Lactobacillus rhamnosus jylr-219 for preventing osteoporosis, bacterial agent and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a *Lactobacillus rhamnosus* JYLR-219 strain for preventing osteoporosis, its inoculum, and its application. Lactobacillus rhamnosus Lactobacillus rhamnosus JYLR-219 was deposited on July 8, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.18097. This Lactobacillus rhamnosus JYLR-219 can be used to prepare products for the prevention of osteoporosis. Lactobacillus rhamnosus JYLR-219 has a strong gastrointestinal survival rate and intestinal colonization ability. It can increase bone density by increasing osteoblast density and decreasing osteoclast density, thereby preventing osteoporosis. This invention has the advantages of being safe, effective, and without side effects.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a Lactobacillus rhamnosus JYLR-219 strain for preventing osteoporosis, its bacterial agents, and its applications. Background Technology

[0002] Osteoporosis is a systemic bone disease characterized by low bone mass, damage to bone microstructure, increased bone fragility, and susceptibility to fractures. Primary osteoporosis is most common in postmenopausal women and the elderly. Its pathogenesis is mainly due to osteoclast-dominated bone resorption exceeding osteoblast-dominated bone formation, resulting in decreased bone density and increased susceptibility to fractures and disability.

[0003] Currently, common treatments for osteoporosis include dietary supplements, health supplements, and medication. First, dietary supplementation, such as high intake of meat and eggs, increases calcium intake to prevent osteoporosis. However, with the prevalence of "diseases of affluence" in modern society, high intake of meat and eggs often leads to high blood pressure, high cholesterol, and high blood sugar. Second, supplementation can be achieved through large doses of health supplements, such as calcium tablets, glucosamine, and chondroitin. However, the quality of health supplements on the market varies greatly, often with exaggerated claims. Furthermore, the digestive and absorption systems of older adults are significantly weakened, making absorption difficult and potentially causing problems like constipation and other digestive issues. Moreover, these supplements are expensive, making long-term use unaffordable for the average consumer. Third, medication can be used, such as vitamin D, calcium supplements, and anti-resorption drugs. However, most osteoporosis medications have side effects, potentially damaging liver and kidney function, digestive function, or affecting endocrine metabolism, making long-term use unsuitable. Summary of the Invention

[0004] To address the shortcomings of the aforementioned osteoporosis treatments, this invention provides a novel approach to prevent osteoporosis using Lactobacillus rhamnosus JYLR-219, its bacterial agent, and its application. This approach offers advantages such as no side effects and safety and effectiveness.

[0005] In a first aspect, the present invention provides a *Lactobacillus rhamnosus* JYLR-219 for preventing osteoporosis, wherein *Lactobacillus rhamnosus* ( Lactobacillus rhamnosus JYLR-219 was deposited on July 8, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.18097.

[0006] In a second aspect, the present invention provides a bacterial agent comprising Lactobacillus rhamnosus JYLR-219 as described in claim 1.

[0007] Furthermore, the bacterial agent includes freeze-dried bacterial powder of Lactobacillus rhamnosus JYLR-219.

[0008] Furthermore, the preparation method of the freeze-dried bacterial powder includes the following steps:

[0009] (1) Take the preserved Lactobacillus rhamnosus JYLR-219 and activate it on MRS plate medium. Inoculate the activated Lactobacillus rhamnosus into MRS liquid medium at an inoculation rate of 1% and culture for 24 hours to obtain bacterial solution.

[0010] (2) After centrifuging the bacterial culture, collect the bacterial cells, wash them with sterile physiological saline, and resuspend them in 15% reconstituted skim milk to obtain a suspension; adjust the concentration of Lactobacillus rhamnosus JYLR-219 in the suspension to 1.0~2.0×10⁻⁶. 10 The bacterial suspension was obtained by lyophilizing the bacterial suspension to obtain lyophilized bacterial powder.

