Phytobacterium plantarum for promoting skeletal development in growth period and application and product thereof

Abstract

The invention provides a plant lactobacillus for promoting skeletal development in a growth period as well as application and a product of the plant lactobacillus, and belongs to the technical field of microorganisms. The invention provides a lactobacillus plantarum BL25 and a probiotic preparation thereof, the preservation number of the lactobacillus plantarum BL25 is CGMCC (China General Microbiological Culture Collection Center) No.33117, stachyose is adopted as prebiotics, and compared with other prebiotics, the lactobacillus plantarum BL25 can obviously promote the activity of the lactobacillus plantarum BL25. The plant lactobacillus BL25 and the probiotic preparation thereof have the effect of resisting the severe environment of gastrointestinal tracts, and can increase the length of femur and bone density, promote the development of a bone microstructure, improve bone metabolism, increase osteogenesis markers and reduce osteoclast markers, so that the skeletal development in the life growth period is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Lactobacillus plantarum for promoting bone development during growth period and an application and a product thereof. Background Art

[0002] Bones are an important structural component of the human body, carrying multiple functions such as supporting the body, protecting organs, coordinating movements, and storing minerals. Infancy to childhood is a critical period for bone development, and bone health during this period has a decisive impact on the long-term health of individuals. First, the rapid increase in bone density, the continuous increase in bone length and hardness, and the formation of bone and the deposition of minerals are mainly completed in this process. Adequate nutrition intake and moderate exercise during this period are important factors in promoting the healthy development of bones. Secondly, strong bone health in childhood can prevent bone diseases such as osteoporosis in adulthood. A large number of studies have shown that the peak bone mass (PBM) reached by the body before and after puberty is an important indicator for predicting the risk of osteoporosis. The formation of PBM is mainly concentrated in childhood to adolescence. Therefore, optimizing bone development during this period will help increase peak bone mass and reduce the risk of bone diseases in adulthood.

[0003] Studies have found that environmental factors such as the gut microbiome also play a key role in the formation and development of bones. The gut microbiome, the bacterial community in the intestine, is an important player in human health. They are not only involved in the digestion of food and the acquisition of energy, but also have close interactions with the host's immune system and endocrine system. Moreover, changes in intestinal microorganisms during this period may have a profound impact on the individual's subsequent bone health. However, the gut microbiome includes thousands of microorganisms, each of which may have unique interactions with other microorganisms and the host system.

[0004] Lactobacillus plantarum is an important probiotic. First, it can optimize the intestinal microbial environment and help maintain the balance of intestinal flora. Secondly, Lactobacillus plantarum can enhance the human immune function, such as improving the phagocytic ability of white blood cells, stimulating immune response, and has a positive auxiliary effect on resisting diseases. Furthermore, Lactobacillus plantarum also improves digestive function, helps break down proteins and complex sugars, improves utilization, and allows the human body to obtain more nutrition from the diet. In addition, Lactobacillus plantarum can resist pathogenic microorganisms and strengthen the body's protective mechanism by producing substances with antibacterial effects, such as lactic acid and hydrogen peroxide. However, there is currently no report on Lactobacillus plantarum promoting bone development. Therefore, it is necessary to screen a strain of Lactobacillus plantarum that can promote bone development during the growth period of life. Summary of the invention

[0005] In view of the above shortcomings, the present invention provides a plant lactobacillus for promoting bone development during growth period and its application and product. The present invention provides a plant lactobacillus ( Lactiplantibacillus plantarum ) BL25 and its probiotic preparation, the deposit number of the plant lactobacillus BL25 is CGMCC No.33117, stachyose is used as a prebiotic, and the activity of plant lactobacillus BL25 can be significantly promoted compared with other prebiotics. The plant lactobacillus BL25 and its probiotic preparation have the effect of resisting the harsh environment of the gastrointestinal tract, can increase the length and bone density of the femur, promote the development of bone microstructure, improve bone metabolism, increase osteogenic markers and reduce osteoclast markers, thereby promoting bone development during the growth period of life.

