A strain of Lactobacillus plantarum for promoting bone development during growth period and its application and products

Abstract

The present invention provides a plant lactobacillus that promotes bone development during growth period and its application and products, belonging to the field of microbial technology. Lactiplantibacillus plantarum BL25 and its probiotic preparation. The Lactobacillus plantarum BL25, deposited under CGMCC No. 33117, uses stachyose as a prebiotic, which significantly boosts the activity of Lactobacillus plantarum BL25 compared to other prebiotics. These Lactobacillus plantarum BL25 and its probiotic preparation are effective in resisting the harsh gastrointestinal environment, increasing femoral length and bone density, promoting the development of bone microstructure, improving bone metabolism, increasing osteoblastic markers, and reducing osteoclast markers, thereby promoting bone development during the growth period.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Lactobacillus plantarum strain for promoting bone development during growth period, and applications and products thereof. Background Art

[0002] Bones are a vital structural component of the human body, carrying out multiple functions, including supporting the body, protecting organs, coordinating movement, and storing minerals. The period from infancy to childhood is a critical period for bone development, and bone health during this period has a decisive impact on a person's long-term health. First, bone density rapidly increases, and bones continue to grow in length and strength. Bone formation and mineral deposition primarily occur during this process. Adequate nutrition and moderate exercise during this period are crucial factors in promoting healthy bone development. Second, strong bone health during childhood can prevent bone diseases such as osteoporosis in adulthood. Numerous studies have shown that peak bone mass (PBM), achieved around puberty, is a key predictor of osteoporosis risk. PBM formation is primarily concentrated during childhood and adolescence. Therefore, optimizing bone development during this period can help increase peak bone mass, thereby reducing the risk of bone diseases in adulthood.

[0003] Research has found that environmental factors, such as the gut microbiome, also play a key role in bone formation and development. The gut microbiome, or the bacterial community in the intestines, plays a crucial role in human health. They not only participate in food digestion and energy acquisition but also closely interact with the host's immune and endocrine systems. Furthermore, changes in gut microbes during this period can have profound effects on an individual's subsequent bone health. However, the gut microbiome includes thousands of microbial species, each of which may have unique interactions with other microbes and the host system.

[0004] Lactobacillus plantarum is an important probiotic. First, it can optimize the intestinal microbial environment and help maintain a balanced intestinal flora. Second, Lactobacillus plantarum can enhance the human immune system, such as by improving the phagocytic capacity of white blood cells and stimulating the immune response, playing a positive auxiliary role in resisting disease. Furthermore, Lactobacillus plantarum improves digestion, helping to break down proteins and complex carbohydrates, increasing their utilization and allowing the body to obtain more nutrients from the diet. Furthermore, Lactobacillus plantarum can resist pathogens and strengthen the body's protective mechanisms by producing antimicrobial substances such as lactic acid and hydrogen peroxide. However, there are currently no reports of Lactobacillus plantarum promoting bone development. Therefore, it is necessary to identify a strain of Lactobacillus plantarum that can promote bone development during the growth period. Summary of the Invention

[0005] In view of the above shortcomings, the present invention provides a plant lactobacillus that promotes bone development during growth period and its application and products. The present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum BL25 and its probiotic preparation. The Lactobacillus plantarum BL25, deposited under CGMCC No. 33117, uses stachyose as a prebiotic, which significantly boosts the activity of Lactobacillus plantarum BL25 compared to other prebiotics. These Lactobacillus plantarum BL25 and its probiotic preparation are effective in resisting the harsh gastrointestinal environment, increasing femoral length and bone density, promoting the development of bone microstructure, improving bone metabolism, increasing osteoblastic markers, and reducing osteoclast markers, thereby promoting bone development during the growth period.

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

[0007] In a first aspect, the present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum ), the plant lactobacillus is plant lactobacillus BL25, and its deposit number is CGMCC No.33117.

[0008] In a second aspect, the present invention provides a probiotic preparation comprising one or more of the above-mentioned Lactobacillus plantarum bacteria, fermentation broth, fermentation broth precipitate, and freeze-dried powder.

[0009] Specifically, the probiotic preparation further includes nutritional additives that are nutritionally acceptable.