[0011] Furthermore, the method for preparing the MRS plate culture medium in step (1) is as follows: 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of Tween-80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar, and 1000mL of distilled water are mixed, the pH is set to natural, the mixture is stirred, and sterilized at 121℃ and 0.1MPa for 20min. The sterilized mixture is then poured into a petri dish, cooled, and set aside for use.

[0012] Thirdly, the present invention provides the application of Lactobacillus rhamnosus JYLR-219 as described in claim 1 in the preparation of products that enhance bone density and prevent osteoporosis.

[0013] Fourthly, the present invention provides the application of the bacterial agent as described in claim 2 in the preparation of products that enhance bone density and prevent osteoporosis.

[0014] The beneficial effects of this invention are as follows:

[0015] 1. The present invention provides a *Lactobacillus rhamnosus* JYLR-219 strain for preventing osteoporosis, primarily for use in individuals with osteoporosis to address low bone density. This strain exhibits strong gastrointestinal survival rate and intestinal colonization ability, significantly increasing osteoblast density and markedly reducing osteoclast density, thereby enhancing bone density and preventing osteoporosis.

[0016] 2. This invention utilizes the preserved strain Lactobacillus rhamnosus JYLR-219 to produce an oral product, which is convenient for clinical treatment of osteoporosis, safe and effective, and has no side effects. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 These are the microscopic observation results of osteoblasts in the control group and experimental group mice in Example 5 of this invention.

[0019] Figure 2 These are the microscopic observation results of osteoclasts in the control group and experimental group mice in Example 6 of this invention. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0021] Example 1: Screening, purification and identification of Lactobacillus rhamnosus JYLR-219

[0022] 1. Source of bacteria

[0023] The sample was collected on April 23, 2018, from homemade fermented yogurt made by herdsmen in Ili Prefecture, Xinjiang.

[0024] 2. Screening of strains

[0025] (1) Take samples of yogurt and dilute them in a 1000-fold gradient. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 The numbers are 1#, 2#, 3#, 4#, 5#, 6#, and 7#, respectively, and are ready for use;

[0026] (2) Preparation of MRS plate culture medium: Mix 10g peptone, 5g beef powder, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween-80, 0.05g manganese sulfate tetrahydrate, 2g triammonium citrate, 20g glucose, 0.2g magnesium sulfate heptahydrate, 15g agar, and 1000mL distilled water. After stirring the bacterial solution at natural pH, sterilize at 121℃ and 0.1MPa for 20min. Pour the sterilized culture medium into petri dishes and let it cool before use.

[0027] (3) Culture: Take 0.1 mL of solutions 1#, 2#, 3#, 4#, 5#, 6# and 7# respectively and spread them on MRS plate culture medium using a spreader. Culture at 37℃ under anaerobic conditions for 48 h.

[0028] (4) Selecting colonies: Select colonies with a diameter of 1-2 mm, round shape, neat edges, slightly white color and raised center.

[0029] (5) Isolation and purification: Based on the colony characteristics in step (4), five single colonies were picked and inoculated onto MRS agar plates using the streak method. The plates were cultured at 37°C under anaerobic conditions for 48 hours. Single colonies were then picked and stored in glycerol tubes at -70°C.

[0030] 2. Identification

[0031] The isolated and purified single colonies were sent for identification by Sangon Biotech (Shanghai) Co., Ltd.

[0032] (1) The primers used in the identification process are as follows:

[0033] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0034] 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0035] (2) The gene sequences obtained are as follows:

[0036]

[0037] (3) Upon identification, the bacterium was found to be Lactobacillus rhamnosus (Lactobacillus rhamnosus). Lactobacillus rhamnosus The strain was named *Lactobacillus rhamnosus* JYLR-219 and deposited with the China General Microbiological Culture Collection Center. The deposit information is as follows:

[0038] Lactobacillus rhamnosus ( Lactobacillus rhamnosus JYLR-219 was deposited on July 8, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC NO.18097.

