methods were strictly performed according to the kit instructions for each indicator.

[0128] The results of antioxidant index determination for commercially fermented cheese and compound fermented cheese during the post-ripening process are shown in Table 5.

[0129] Table 5. Results of antioxidant index determination for commercially fermented cheese and compound fermented cheese.

[0130]

[0131] Note: Different capital letters in the same column indicate differences in cheese at different ripening stages (P < 0.05); different lowercase letters in the same row indicate significant differences in cheese at the same ripening stage (P < 0.05).

[0132] As shown in Table 5, during the post-ripening period, the DPPH radical, ABTS cationic radical, and hydroxyl radical scavenging abilities of the compound fermented cheese were significantly stronger than those of the commercially fermented cheese (P < 0.05). Specifically, at 7 days of fermentation, the ABTS radical scavenging rate of the compound fermented cheese (35.08%) was 2.5 times that of the control group, and the hydroxyl radical scavenging rate (35.37%) was 2.3 times that of the control group. At 14 days of fermentation, the hydroxyl radical scavenging rate of the compound fermented cheese (30.22%) was 1.6 times that of the control group, and the DPPH radical scavenging rate (14.60%) was 1.8 times that of the control group. At 21 days of fermentation, the ABTS radical scavenging rate of the compound fermented cheese (17.49%) was 2.7 times that of the control group, the hydroxyl radical scavenging rate (25.34%) was 2.3 times that of the control group, and the DPPH radical scavenging rate (16.30%) was 2.7 times that of the control group. The results show that the addition of *Lactobacillus plantarum* XZFMCC101.23155 significantly increased the antioxidant activity of mozzarella cheese, and the antioxidant activity initially increased and then decreased during fermentation. In conclusion, the screened *Lactobacillus plantarum* XZFMCC101.23155 can significantly enhance the antioxidant activity of mozzarella cheese.

[0133] Example 13: Comparison of Metabolite Differences in Cheese Based on Non-Targeted Metabolomics

[0134] This study used commercially fermented cheese and compound fermented cheese aged 21 days after ripening as research subjects, comparing metabolite differences based on non-targeted metabolomics. Figure 11As can be seen, KEGG pathway analysis revealed five main metabolic pathways, in order of frequency: ① alanine, aspartate, and glutamate metabolism; ② valine, leucine, and isoleucine biosynthesis; ③ phenylalanine, tyrosine, and tryptophan biosynthesis; ④ arginine and proline metabolism; and ⑤ phenylalanine metabolism. See the detailed metabolic pathway diagram below. Figures 12-16 .

[0135] The association with antioxidant activity is reflected in the following aspects: the alanine-glucose cycle can transport amino acids between muscles and the liver, reducing ammonia toxicity (high ammonia levels can induce oxidative stress); glutamate is an essential component for the synthesis of glutathione (one of the most important antioxidants in cells), and glutathione exerts its antioxidant effect directly by scavenging reactive oxygen species (ROS) and repairing oxidative damage; leucine promotes protein synthesis and cell growth by activating the mTORC1 pathway (mammalian target of rapamycin complex 1), and moderate mTOR signaling can enhance defense by inducing the expression of antioxidant genes (such as the Nrf2 target gene); phenylalanine is catalyzed by phenylalanine hydroxylase to produce tyrosine, which is converted into dopamine (with free radical scavenging ability) and melanin (which reduces oxidative damage by chelating metal ions); arginine generates NO through nitric oxide synthase (NOS), and low concentrations of NO have antioxidant effects (such as inhibiting lipid peroxidation); proline acts as a compatible solute in plant and some animal cells, protecting proteins and membrane structures from ROS damage.

[0136] In conclusion, compared with commercially fermented mozzarella cheese, the compound fermented mozzarella cheese with added Lactobacillus plantarum XZFMCC101.23155, which has antioxidant functions, demonstrated higher antioxidant activity at the amino acid metabolic pathway level.

[0137] Example 14

[0138] After the compound fermented cheese prepared in Example 13 was completed, it was tested by Chengdu Huace Testing Technology Co., Ltd., and the test results are shown in Table 6.

[0139] Table 6. Detection results of compound fermented cheese

[0140]

[0141]

[0142]

[0143] The tests showed that the compound fermented cheese met the requirements of GB5420-2021 "National Food Safety Standard for Cheese".

[0144] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Lactobacillus plantarum (Lactiplantibacillus plantarum) XZFMCC101.23155, characterized in that The deposit number of the Lactobacillus plantarum XZFMCC101.23155 is GDMCC No: 65783.

2. A probiotic, characterized in that It comprises the plant lactobacillus XZFMCC101.23155 described in claim 1.

3. The probiotic according to claim 2, characterized in that The dosage forms of the probiotics include granules, capsules, liquids, tablets or powders.

4. The method for preparing the probiotic according to claim 2 or 3, characterized in that: include: Lactobacillus plantarum XZFMCC101.23155 is cultured in a culture medium to obtain a probiotic.

5. The preparation method according to claim 4, characterized in that The culture temperature is 30-37° C.; the culture time is 12-48 hours.

6. Use of the Lactobacillus plantarum XZFMCC101.23155 according to claim 1, the probiotic according to claim 2 or 3, or the probiotic prepared by the preparation method according to claim 4 or 5 in preparing an antioxidant product and / or improving the antioxidant activity of a product.

7. The use according to claim 6, characterized in that The antioxidant includes one or more of the following (1) to (7): (1) Improve DPPH scavenging ability; (2) Improve the ABTS cationic radical scavenging ability; (3) Improve the ability to scavenge hydroxyl radicals; (4) Improve total antioxidant capacity; (5) Reduce malondialdehyde content; (6) Increase superoxide dismutase content; (7) Increase glutathione content.

8. The use according to claim 6, characterized in that The products include medicines, cosmetics or foods.

9. A mozzarella cheese, characterized in that: The active ingredients include the Lactobacillus plantarum XZFMCC101.23155 according to claim 1, the probiotic according to claim 2 or 3, or the probiotic prepared by the preparation method according to claim 4 or 5.

10. The method for preparing mozzarella cheese according to claim 9, characterized in that: include: The Lactobacillus plantarum XZFMCC101.23155 and / or the probiotic agent are compounded with lactic acid bacteria as a starter to ferment milk to prepare cheese, thereby obtaining mozzarella cheese.

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