[0010] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0011] More preferably, the nutritional additive is a prebiotic.

[0012] Still more preferably, the prebiotic is stachyose.

[0013] The preparation method of the probiotic preparation comprises the following steps: inoculating Lactobacillus plantarum BL25 into a culture medium containing nutrient additives, culturing the culture medium, and then freeze-drying the culture medium.

[0014] Specifically, the culture medium containing nutrient additives includes: MRS culture medium, TPY culture medium, MC culture medium or BCP culture medium.

[0015] Preferably, the culture medium containing nutrient additives comprises MRS culture medium.

[0016] Specifically, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0017] Preferably, the nutritional additive is a prebiotic.

[0018] More preferably, the prebiotic is stachyose.

[0019] Specifically, the content of the nutrient additive in the culture medium is 0.5%-2% w / v.

[0020] Preferably, the content of the nutrient additive in the culture medium is 1% w / v.

[0021] Specifically, the culture medium containing nutrient additives is sterilized by filtering membrane before use.

[0022] Preferably, the filter membrane is a 0.2 μm filter membrane.

[0023] Specifically, the Lactobacillus plantarum BL25 is a suspension of Lactobacillus plantarum BL25.

[0024] More specifically, the method for preparing the suspension of Lactobacillus plantarum BL25 comprises: inoculating Lactobacillus plantarum BL25 into a culture medium for incubation, centrifuging after the incubation is complete, collecting the precipitate, and resuspending to obtain a suspension.

[0025] Preferably, the culture medium includes but is not limited to: MRS medium, TPY medium, MC medium or BCP medium.

[0026] More preferably, the culture medium is MRS culture medium.

[0027] Preferably, the incubation condition is 35-39° C. for 12-48 hours.

[0028] More preferably, the incubation condition is 37° C. for 24 hours.

[0029] Preferably, the centrifugation is performed at 1000-3000 rpm for 5-15 min.

[0030] More preferably, the centrifugation is performed at 2000 rpm for 10 min.

[0031] Preferably, the suspension contains at least 10 7 CFU / mL of Lactobacillus plantarum BL25.

[0032] More preferably, the suspension contains 10 7 -10 12 CFU / mL of Lactobacillus plantarum BL25.

[0033] In a third aspect, the present invention provides the use of the above-mentioned Lactobacillus plantarum or probiotic preparation in any of the following aspects: preparing a health product for improving bone density; or preparing a drug for promoting bone development during growth and alleviating osteoporosis; or preparing a probiotic food, health product or drug that can colonize the intestine.

[0034] In a fourth aspect, the present invention provides a product comprising the aforementioned Lactobacillus plantarum or probiotic preparation, wherein the product includes food, health care product or medicine.

[0035] Specifically, the product contains at least 1×10 7 CFU / mL of Lactobacillus plantarum BL25.

[0036] Preferably, the product contains 1×10 7 -1×10 12 CFU / mL of Lactobacillus plantarum BL25.

[0037] Specifically, the food also includes nutritional additives that are nutritionally acceptable.

[0038] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0039] Preferably, the food includes candy flakes, soy milk, yogurt, canned food, biscuits, chocolate, cakes, cream, cheese, milk powder, formula milk powder, ice cream, jam, puree, candied fruit, preserved fruit, bread, egg rolls, protein drinks, solid drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee or puffed food.

[0040] Preferably, the food comprises human food or animal food.

[0041] Specifically, the health care product also includes nutritional additives that are nutritionally acceptable.

[0042] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0043] Preferably, the dosage form of the health care product includes tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

[0044] Specifically, the drug further includes one or more physiologically acceptable adjuvants or pharmaceutically acceptable excipients.

[0045] Preferably, the physiologically acceptable excipients include but are not limited to: erythritol, D-mannitol, fumaric acid, glycerin, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate or dextrin.

[0046] Preferably, the pharmaceutically acceptable excipients include, but are not limited to, solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0047] The present invention has the following beneficial effects:

[0048] (1) The present invention provides a Lactobacillus plantarum BL25, with a deposit number of CGMCC No. 33117. The mixed prebiotic preparation of Lactobacillus plantarum BL25 and stachyose can help BL25 resist the harsh environment of the gastrointestinal tract and promote colonization.