[0039] Example 2 Preparation of Lactobacillus rhamnosus JYLR-219 product

[0040] (1) Preparation of MRS liquid culture medium: Mix 10g peptone, 5g beef powder, 5g sodium acetate trihydrate, 2g dipotassium hydrogen phosphate heptahydrate, 1mL Tween 80, 0.05g manganese sulfate tetrahydrate, 2g triammonium citrate, 20g glucose, 0.2g magnesium sulfate heptahydrate, and 1000mL distilled water, adjust the pH to 6.8, stir the bacterial solution, and sterilize at 121℃ and 0.1MPa for 20min.

[0041] (2) Take the preserved Lactobacillus rhamnosus JYLR-219 and activate it on the MRS plate medium prepared in Example 1. Inoculate the activated Lactobacillus rhamnosus into the MRS liquid medium at an inoculation rate of 1% and then culture it at 37°C for 24 hours to obtain the bacterial solution.

[0042] (3) After centrifuging the bacterial culture, collect the bacterial cells, wash them with sterile physiological saline, and resuspend them in 15% reconstituted skim milk to obtain a suspension; adjust the concentration of the suspension to 1.0~2.0×10⁻⁶. 10 The bacterial suspension was obtained by measuring cfu / mL, and then freeze-drying the bacterial suspension to obtain freeze-dried bacterial powder.

[0043] (4) The freeze-dried bacterial powder was mixed with isomaltooligosaccharide to prepare Lactobacillus rhamnosus JYLR-219 bacterial agent, wherein the isomaltooligosaccharide was purchased from Baolingbao Biotechnology Co., Ltd.

[0044] In this embodiment, the number of bacterial cells in the prepared bacterial agent is 1×10⁻⁶. 10 cfu / g, 1.5×10 10 cfu / g.

[0045] Example 3: Resistance of Lactobacillus rhamnosus JYLR-219 to gastrointestinal digestive fluids

[0046] 1. Preparation and handling of gastric juice

[0047] (1) Prepare artificial gastric juice: Take 16.4 mL of 9.5% hydrochloric acid solution, dilute it with distilled water to pH 1.5, add 1.0 g of pepsin per 100 mL, mix well and filter with a 0.22 μm sterile filter membrane. Prepare and use immediately.

[0048] (2) Weigh out 12 portions of Lactobacillus rhamnosus JYLR-219 cells prepared in Example 2, the number of which is 1.5 × 10⁻⁶. 10 The product with cfu / g was used as a sample, with each sample weighing 1g. The sample was transferred to a preheated test tube containing 9mL of simulated gastric fluid and treated at 37℃ and 80r / min for 1h, 2h, 3h and 4h, respectively, with 3 replicates for each treatment.

[0049] 2. Preparation and handling of intestinal fluid

[0050] (1) Preparation of artificial intestinal fluid: Dissolve 6.8g of KH2PO4 in 500mL of distilled water, add 3g of bile salt and 10g of trypsin, adjust the pH of the solution to 6.8 with NaOH solution of 4g / L, make up to 1L with distilled water, mix well and filter with 0.22μm sterile filter membrane, and use immediately after preparation.

[0051] (2) Weigh out 12 portions of Lactobacillus rhamnosus JYLR-219 cells prepared in Example 2, with a cell count of 1×10⁻⁶. 10 The product with cfu / g was used as a sample. Each sample weighed 1g and was transferred to a test tube containing 9mL of simulated intestinal fluid. The samples were treated at 37℃ and 80r / min for 1h, 2h, 3h and 4h, respectively, with 3 replicates for each treatment.

[0052] 3. Statistics and Analysis

[0053] The treated sample solutions were serially diluted, viable bacteria were counted using the pour method, and the survival rate was calculated.

[0054] Survival rate = (Number of bacteria after treatment / Original number of bacteria) × 100%

[0055] Table 1. Survival rate of JYLR-219 under simulated gastrointestinal environment in vitro

[0056]

[0057] The survival rate data of strain JYLR-219 in the in vitro simulated gastrointestinal environment are shown in Table 1. It can be seen that Lactobacillus rhamnosus JYLR-219 has a very high survival rate in the in vitro simulated gastrointestinal environment. After 4 hours in gastric and intestinal fluids, the survival rate is still above 90%, which lays the foundation for subsequent colonization of the intestine and the exertion of its functional role.