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

[0007] In a second aspect, the present invention provides a probiotic preparation, which comprises one or more of the above-mentioned Lactobacillus plantarum bacteria, fermentation broth, fermentation broth precipitate, and freeze-dried powder.

[0008] Specifically, the probiotic preparation also includes nutritional additives that are nutritionally acceptable.

[0009] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0010] More preferably, the nutritional additive is a prebiotic.

[0011] Still more preferably, the prebiotic is stachyose.

[0012] The preparation method of the probiotic preparation comprises: inoculating plant lactobacillus BL25 into a culture medium containing nutrient additives, culturing the culture medium and then freeze-drying the culture medium.

[0013] Specifically, the culture medium containing nutrient additives includes: MRS culture medium, TPY culture medium, MC culture medium or BCP culture medium.

[0014] Preferably, the culture medium containing nutrient additives comprises MRS medium.

[0015] Specifically, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0016] Preferably, the nutritional additive is a prebiotic.

[0017] More preferably, the prebiotic is stachyose.

[0018] Specifically, the content of the nutrient additive in the culture medium is 0.5%-2% w / v.

[0019] Preferably, the content of the nutrient additive in the culture medium is 1% w / v.

[0020] Specifically, the culture medium containing the nutrient additives is sterilized by filtering membrane before use.

[0021] Preferably, the filter membrane is a 0.2 μm filter membrane.

[0022] Specifically, the Lactobacillus plantarum BL25 is a suspension of Lactobacillus plantarum BL25.

[0023] 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 completed, collecting the precipitate, and resuspending to obtain a suspension.

[0024] Preferably, the culture medium includes, but is not limited to, MRS medium, TPY medium, MC medium or BCP medium.

[0025] Further preferably, the culture medium is MRS culture medium.

[0026] Preferably, the incubation condition is 35-39° C. for 12-48 h.

[0027] More preferably, the incubation condition is 37° C. for 24 h.

[0028] Preferably, the centrifugation is performed at 1000-3000 rpm for 5-15 min.

[0029] More preferably, the centrifugation is performed at 2000 rpm for 10 min.

[0030] Preferably, the suspension contains at least 10 7 CFU / mL of Lactobacillus plantarum BL25.

[0031] More preferably, the suspension contains 10 7 -10 12 CFU / mL of Lactobacillus plantarum BL25.

[0032] 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 in the intestine.

[0033] In a fourth aspect, the present invention provides a product comprising the above-mentioned Lactobacillus plantarum or probiotic preparation, wherein the product includes food, health care product or medicine.

[0034] Specifically, the product contains at least 1×10 7 CFU / mL of Lactobacillus plantarum BL25.

[0035] Preferably, the product contains 1×10 7 -1×10 12 CFU / mL of Lactobacillus plantarum BL25.

[0036] Specifically, the food also includes nutritional additives that are nutritionally acceptable.

[0037] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0038] Preferably, the food includes candy flakes, soy milk, yogurt, canned food, biscuits, chocolate, cakes, cream, cheese, sour cream, 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.

[0039] Preferably, the food comprises human food or animal food.

[0040] Specifically, the health care product also includes nutritional additives that are nutritionally acceptable.

[0041] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0042] Preferably, the dosage form of the health care product includes tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

[0043] Specifically, the drug further comprises one or more physiologically acceptable adjuvants or pharmaceutically acceptable excipients.

[0044] Preferably, the physiologically acceptable excipients include, but are not limited to, erythritol, D-mannitol, fumaric acid, glycerol, 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.

[0045] 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.

[0046] The present invention has the following beneficial effects: (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.

[0047] (2) The Lactobacillus plantarum BL25 and the prebiotic preparation thereof of the present invention have the effect of promoting bone development, increasing femur length, and increasing bone density by increasing BV / TV, Tb.N and reducing Tb.Sp.

[0048] (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.