[0049] (2) The Lactobacillus plantarum BL25 and its prebiotic preparation of the present invention have the effect of promoting bone development, increasing femoral length, and increasing bone density by increasing BV / TV, Tb.N, and reducing Tb.Sp.

[0050] (3) The Lactobacillus plantarum BL25 and its prebiotic preparation of the present invention have the effect of increasing bone formation markers and reducing bone resorption markers in mice.

[0051] Preservation Instructions

[0052] Preserved strain: Lactobacillus plantarum BL25

[0053] Classification name: Lactobacillus plantarum Lactiplantibacillus plantarum ;

[0054] Deposit number: CGMCC No.33117;

[0055] Deposit date: December 18, 2024;

[0056] Depository: General Microbiology Center, China Culture Collection Administration;

[0057] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The promoting effects of different prebiotics on the activity of Lactobacillus plantarum BL25; ** indicates P < 0.01 compared with the model group.

[0059] Figure 2 A comparison of the protein content of the model diet and the standard diet.

[0060] Figure 3 The effect of each treatment group on femur length; * indicates P < 0.05 compared with the model group; ** indicates P < 0.01 compared with the model group; *** indicates P < 0.001 compared with the model group.

[0061] Figure 4 Representative Micro CT images of each treatment group.

[0062] Figure 5 Figure 2 is an analysis of bone density and bone microstructure in rats. A in the figure is a quantitative analysis of BMD; B is the number of trabeculae Tb.N; C is the thickness of trabeculae Tb.Th; D is the separation of trabeculae Tb.Sp; * indicates P < 0.05 compared with the model group; ** indicates P < 0.01 compared with the model group; *** indicates P < 0.001 compared with the model group.

[0063] Figure 6 Representative images of Gloder staining and TRAP staining in each treatment group.

[0064] Figure 7 Figure 3 shows the effects of each treatment group on the levels of bone formation markers; A is the level of type I procollagen N-terminal propeptide (PINP) in serum; B is the level of alkaline phosphatase (ALP); * indicates P < 0.05 compared with the model group; ** indicates P < 0.01 compared with the model group; *** indicates P < 0.001 compared with the model group.

[0065] Figure 8 Figure 3 shows the effects of each treatment group on the levels of bone resorption markers; A is the level of type I collagen cross-linked C-terminal peptide (CTX-1) in serum; B is the level of acid phosphatase (TRACP); ** indicates P < 0.01 compared with the model group; *** indicates P < 0.001 compared with the model group. DETAILED DESCRIPTION

[0066] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, the methods are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are conventional products that can be purchased commercially. The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0067] Example 1 Materials and Methods

[0068] 1. Experimental strains

[0069] The plant lactobacillus BL25 of the present invention is isolated from traditional yogurt in Xiahe County, Gannan Tibetan Autonomous Prefecture, and was deposited in the General Microbiology Center of the China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No. 33117.

[0070] 2. Experimental strain culture medium

[0071] MRS medium: 12 g peptone, 6 g yeast extract, 6 g beef extract, 18 g glucose, 5 g sodium acetate, 2.15 g ammonium citrate, 1 g Tween, 0.58 g magnesium sulfate, 0.05 g manganese sulfate, 2 g dipotassium hydrogen phosphate. Use pure water to make up to 1 L, adjust the pH to 6.5, and sterilize in a high-pressure steam autoclave at 121°C for 15 min.

[0072] 3. Data processing

[0073] The experimental data were expressed as mean ± standard deviation and repeated at least 3 times. SPSS18.0 software was used to perform one-way ANOVA on the data between different groups. The Spearman correlation coefficient was used to evaluate the correlation between two statistical variables, and the graphs were drawn using GraphPad Prism 5.0 and Origin 9.0 software. P < 0.05 was considered statistically significant.

[0074] Example 2 Microbial Activation and Preparation of Bacterial Suspension

[0075] Lactobacillus plantarum BL25 was stored at -80°C in 20% v / v glycerol. Before the experiment, Lactobacillus plantarum BL25 was inoculated into sterile MRS medium at 18°C and incubated in a constant temperature incubator at 37°C for 24 h. After incubation, Lactobacillus plantarum BL25 was centrifuged at 2000 rpm for 10 min, the medium was removed, and the pellet was harvested and washed twice with sterile phosphate buffered saline (PBS). The washed pellet was diluted with PBS to a cell concentration of 10 7 CFU / mL of Lactobacillus plantarum BL25 suspension.