[0058] Example 4: Adhesion rate control experiment of Lactobacillus rhamnosus JYLR-219 to Caco-2

[0059] (1) Test reagents:

[0060] DMEM culture medium, purchased from Gibco;

[0061] MRS plate culture medium was prepared using the same method as in Example 1;

[0062] MRS liquid culture medium was prepared using the same method as in Example 2.

[0063] Weigh 0.27g KH2PO4, 1.42g Na2HPO4, 8g NaCl, and 0.2g KCl, add about 800mL of deionized water and stir thoroughly to dissolve. Adjust the pH to 7.4 with concentrated hydrochloric acid, add deionized water to bring the volume to 1L, and sterilize at 121℃ for 20min to obtain sterile PBS buffer with pH=7.4.

[0064] (2) Experimental preparation:

[0065] Caco-2 cells were placed in DMEM medium containing 10% heat-inactivated newborn calf serum and penicillin (100 U / mL) and streptomycin (1.0 μg / mL) and incubated at 37°C, 5% CO2, and 95% relative humidity in a carbon dioxide incubator. The medium was changed daily, and the cells were passaged every 3 days. After 18 days, the cells were seeded into 24-well plates at a density of approximately 5 × 10⁶ cells / well. 5 cfu / mL, and after the cells grow into a monolayer, an adhesion assay is performed;

[0066] The preserved Lactobacillus rhamnosus JYLR-219 was activated on MRS plate medium. The activated Lactobacillus rhamnosus JYLR-219 was inoculated into MRS liquid medium at a volume fraction of 1% and cultured at 37℃ for 12h to obtain the fermentation broth of the strain.

[0067] The bacterial cells were collected by centrifugation of the fermentation broth (4000 r / min, 4℃, 10 min), and washed three times with sterile PBS buffer. The bacterial cells were then resuspended in sterile PBS buffer to obtain a concentration of 2 × 10⁻⁶. 8 JYLR-219 bacterial suspension at cfu / mL.

[0068] (3) Experimental procedures:

[0069] The Caco-2 cells that had grown into a monolayer were washed twice with sterile PBS buffer. 0.5 mL of JYLR-219 bacterial suspension and 0.5 mL of fresh DMEM culture medium were added to each well. The cells were incubated for 1 h in a carbon dioxide incubator at 37°C, 5% CO2 and 95% relative humidity. The cells were then washed five times with sterile PBS buffer to remove any unattached bacteria.

[0070] The rinsing solution was serially diluted, and viable bacteria were counted using the pouring method. The adhesion rate was calculated as follows: Adhesion rate = (initial bacterial count - bacterial count after rinsing) / initial bacterial count × 100%.

[0071] After rinsing, the cells were fixed with formaldehyde, Gram-stained, and examined under a microscope. The number of bacteria adhering to 100 cells in 20 random fields of view was counted under an inverted microscope. Using cell culture without bacterial suspension as a control, the average number of bacteria adhering to each cell was calculated. Each treatment was performed in triplicate.

[0072] Table 2. Adhesion rate and number of JYLR-219 cells on Caco-2 cells

[0073] project Adhesion rate Number of adhesions result 85% 692

[0074] Table 2 shows the adhesion rate and number of cells adhered to Caco-2 cells by strain JYLR-219, indicating that Lactobacillus rhamnosus JYLR-219 has a strong ability to colonize cells.

[0075] Example 5: Effects of Lactobacillus rhamnosus JYLR-219 on mouse osteoblasts

[0076] Osteoblasts are the main functional cells in bone formation, responsible for the synthesis, secretion, and mineralization of the bone matrix. Bone is constantly being rebuilt, and osteoblasts are key to maintaining normal bone mass.

[0077] Forty inbred male mice were randomly divided into two groups (control group and experimental group, 20 mice each) after a 5-day acclimatization period. The animals were housed at an ambient temperature of 21±2 ℃ and humidity of 30%–70%, with 12-hour light alternation, and free access to water and feed. Bedding was changed every three days, and feed and water were replenished as needed.