[0049] Collection Instructions Preserved strain: Lactobacillus plantarum BL25 Classification name: Lactobacillus plantarum Lactiplantibacillus plantarum ; Deposit number: CGMCC No.33117; Deposit date: December 18, 2024; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1The promoting effects of different prebiotics on the activity of Lactobacillus plantarum BL25; ** indicates P < 0.01 compared with the model group.

[0051] Figure 2 The figure shows a comparison of the protein content of the model diet and the standard diet.

[0052] 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.

[0053] Figure 4 Representative Micro CT images of each treatment group.

[0054] Figure 5 Analysis of bone density and bone microstructure in rats; A in the figure is 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.

[0055] Figure 6 Representative images of Gloder staining and TRAP staining of each treatment group.

[0056] Figure 7 The figure shows the effect of each treatment group on the content of bone formation markers; A in the figure is the content of type I procollagen N-terminal propeptide (PINP) in serum; B is the content 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.

[0057] Figure 8 The figure shows the effect of each treatment group on the content of bone resorption markers; A in the figure is the content of type I collagen cross-linked C-terminal peptide (CTX-1) in serum; B is the content of acid phosphatase (TRACP); ** indicates P < 0.01 compared with the model group; *** indicates P < 0.001 compared with the model group. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all 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.

[0059] Example 1 Materials and Methods 1. Experimental strains 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 China Culture Collection Administration on December 18, 2024, with the deposit number CGMCC No.33117.

[0060] 2. Experimental strain culture medium MRS medium: 12g peptone, 6g yeast extract, 6g beef extract, 18g glucose, 5g sodium acetate, 2.15g ammonium citrate, 1g Tween, 0.58g magnesium sulfate, 0.05g manganese sulfate, 2g dipotassium hydrogen phosphate, make up to 1L with pure water, adjust the pH to 6.5, sterilize in a high-pressure steam autoclave at 121°C for 15min for later use.

[0061] 3. Data processing 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 GraphPad Prism 5.0 and Origin 9.0 software were used for plotting. P < 0.05 was considered statistically significant.

[0062] Example 2 Activation of microorganisms and preparation of bacterial suspension Lactobacillus plantarum BL25 was stored at -80°C in 20% v / v glycerol. Before the experiment, Lactobacillus plantarum BL25 was inoculated in 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 precipitate was harvested and washed twice with sterile phosphate buffered saline (PBS). The washed precipitate was diluted with PBS to a cell concentration of 10 7 CFU / mL of Lactobacillus plantarum BL25 suspension.

[0063] Example 3 Effect of prebiotics on bacterial activity The suspension of Lactobacillus plantarum BL25 was 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 sterile operation at a 5% inoculation rate, 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.

[0064] In order to verify the effect of prebiotic stachyose on the activity of Lactobacillus plantarum BL25, 1% stachyose was added to MRS medium for cultivation. The results are as follows: Figure 2 As shown, the number of viable bacteria in the MRS 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 ordinary MRS medium (p<0.05) and 8.79±0.13 Log of CFU / mL in the prebiotic inulin MRS 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.

[0065] Example 4 Simulation of gastrointestinal digestion of prebiotic preparations The suspension of Lactobacillus plantarum BL25 was inoculated into MRS liquid medium, MRS medium containing 1% w / v stachyose, and MRS medium containing 1% w / v inulin at a 5% inoculum size under aseptic operation and cultured at 37°C for 24 hours, and the initial pH of the medium was adjusted to 5.9. Freeze-dried bacterial powder was prepared by freeze drying, and freeze-dried bacterial powder of Lactobacillus plantarum BL25, stachyose prebiotic preparation, and inulin prebiotic preparation were obtained respectively. The blank group was freeze-dried bacterial powder of Lactobacillus plantarum BL25 (BL25), the experimental group was stachyose prebiotic preparation (BL25+stachyose), and the inulin prebiotic preparation (BL25+inulin) was used as the control scheme.

[0066] Artificial saliva fluid (SSF) was prepared by dissolving 12.0 mg NaCl, 15.0 mg KCl, 100.0 mg mucin, and 7.5 mg α-amylase in 50.0 mL distilled water.