[0076] Example 3 Effect of prebiotics on bacterial activity

[0077] The Lactobacillus plantarum BL25 suspension was aseptically inoculated into MRS liquid culture medium, MRS culture medium containing 1% w / v stachyose, and MRS culture medium containing 1% w / v inulin at a 5% inoculum size and cultured at 37°C for 24 h. The initial pH of the culture medium was adjusted to 5.9, and the viable bacteria were counted to determine the activity in log CFU / mL.

[0078] In order to verify the promoting effect of prebiotic stachyose on the activity of Lactobacillus plantarum BL25, 1% stachyose was added to MRS medium for cultivation. The results are shown in the figure. Figure 2 As shown in the results, the number of viable bacteria in the MRS culture medium containing stachyose reached 10.03±0.15 Log of CFU / mL, which was significantly higher than 8.08±0.21 Log of CFU / mL in the ordinary MRS culture medium (p<0.05) and 8.79±0.13 Log of CFU / mL in the prebiotic inulin MRS culture medium (p<0.05), indicating that the prebiotic ingredient stachyose of the present invention has a significant bacteriostatic activity effect and is better than the bacteriostatic activity effect of inulin.

[0079] Example 4 Simulation of gastrointestinal digestion of prebiotic preparations

[0080] A suspension of Lactobacillus plantarum BL25 was aseptically inoculated at a 5% inoculum into MRS liquid medium, MRS medium containing 1% w / v stachyose, and MRS medium containing 1% w / v inulin, respectively. The cultures were incubated at 37°C for 24 hours, with the initial pH of the medium adjusted to 5.9. Freeze-dried bacterial powders were prepared by freeze-drying to produce freeze-dried Lactobacillus plantarum BL25 powder, a stachyose prebiotic formulation, and an inulin prebiotic formulation, respectively. A blank control group received freeze-dried Lactobacillus plantarum BL25 powder (BL25), while experimental groups received the stachyose prebiotic formulation (BL25+stachyose) and an inulin prebiotic formulation (BL25+inulin) as controls.

[0081] Artificial saliva (SSF) was prepared by dissolving 12.0 mg of NaCl, 15.0 mg of KCl, 100.0 mg of mucin, and 7.5 mg of α-amylase in 50.0 mL of distilled water.

[0082] Artificial gastric fluid (SGF) was prepared by mixing 12.0 mg of NaCl, 15.0 mg of KCl, and 100.0 mg of mucin with 7.5 mg of α-amylase from human saliva. The mixture was dissolved in 50.0 mL of distilled water, and 0.1 M HCl was added to adjust the pH to 3.0 to obtain artificial gastric fluid (SGF).

[0083] Artificial intestinal fluid (SIF) was prepared by dissolving 4.0 mg / mL bile salts, 2.0 mg / mL trypsin, and 1.0 mg / mL lipase in 8.0 M PBS adjusted to pH 1 containing 0.1 M HCl.

[0084] 2 g of each preparation was inoculated into a shake flask containing 20 mL of SSF and incubated in a 37°C incubator for 2 minutes. The saliva-digested preparation was then centrifuged (1000 rpm for 2 minutes) and added to a shake flask containing 20 mL of SGF, which was then incubated in a constant-temperature shaker at 37°C, 3 rpm, for 2 hours. The gastric juice-digested preparation was then centrifuged (1000 rpm for 2 minutes) and added to a shake flask containing 20 mL of SIF, which was then incubated in a constant-temperature shaker at 37°C, 2 rpm, for 3 hours. Each preparation was then harvested. At the end of each step (SIF, SGF, and SIF), viability was determined using a viability counter (log CFU / mL).

[0085] In order to understand the effect of Lactobacillus plantarum BL25 combined with prebiotics on resisting gastrointestinal digestion, in vitro gastrointestinal digestion simulation was performed. The results of each group are shown in Table 1.

[0086] Table 1 Effects of prebiotic preparations on simulated gastrointestinal digestion

[0087]

[0088] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate significant differences (P<0.05).