[0078] The number of Lactobacillus rhamnosus JYLR-219 cells obtained in Example 2 was 1×10⁻⁶. 10 For products containing CFU / g, dilute with water to make 1×10. 9 Probiotic liquid with cfu / mL.

[0079] The experimental group was administered 2 mL of probiotic solution by gavage daily, while the control group received an equal volume of sterile saline. This gavage treatment continued for 7 days. Then, one mouse from each group was randomly selected, sacrificed, and the femur bone of the right hind leg was examined for cellular status. The experiment continued for another 7 days. After 7 days, one mouse from each group was again randomly selected, sacrificed, and the femur bone of the right hind leg was examined for cellular status.

[0080] Table 3. MTT assay for osteoblast proliferation (OD 490nm)

[0081] Day 7 Day 14 control group 0.4637±0.0159 0.5010±0.0218 experimental group 0.6029±0.0238** 0.7462±0.0199**

[0082] Note: * indicates a significant difference (p<0.05), ** indicates an extremely significant difference (p<0.01).

[0083] In this study, the experimental group was Lactobacillus rhamnosus JYLR-219. As shown in Table 3, the experimental group significantly promoted osteoblast proliferation (p<0.01), while the control group showed no difference. This indicates that the gavage administration of Lactobacillus rhamnosus JYLR-219 to the experimental group mice did indeed enhance osteoblast proliferation.

[0084] Depend on Figure 1 The proliferation of osteoblasts in mice after administration of *Lactobacillus rhamnosus* JYLR-219 can be observed. In the figure, a represents osteoblasts from the control group after 7 days, b represents osteoblasts from the experimental group after 7 days, c represents osteoblasts from the control group after 14 days, and d represents osteoblasts from the experimental group after 14 days. Figure 1 It can be seen that after 7 days, the osteoblast density in the control group mice was lower than that in the experimental group mice, indicating that administration of Lactobacillus rhamnosus JYLR-219 increased the osteoblast density in mice. Comparing the control group mice after 7 days and after 14 days, the osteoblast density remained essentially unchanged. However, comparing the control group mice after 14 days and the experimental group mice, it is clear that the osteoblast density in the experimental group mice was significantly higher than that in the control group mice after 14 days, indicating that administration of Lactobacillus rhamnosus JYLR-219 increased the osteoblast density in mice. Comparing the experimental group mice after 7 days and after 14 days, with continued administration of Lactobacillus rhamnosus JYLR-219, the osteoblast density in mice further increased, demonstrating that administration of strain JYLR-219 can enhance bone density and prevent osteoporosis.

[0085] Example 6: Effects of Lactobacillus rhamnosus JYLR-219 on mouse osteoclasts

[0086] Osteoclasts are composed of multinucleated giant cells, 100 μm in diameter, containing 2–50 tightly packed nuclei. They are mainly distributed on the bone surface and around intraosseous vascular channels. They are formed by the fusion of multiple mononuclear cells, with basophilic cytoplasm that gradually becomes eosinophilic as the cells age. Osteoclasts release lactic acid and citric acid locally. Under acidic conditions, inorganic minerals within the bone are pinocytotically, forming pinocytic or phagocytic vesicles within the fold matrix. Inside the osteoclast, inorganic matter is degraded and released into the bloodstream as calcium ions. Therefore, the osteoclast content significantly affects bone calcium content and plays a crucial role in bone density and osteoporosis formation.

[0087] In Example 5 above, osteoclasts were also subjected to MTT assay, and the results are shown in Table 4 below.

[0088] Table 4. MTT assay for osteoclast proliferation (OD 490nm)

[0089] Day 7 Day 14 control group 1.2396±0.0360 1.7869±0.0181 experimental group 1.2166±0.0047* 1.2524±0.0306**

[0090] Note: * indicates a significant difference (p<0.05), ** indicates an extremely significant difference (p<0.01).

