Sublactobacillus paracasei mg-b200 capable of alleviating osteoporosis and use thereof

Abstract

The present application relates to the field of probiotics, and particularly relates to a paracasei MG-B200 capable of relieving osteoporosis and application thereof. The paracasei MG-B200 in the present application has a preservation number of CGMCC No.38263, a preservation time of 2026-04-15, and is preserved in the China General Microbiological Culture Collection Center. The paracasei MG-B200 in the present application has a significant ability to promote alkaline phosphatase secretion. In addition, the paracasei MG-B200 can significantly increase the bone density, bone volume fraction, trabecular number and thickness of the femur and significantly reduce the trabecular separation degree.

Description

Technical Field

[0001] This invention relates to the field of probiotics, and in particular to a type of Lactobacillus paracasei MG-B200 that can alleviate osteoporosis and its uses. Background Technology

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and deterioration of bone microstructure, leading to increased bone fragility and a higher risk of fractures. Bone mineral density (BMD), a key indicator for assessing the calcium and mineral content of bone, is a crucial diagnostic criterion. It is the most common skeletal disorder, particularly affecting women over 50, with approximately half of all osteoporosis patients experiencing a hip, wrist, or vertebral fracture at some point in their lives. As a dynamic living tissue, bone maintains its self-renewal through a balance between osteoblast (OB)-mediated bone formation and osteoclast (OCL)-mediated bone resorption. Osteoporosis occurs when bone formation is insufficient to compensate for bone resorption, or when old bone is excessively absorbed. Calcium is the core mineral for bone mineralization, and its effective utilization depends on vitamin D; insufficient intake or malabsorption in the intestines leads to increasingly fragile bones. Decreased estrogen levels after menopause and decreased testosterone levels in older men are major endocrine factors contributing to accelerated bone loss. Recent studies have found that Lactobacillus paracasei, as a probiotic, can regulate the gut microbiota, enhance the bioavailability of calcium and vitamin D, and may affect bone metabolism through immune regulation, thus opening up new avenues for the prevention and treatment of osteoporosis.

[0003] Bone remodeling is a collaborative process involving osteoblast-led bone formation and osteoclast-mediated bone resorption, with its balance precisely regulated by multiple molecules. Macrophage colony-stimulating factor (M-CSF) promotes the proliferation of osteoclast precursors and upregulates the expression of their surface RANK receptors, creating conditions for RANK ligand (RANKL) binding and thus activating the osteoclast differentiation signaling cascade. Osteoporosis, acting as a decoy receptor for RANKL, can block this process and maintain the homeostasis of bone resorption and formation. Osteoporosis arises from an imbalance in bone remodeling, which may manifest as excessive bone resorption, osteoclasts forming excessively deep resorption lacunae, or insufficient osteoblast repair function. Central bones (such as vertebrae) are more prone to bone loss and fractures due to their higher remodeling activity. The link between inflammation and bone loss has been widely established: in autoimmune diseases, inflammatory cytokines secreted by activated T cells can drive osteoclast activation; epidemiological evidence shows that mild systemic inflammation (marked by a slight increase in hsCRP) is associated with low bone mineral density, high bone resorption, and an increased risk of fracture. Postmenopausal estrogen deficiency leads to weakened immunosuppression, promoting increased production of osteoclast-promoting cytokines such as TNF-α and IL-1, and prolonging osteoclast lifespan, thereby accelerating bone loss. Lactobacillus paracasei can reduce systemic low-grade inflammation by regulating gut microbiota composition, enhancing intestinal barrier function, and reducing endotoxin entry into the bloodstream, thereby inhibiting the production of inflammatory cytokines, restoring bone remodeling balance, reducing osteoclast activity and bone resorption, and has potential therapeutic value for postmenopausal osteoporosis and other types of bone loss.

[0004] However, although existing technologies have preliminarily verified that Lactobacillus paracasei has the ability to regulate bone metabolism, the function and effect of probiotics are highly correlated with specific strains, and there are often huge differences in effects between different strains. Therefore, finding strains with more significant effects remains an important issue in the field of probiotic applications. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a Lactobacillus paracasei MG-B200 that can alleviate osteoporosis and its uses, in order to solve the problems in the prior art.