[0067] Artificial gastric juice (SGF) was prepared by mixing 12.0 mg NaCl, 15.0 mg KCl, and 100.0 mg mucin with 7.5 mg α-amylase in human saliva, dissolving the mixture in 50.0 mL distilled water, and adding 0.1 M HCl to adjust the pH to 3.0 to obtain artificial gastric juice (SGF).

[0068] Simulated intestinal fluid (SIF) was prepared by placing 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.

[0069] 2 g of each preparation was inoculated into a shake flask containing 20 mL of SSF, incubated in a 37°C incubator for 2 minutes, and then centrifuged (1000 rpm, 2 min) to obtain the preparation after saliva digestion. The preparation after saliva digestion was added to a shake flask containing 20 mL of SGF and placed in a constant temperature shaker (37°C, 3 rpm, 2 h). The preparation digested by gastric juice was obtained by centrifugation (1000 rpm, 2 min) and added to a shake flask containing 20 mL of SIF, placed in a constant temperature shaker (37°C, 2 rpm, 3 h), and then each group of preparations was harvested. At the end of each step (SIF, SGF, and SIF), the survival rate was determined by a viability meter in log CFU / mL.

[0070] In order to understand the effect of Lactobacillus plantarum BL25 combined with prebiotics on resisting gastrointestinal digestion, in vitro gastrointestinal digestion simulation was carried out. The results of each group are shown in Table 1.

[0071] Table 1 Effects of prebiotic preparations on simulated gastrointestinal digestion

[0072] Note: Data are expressed as mean ± standard deviation; different lowercase letters in the same column indicate significant differences (P<0.05).

[0073] After gastrointestinal digestion simulation, BL25, BL25+inulin and BL25+stachyose were all able to resist the harsh environment of the gastrointestinal tract. Compared with the BL25 and BL25+inulin groups, BL25+stachyose was significantly able to resist the harsh environment of the gastrointestinal tract, and the colonization was significantly improved. This suggests that the combination of stachyose and Lactobacillus plantarum BL25 helps to enhance resistance to gastrointestinal digestion. This may be because stachyose is relatively stable and not easily decomposed by gastric acid and intestinal enzymes, and therefore can provide an energy source in the intestine for a long time. Lactobacillus plantarum BL25 can utilize stachyose to survive and reproduce stably in the intestine.

[0074] Example 5 Animal Experiment 1. Experimental methods 1.1 Animal Experiment Design 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 with reference to the research results of Schwarzer et al. on Microbe-mediated intestinal NOD2 stimulation improves linear growth of undernourished infant mice, with slight modifications. Figure 2The difference in nutritional indicators of the two diets was shown. 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%.

[0075] 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 Animal Care and Use Guidelines, with three rats per cage, and standard cages allowed free access to food and water. The ambient temperature was maintained at 22 ± 2 °C to 45 ± 5%, with a 12-h light-off / dark cycle. The Animal Ethics Committee of Northeast Agricultural University approved this study with approval number NEUAEC20230424.

[0076] All rats first ate standard diet freely for one week to adapt and observe. After the adaptation period, the rats were divided into three groups: control group (C), model group (M), BL25 group (BL25), and BL25+stachyose group (BL25+stachyose). There were 6 rats in each group. After the adaptation period, all rats except the control group ate model diet for 4 weeks. During the model diet period, the BL25 group and BL25+stachyose group were gavaged with 1g of plantarum Lactobacillus BL25 freeze-dried bacterial powder or stachyose prebiotic preparation (dissolved in PBS) every day, and the blank combination model group was gavaged with corresponding physiological saline.

[0077] 1.2 Tissue sample collection After the last oral gavage, the rats were fasted for 12 h. The rats were then weighed and anesthetized for the next step. Blood samples were collected from the rats before they were humanely euthanized. The rat organs were isolated to calculate the organ index, and the colon contents were collected for genomic and non-targeted metabolomics analysis. The left and right femurs of the rats were collected and the length was measured using a vernier caliper for micro-CT imaging and subsequent histomorphometric analysis.