[0091] Table 4 clearly shows that the control group had faster osteoclast proliferation on days 7 and 14 without significant inhibition. However, the mice treated with Lactobacillus rhamnosus JYLR-219 showed a certain difference on day 7. After 14 days of continuous treatment, the osteoclast proliferation in the experimental group mice was as low as 1.2524±0.0306, showing a very significant difference. Therefore, it can be concluded that the number of osteoclasts in the experimental group mice was greatly reduced after taking Lactobacillus rhamnosus JYLR-219, which helps maintain bone density and plays an important role in preventing osteoporosis.

[0092] Depend on Figure 2 This shows the changes in osteoclasts in mice after administration of *Lactobacillus rhamnosus* JYLR-219. In the figure, e represents osteoclasts from the control group after 7 days, f represents osteoclasts from the experimental group after 7 days, g represents osteoclasts from the control group after 14 days, and h represents osteoclasts from the experimental group after 14 days. Figure 2 It can be seen that, compared with the control group mice after 7 days, the number of osteoclasts in the control group mice increased; while compared with the experimental group mice after 7 days, the number of osteoclasts in the experimental group mice decreased; comparing the osteoclast status of the control group mice and the experimental group mice after 14 days, it can be clearly seen that the osteoclast content of mice treated with Lactobacillus rhamnosus JYLR-219 for 14 days was much lower than that of the control group mice.

[0093] Although the invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this invention without departing from the spirit and essence of the invention should be included within the protection scope of this invention.

Claims

1. The application of Lactobacillus rhamnosus JYLR-219 in the preparation of products that enhance bone density and prevent osteoporosis, characterized in that, The Lactobacillus rhamnosus ( Lactobacillus rhamnosus JYLR-219 was deposited on July 8, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.18097. Lactobacillus rhamnosus JYLR-219 can significantly increase osteoblast density and significantly decrease osteoclast density.

2. The application of a bacterial agent containing *Lactobacillus rhamnosus* JYLR-219 in the preparation of products for enhancing bone density and preventing osteoporosis, characterized in that... The Lactobacillus rhamnosus ( Lactobacillus rhamnosus JYLR-219 was deposited on July 8, 2019, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.18097. Lactobacillus rhamnosus JYLR-219 can significantly increase osteoblast density and significantly decrease osteoclast density.

3. The application as described in claim 2, characterized in that, The bacterial agent includes freeze-dried Lactobacillus rhamnosus JYLR-219 bacterial powder.

4. The application as described in claim 3, characterized in that, The method for preparing the freeze-dried bacterial powder includes the following steps: (1) Take the preserved Lactobacillus rhamnosus JYLR-219 and activate it on MRS plate medium. Inoculate the activated Lactobacillus rhamnosus into MRS liquid medium at an inoculation rate of 1% and culture for 24 hours to obtain bacterial solution. (2) After centrifuging the bacterial culture, collect the bacterial cells, wash them with sterile physiological saline, and resuspend them in 15% reconstituted skim milk to obtain a suspension; adjust the concentration of Lactobacillus rhamnosus JYLR-219 in the suspension to 1.0~2.0×10⁻⁶. 10 The bacterial suspension was obtained by lyophilizing the bacterial suspension to obtain lyophilized bacterial powder.

5. The application as described in claim 4, characterized in that, The method for preparing the MRS plate culture medium in step (1) is as follows: Mix 10g of peptone, 5g of beef powder, 5g of sodium acetate trihydrate, 2g of dipotassium hydrogen phosphate heptahydrate, 1mL of Tween-80, 0.05g of manganese sulfate tetrahydrate, 2g of triammonium citrate, 20g of glucose, 0.2g of magnesium sulfate heptahydrate, 15g of agar, and 1000mL of distilled water. Set the pH to natural, stir the mixture, and sterilize it at 121℃ and 0.1MPa for 20min. Pour the sterilized mixture into a petri dish, let it cool, and set aside for use.

6. The application as described in claim 3, characterized in that, The microbial agent also includes isomaltooligosaccharide.

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