[0089] After simulated gastrointestinal digestion, BL25, BL25 + inulin, and BL25 + stachyose were all able to withstand the harsh gastrointestinal environment. Compared to the BL25 and BL25 + inulin groups, BL25 + stachyose significantly improved its colonization and significantly resisted the harsh gastrointestinal environment. This suggests that the combination of stachyose and Lactobacillus plantarum BL25 helps enhance resistance to gastrointestinal digestion. This may be because stachyose is relatively stable and not easily broken down by gastric acid and intestinal enzymes, thus providing a long-term energy source in the intestine. Lactobacillus plantarum BL25 is able to utilize stachyose, allowing it to survive and reproduce stably in the intestine.

[0090] Example 5 Animal Experiment

[0091] 1. Experimental methods

[0092] 1.1 Animal Experiment Design

[0093] Standard diets and model diets (low-fat, low-protein) were purchased or designed and produced by Beijing Keao Xieli Feed Co., Ltd. The fat and protein content of the model diet was designed based on the research results of Schwarzer et al. (Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice), with slight modifications. Figure 2 The results showed that the crude protein content of the standard diet was 18.63% and the fat content was 4.8%, while the crude protein content of the low-fat and low-protein diet was 5.2% and the fat content was 2.3%.

[0094] Healthy female Sprague-Dawley (SD) rats (60.0 ± 10.0 g, 3 weeks old, n = 42) were purchased from Harbin Medical University, China. Rats were housed in accordance with the guidelines for animal care and use, with three rats per cage and free access to food and water. The ambient temperature was maintained at 22 ± 2°C to 45 ± 5%, with a 12-h lights-on / dark cycle. This study was approved by the Animal Ethics Committee of Northeast Agricultural University, approval number NEUAEC20230424.

[0095] All rats were first fed a standard diet ad libitum for one week to allow for acclimation and observation. After the acclimation period, the rats were divided into three groups: a control group (C), a model group (M), a BL25 group (BL25), and a BL25 + Stachyose group (BL25 + Stachyose). Each group consisted of six rats. After the acclimation period, all rats except the control group were fed a model diet for four weeks. During the model diet period, the BL25 and BL25 + Stachyose groups were gavaged daily with 1 g of Lactobacillus plantarum BL25 freeze-dried powder or a Stachyose prebiotic preparation (dissolved in PBS). The blank combination model group was gavaged with the corresponding saline solution.

[0096] 1.2 Tissue Sample Collection

[0097] After the final oral gavage, the rats were fasted for 12 hours. The rats were then weighed and anesthetized for further manipulation. Blood samples were collected before humane euthanasia. Organs were isolated to calculate organ indices, and colonic contents were collected for genomic and untargeted metabolomics analyses. Left and right femurs were harvested and their lengths measured using a vernier caliper for microCT imaging and subsequent histomorphometric analysis.

[0098] 1.3 Micro-CT

[0099] The right femur of the rat was scanned using a microcomputed tomography system (SCANCO MEDICAL μCT-50, Switzerland). The region of interest (ROI) volume was selected, and computer software automatically reconstructed a three-dimensional image of the bone tissue. The main parameters were as follows: bone volume / tissue volume (BV / TV) (%), trabecular number (Tb.N) (mm-1), trabecular spacing (Tb.Sp) (mm), trabecular N (mm-1), trabecular spacing (Tb.Sp) (mm), and trabecular thickness (Tb.Th) (mm).

[0100] 1.4 Histopathological and bone morphometric analysis

[0101] After microCT imaging, femoral specimens were fixed with 4% paraformaldehyde, decalcified, trimmed, dehydrated, embedded, sectioned, stained (Goldner and Trap), and sealed according to the pathology laboratory's standard operating procedures. Specimens that passed microscopic examination were scanned using a PANNORAMIC (3DHISTECH, Hungary) panoramic slide scanner. SlideViewer 2.5 scanning software was used to select target areas of tissue for imaging at 400x magnification. The tissue was imaged to fill the entire field of view, ensuring consistent background illumination for each image. Three images were generated using the same method. Quantitative histomorphometric measurements were performed on the generated images using Image-Pro Plus 6.0 (MediaCybemetics, USA). Key parameters examined included Goldner staining for osteoclast number (N.Ob / BS), osteoclast surface area (Ob.S / BS), and osteoclast index (N.Ob / Ob.Pm). Trapping staining was used to analyze the number of osteoclasts (N.Oc / BS), osteoclast surface (Oc.S / BS), and osteoclast index (N.Oc / Oc.Pm).