[0078] 1.3 Micro CT The right femur of rats was scanned using a micro-computed tomography system (SCANCO MEDICAL μCT-50, Switzerland), and the volume of the region of interest (ROI) was selected, and the computer software automatically reconstructed the 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) and trabecular N (mm-1), trabecular spacing (Tb.Sp) (mm) and trabecular thickness (Tb.Th) (mm).

[0079] 1.4 Histopathological and bone morphometric analysis After micro-CT imaging, femoral specimens were fixed with 4% paraformaldehyde, decalcified, trimmed, dehydrated, embedded, sliced, stained (Goldner and Trap), and sealed according to the SOP procedures of the pathology laboratory. After microscopic examination, the specimens were scanned using a PANNORAMIC (3DHISTECH, Hungary) panoramic slide scanner, and the target area of ​​the tissue was selected for imaging at 400 times using SlideViewer 2.5 scanning software. During imaging, the tissue was made to fill the entire field of view as much as possible, and the background light of each photo was ensured to be consistent. Three images were generated according to the above method. The generated images were used for quantitative tissue morphology measurement using Image-Pro Plus 6.0 (MediaCybemetics, USA). The main detection parameters were as follows: Goldner staining was used to analyze the number of osteoclasts (N.Ob / BS), osteoclast surface (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).

[0080] 1.5 Analysis of osteogenic markers Weigh an appropriate amount of serum and measure bone formation markers (PINP, ALP) in rat serum samples using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0081] 1.6 Analysis of osteoclast markers An appropriate amount of serum was weighed and the bone resorption markers (CTX-1, TRACP) in rat serum samples were determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions.

[0082] 2. Experimental results 2.1 Femur length The femur length of each group Figure 3 As shown in the figure, the femur length of the model group was significantly reduced, which indicates that the model treatment can effectively simulate the phenomenon of impaired bone development. After intervention with Lactobacillus plantarum BL25, the femur length increased significantly (p<0.05). Interestingly, the femur length of the BL25+stachyose group increased most significantly. This shows that BL25 has the effect of increasing bone development during the growth period.

[0083] 2.2 Bone density and bone microstructure To evaluate the effect of Lactobacillus plantarum BL25 on bone development during the growth period, femurs were scanned by Micro-CT to analyze bone density and bone microstructure. Figure 4As shown in Figure 2, μCT analysis showed that the bone structure of the M group fed with the model diet was significantly changed compared with the control group. These changes included increased trabecular spacing, decreased trabecular number, thinning of cortical bone, increased number of bone marrow cavity cavities, and the presence of fractures and discontinuous reticular structures. These findings indicate that the model diet successfully mimics the phenotype of impaired bone development. It is worth noting that BL25, especially BL25+stachyose treatment, can improve the above conditions and promote bone development during growth.

[0084] In order to more accurately understand the bone development level of each group, trabecular structure and bone density were quantitatively analyzed. The results of quantitative analysis are shown in Figure 5 Compared with the control group, the BV / TV, TB.N, Tb.Th and BMD values ​​of the M group were significantly lower, while Tb.Sp was significantly higher (p<0.05). Lactobacillus plantarum BL25 can effectively improve bone development, which is reflected in the significant increase of BV / TV, Tb.N and BMD and the decrease of Tb.Sp. In the BL25+stachyose group after adding stachyose, its bone development promoting effect was more significant (p<0.05).