[0006] To achieve the above and other related objectives, this invention provides a *Lactaseibacillus paracasei* MG-B200 that can alleviate osteoporosis. Its accession number is CGMCC No. 38263, and its accession date is April 15, 2026. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing.

[0007] Preferably, the *Lactobacillus paracasei* MG-B200 comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID No. 1.

[0008] The present invention also provides a microbial agent containing the aforementioned *Lactobacillus paracasei* MG-B200.

[0009] The present invention also provides the use of the aforementioned Lactobacillus paracasei MG-B200 or the aforementioned bacterial agent in the preparation of products for the prevention and treatment of bone diseases or for the improvement of bone disease symptoms.

[0010] As described above, the present invention provides a strain of *Lactobacillus paracasei* MG-B200 that can alleviate osteoporosis and its uses, which have the following beneficial effects:

[0011] The *Lactobacillus paracasei* MG-B200 provided by this invention has the ability to significantly promote alkaline phosphatase secretion and bone mineralization. Furthermore, *Lactobacillus paracasei* MG-B200 can significantly increase the bone mineral density, bone volume fraction, number and thickness of trabeculae in the femur and significantly reduce trabecular separation, indicating that it can be effectively used to improve osteoporosis. Attached Figure Description

[0012] Figure 1 The diagram shows the effect of Lactobacillus paracasei MG-B200 on alkaline phosphatase secretion by MC3T3-E1 in this invention (compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0013] Figure 2 The diagram shows the anti-inflammatory results of Lactobacillus paracasei MG-B200 in this invention (compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0014] Figure 3 This diagram illustrates the effect of Lactobacillus paracasei MG-B200 on body weight in an osteoporosis model rat during the experiment.

[0015] Figure 4 The diagram shows the effects of Lactobacillus paracasei MG-B200 on IL-6 and TNF-α in the colon of osteoporosis model rats in this invention (compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0016] Figure 5The diagram shows the effect of Lactobacillus paracasei MG-B200 on the intestinal flora of osteoporosis model rats in this invention (compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0017] Figure 6 The diagram shows the effect of Lactobacillus paracasei MG-B200 on the micro-CT bone morphology of the femoral tissue of osteoporotic model rats (compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Detailed Implementation

[0018] This application provides a *Lactaseibacillus paracasei* MG-B200 that can alleviate osteoporosis. Its accession number is CGMCC No. 38263, the accession date is 2026-04-15, and it is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, Beijing.

[0019] In some specific embodiments, the *Lactobacillus paracasei* MG-B200 comprises a DNA fragment with a nucleotide sequence as shown in SEQ ID NO. 1.

[0020] In some specific embodiments, the *Lactobacillus paracasei* MG-B200 contains a DNA fragment with a nucleotide sequence similarity of 50% or more to that shown in SEQ ID No. 1. Specifically, the *Lactobacillus paracasei* MG-B200 comprises a DNA fragment with a nucleotide sequence similarity of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 84%, 86%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more to that of SEQ ID No. 1. Preferably, the *Lactobacillus paracasei* MG-B200 comprises a DNA fragment with a nucleotide sequence similarity of 90% or more to that of SEQ ID No. 1; more preferably, the *Lactobacillus paracasei* MG-B200 comprises a DNA fragment with a nucleotide sequence similarity of 99% or more to that of SEQ ID No. 1. Specifically, the DNA fragments contained in *Lactobacillus paracasei* MG-B200 can be obtained by nucleotide substitution, deletion, insertion, or homologous recombination of nucleic acid fragments on the DNA strand with the nucleotide sequence shown in SEQ ID No. 1.

[0021] In some specific embodiments, the *Lactobacillus paracasei* MG-B200 is isolated from the saliva of healthy human individuals.

[0022] In some specific embodiments, the *Lactobacillus paracasei* MG-B200 has one or more of the following functions:

[0023] 1) Increase alkaline phosphatase levels;

[0024] 2) Reduce the level of inflammatory factors;

[0025] 3) Increase bone density, bone volume fraction, or the number and thickness of trabeculae.