[0102] 1.5 Analysis of osteoblastic markers

[0103] An appropriate amount of serum was weighed and the bone formation markers (PINP, ALP) in the rat serum samples were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0104] 1.6 Analysis of osteoclast markers

[0105] An appropriate amount of serum was weighed and the bone resorption markers (CTX-1, TRACP) in the rat serum samples were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0106] 2. Experimental results

[0107] 2.1 Femoral length

[0108] The femur length of each group Figure 3As shown, the femur length of the model group was significantly reduced, indicating that the model treatment can effectively simulate the phenomenon of impaired bone development. After intervention with Lactobacillus plantarum BL25, femur length increased significantly (p<0.05). Interestingly, the femur length increase was most significant in the BL25+stachyose group. This shows that BL25 has the effect of promoting bone development during growth.

[0109] 2.2 Bone density and bone microstructure

[0110] To evaluate the effects of Lactobacillus plantarum BL25 on bone development during growth, femurs were scanned with Micro-CT to analyze bone density and bone microstructure. Figure 4 As shown, μCT analysis revealed significant changes in bone structure in the M group fed the model diet compared to the control group. These changes included increased trabecular spacing, decreased trabecular number, thinning of cortical bone, an increase in the number of bone marrow cavities, and the presence of fractures and discontinuous reticular structures. These findings indicate that the model diet successfully mimics the phenotype of impaired skeletal development. Notably, BL25, and particularly BL25 + stachyose treatment, ameliorated these conditions and promoted skeletal development during growth.

[0111] In order to more accurately understand the bone development level of each group, trabecular structure and bone density were quantitatively analyzed. The quantitative analysis results are shown in Figure 5 Compared with the control group, the M group had significantly lower BV / TV, TB.N, Tb.Th, and BMD values, while significantly higher Tb.Sp (p<0.05). Lactobacillus plantarum BL25 effectively improved bone development, as evidenced by significant increases in BV / TV, Tb.N, and BMD, and a decrease in Tb.Sp. The bone-promoting effect was even more pronounced in the BL25+stachyose group, which also included stachyose (p<0.05).

[0112] 2.3 Bone histopathology

[0113] Bone metabolism is intricately regulated by the balance between osteoblasts, responsible for bone formation, and osteoclasts, responsible for bone resorption. Any alteration in the balance between these two processes can have a significant impact on bone metabolism. To evaluate the effects of BL25 on bone resorption and formation, we analyzed the right femur (Figure 2) after microCT examination using Goldner and Trap staining techniques. Figure 6), and osteoblast and osteoclast activity was assessed using bone histomorphometry. Goldner and Trap staining revealed that in the model group, trabeculae appeared sparse and irregularly arranged. Furthermore, the color and transparency of the bone matrix decreased compared to the control group, indicating skeletal hypoplasia. Notably, compared to the model group, the BL25 group and the BL25 + Stachyose group showed significant new bone tissue, appearing orange-red. The BL25 + Stachyose group showed the greatest effect in improving bone metabolism.

[0114] 2.4 Osteogenesis markers

[0115] Bone formation markers are biomarkers used to assess bone metabolic activity and reflect the extent of bone formation. Procollagen type I N-telopeptide (PINP) and alkaline phosphatase (ALP) are two commonly used bone formation markers. Procollagen type I N-telopeptide (PINP): PINP is a byproduct of collagen type I synthesis. Its concentration can reflect the rate of collagen synthesis and the extent of bone formation activity. PINP measurement is also less affected by the patient's diet and lifestyle and exhibits good stability. Therefore, PINP can serve as an important indicator for evaluating bone metabolic activity, particularly bone formation. Alkaline phosphatase (ALP): Alkaline phosphatase is an enzyme present in various cells, with particularly high levels in bone cells. ALP is involved in the process of bone mineralization, and changes in its concentration can reflect the extent of bone formation and mineralization. Therefore, ALP is often used as an indicator for evaluating bone metabolic activity, particularly bone formation.