[0085] 2.3 Bone histopathology Bone metabolism is regulated by a complex balance between osteoblasts, which are responsible for bone formation, and osteoclasts, which are responsible for bone resorption. Any change in the balance between these two processes will have a significant impact on bone metabolism. To evaluate the effect of BL25 on bone resorption and bone formation, the present invention analyzed the right femur ( Figure 6 ), and the activity of osteoblasts and osteoclasts was evaluated by bone morphometry. Goldner and Trap staining showed that in the model group, trabeculae appeared sparse and irregularly arranged. In addition, the color transparency of the bone matrix decreased compared with the control group, indicating skeletal dysplasia. It is worth noting that compared with the model group, new bone tissue appeared significantly in the BL25 group and the BL25+stachyose group, showing an orange-red color, and the BL25+stachyose group had the best effect in improving bone metabolism.

[0086] 2.4 Osteogenesis markers Bone formation markers are biomarkers used to evaluate bone metabolic activity and can reflect the degree of bone formation. Among them, procollagen type I N-terminal peptide (PINP) and alkaline phosphatase (ALP) are two commonly used bone formation markers. Procollagen type I N-terminal peptide (PINP): PINP is a byproduct of collagen type I synthesis, and its concentration can reflect the synthesis rate of bone collagen and the degree of bone formation activity. At the same time, the determination of PINP is less affected by the patient's diet and lifestyle, and has good stability. Therefore, PINP can be used as an important indicator for evaluating bone metabolic activity, especially bone formation activity. Alkaline phosphatase (ALP): Alkaline phosphatase is an enzyme present in many cells, and its content is particularly high in osteocytes. ALP is involved in the process of bone mineralization, and changes in its concentration can reflect the degree of bone formation and mineralization. Therefore, ALP is often used as an indicator for evaluating bone metabolic activity, especially bone formation activity.

[0087] 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 reduced (p<0.05), indicating that the bone formation in the model group was significantly lower than that in the control group. After intervention with BL25, the levels of PINP and ALP were significantly increased, indicating that Lactobacillus plantarum BL25 has the effect of increasing bone formation. Interestingly, the levels of PINP and ALP in BL25+stachyose were significantly higher than those in the model group and BL25 (p<0.05), which may be because stachyose promoted the colonization of Lactobacillus plantarum BL25 in the intestine, and better played its role in promoting bone formation.

[0088] 2.5 Osteoclast markers CTX-1 and TRACP are two commonly used bone resorption markers that can reflect the degree of activity of bone tissue being absorbed by osteoclasts, thereby evaluating the state of bone metabolism. CTX-1 (C-terminal carboxyl-terminal peptide of bone transformation): CTX-1 is a peptide produced by the decomposition of type I collagen. When osteoclast activity is enhanced and bone tissue is absorbed faster, the level of CTX-1 in the blood will increase relatively. Therefore, measuring the level of CTX-1 in the blood can indirectly reflect the degree of bone absorption, which can be used for risk assessment of bone metabolic diseases such as osteoporosis, and can also be used to monitor the effect of treatment. TRACP is mainly produced by osteoclasts in the human body, and the level of TRACP increases during the process of bone tissue absorption. Similar to CTX-1, changes in TRACP levels can also reflect the state of bone metabolism, especially the degree of bone absorption.

[0089] Bone resorption markers in rat serum were determined by enzyme-linked immunosorbent assay. Figure 8As shown in the figure, compared with the control group, the levels of CTX-1 and TRACP in the model group were significantly increased (p<0.05), indicating that the bone resorption in the model group was significantly higher than that in the blank group. After BL25 intervention, the levels of CTX-1 and TRACP were significantly reduced (p<0.05), indicating that Lactobacillus plantarum BL25 has the effect of reducing bone resorption. The levels of CTX-1 and TRACP in BL25+stachyose were significantly lower than those in the model group and BL25 (p<0.001), which may be because stachyose promoted the colonization of Lactobacillus plantarum BL25 in the intestine, and better exerted its effect of inhibiting bone resorption.

[0090] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized in that, The plant lactobacillus is plant lactobacillus 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 plant lactobacillus according to claim 1.

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

4. The probiotic preparation according to claim 3, characterized in that The nutritional additive 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: inoculating plant lactobacillus 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 promoting bone development during growth and alleviating osteoporosis; or preparing a probiotic food, health product or drug that can colonize in the intestine.

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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