[0116] The results of bone markers in each group are shown in Figure 7 Compared with the control group, the levels of PINP and ALP in the model group were significantly lower (p<0.05), indicating that bone formation in the model group was significantly lower than that in the control group. After BL25 intervention, the levels of PINP and ALP were significantly increased, indicating that Lactobacillus plantarum BL25 has the effect of enhancing bone formation. Interestingly, BL25+stachyose significantly increased the levels of PINP and ALP compared with the model group and BL25 (p<0.05). This may be because stachyose promotes the colonization of Lactobacillus plantarum BL25 in the intestine, better exerting its role in promoting bone formation.

[0117] 2.5 Osteoclast markers

[0118] CTX-1 and TRACP are two commonly used bone resorption markers that reflect the degree of bone tissue resorption by osteoclasts, thereby assessing the state of bone metabolism. CTX-1 (C-terminal carboxyl-terminal peptide of bone turnover): CTX-1 is a peptide produced by the breakdown of type I collagen. When osteoclast activity increases and bone tissue resorption is accelerated, CTX-1 levels in the blood will increase relatively. Therefore, measuring CTX-1 levels in the blood can indirectly reflect the degree of bone resorption and can be used for risk assessment of bone metabolic diseases such as osteoporosis, as well as for monitoring treatment efficacy. TRACP is primarily produced by osteoclasts in the human body, and its levels increase during the process of bone resorption. Similar to CTX-1, changes in TRACP levels can also reflect the state of bone metabolism, particularly the degree of bone resorption.

[0119] Bone resorption markers in rat serum were determined by enzyme-linked immunosorbent assay. Figure 8 As shown in the results, compared with the control group, the levels of CTX-1 and TRACP in the model group were significantly increased (p<0.05), indicating that bone resorption in the model group was significantly increased compared with the blank group. After BL25 intervention, the levels of CTX-1 and TRACP were significantly decreased (p<0.05), indicating that Lactobacillus plantarum BL25 has the effect of reducing bone resorption. The BL25+stachyose treatment significantly reduced the levels of CTX-1 and TRACP compared with the model group and BL25 (p<0.001), which may be because stachyose promotes the colonization of Lactobacillus plantarum BL25 in the intestine, better exerting its effect of inhibiting bone resorption.

[0120] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized in that, The Lactobacillus plantarum is Lactobacillus plantarum BL25, and its preservation number is CGMCC No.33117.

2. A probiotic preparation, characterized in that The probiotic preparation comprises one or more of the bacterial bodies, fermentation liquid, fermentation liquid precipitation and freeze-dried powder of the Lactobacillus plantarum according to claim 1.

3. The probiotic preparation according to claim 2, characterized in that The probiotic preparation further comprises nutritional additives that are nutritionally acceptable, and the nutritional additives include one or more of dietary fiber, protein, lipids, minerals, and vitamins.

4. The probiotic preparation according to claim 3, characterized in that The dietary fiber is a prebiotic, and the prebiotic is stachyose.

5. The probiotic preparation according to claim 2, characterized in that The preparation method of the probiotic preparation comprises the following steps: inoculating Lactobacillus plantarum BL25 into a culture medium containing nutrient additives, culturing the culture medium, and then freeze-drying the culture medium.

6. The probiotic preparation according to claim 5, characterized in that The culture medium includes: MRS culture medium, TPY culture medium, MC culture medium or BCP culture medium.

7. Use of the Lactobacillus plantarum according to claim 1 or the probiotic preparation according to any one of claims 2 to 6 in any of the following aspects: preparing a health product for improving bone density; or preparing a drug for alleviating osteoporosis.

8. A product comprising the Lactobacillus plantarum of claim 1 or the probiotic preparation of any one of claims 2 to 6, characterized in that: The products include food, health products or medicines.

9. The product according to claim 8, characterized in that The product contains at least 1×10 7 CFU / mL of Lactobacillus plantarum BL25.

10. The product according to claim 9, characterized in that The product contains 1×10 7 -1×10 12 CFU / mL of Lactobacillus plantarum BL25.

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