[0026] The present invention also provides a microbial agent containing the aforementioned *Lactobacillus paracasei* MG-B200.

[0027] In some specific embodiments, the aforementioned *Lactobacillus paracasei* MG-B200 in the microbial agent exists in one or more of the following forms: live bacteria, inactivated bacteria, fermentation products, or fermentation extracts.

[0028] In some specific embodiments, the microbial agent further comprises *Lactobacillus plantarum*, *Akkermansia muciniphila*, *Lactobacillus acidophilus*, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Lactobacillus reuteri*, *Lactobacillus shelveticus*, *Lactobacillus bulgaricus*, *Bifidobacterium bifidum*, *Bifidobacterium longum*, *Bifidobacterium breve*, *Bacillus subtilis*, *Streptococcus thermophilus*, *Prevotella histicola*, and *Faecalibacterium*. One or more of the following: *Propionibacterium prausnitzii* or *Propionibacterium acidipropionici*.

[0029] In some specific embodiments, the microbial agent further includes one or more of the following: prebiotics, vitamins, minerals, amino acids, polypeptides, emulsifiers, thickeners, sweeteners, flavorings, stabilizers, or preservatives.

[0030] Furthermore, the prebiotic may be selected from one or more of fructooligosaccharides, inulin, galactooligosaccharides, or polydextrose.

[0031] In some specific embodiments, the concentration of *Lactobacillus paracasei* MG-B200 in the bacterial agent can be at least 1 × 10⁻⁶. 8 CFU / g, at least 2×10 8 CFU / g, at least 3×10 8 CFU / g, at least 4×10 8 CFU / g, at least 5×10 8 CFU / g, at least 6×10 8 CFU / g, at least 7×10 8 CFU / g, at least 8×10 8 CFU / g, at least 9×10 8 CFU / g, 1×10 9 CFU / g, at least 2×10 9 CFU / g, at least 3×10 9 CFU / g, at least 4×10 9 CFU / g, at least 5×10 9 CFU / g, at least 6×10 9 CFU / g, at least 7×10 9 CFU / g, at least 8×10 9 CFU / g, at least 9×10 9 CFU / g, at least 1×10 10 CFU / g, at least 2×10 10 CFU / g, at least 3×10 10 CFU / g, at least 4×10 10 CFU / g, at least 5×10 10 CFU / g, at least 6×10 10 CFU / g, at least 7×10 10 CFU / g, at least 8×10 10 CFU / g, at least 9×10 10 CFU / g, or at least 1×10 11 CFU / g or higher.

[0032] In some specific embodiments, the microbial agent may be selected from one or more of the following forms: compressed candy, tablets, powders, granules, powders, oral liquids, drops, gummies, capsules, jellies, solid beverages, or fermented dairy products.

[0033] The present invention also provides the use of the aforementioned Lactobacillus paracasei MG-B200 or the aforementioned bacterial agent in the preparation of products for the prevention and treatment of bone diseases or for the improvement of bone disease symptoms.

[0034] In some specific embodiments, the bone disease is selected from one or more of osteoporosis, osteomalacia, rheumatoid arthritis, degenerative arthritis, osteogenesis imperfecta, osteopenia, bone atrophy, rickets, or fracture.

[0035] In some specific embodiments, the product is a pharmaceutical, health product, or food.

[0036] When the product is a pharmaceutical product, it contains pharmaceutically acceptable excipients. Specifically, the excipients include various excipients and diluents that are not essential active ingredients and do not cause excessive toxicity after administration. The excipients include sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When the excipients are used in an aqueous solution for injection, they are selected from physiological saline, isotonic glucose solution, isotonic D-sorbitol solution, isotonic D-mannose solution, or isotonic D-mannitol or sugar alcohol solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG) and / or nonionic surfactants (Tween 80 or HCO-50).

[0037] In this invention, the term "similarity" is defined as the percentage of identical nucleotide residues in a candidate nucleotide sequence to a reference nucleotide sequence after aligning nucleotide sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine the percentage nucleotide sequence similarity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0038] In this invention, the term "vitamin" refers to a class of trace organic substances that humans and animals must obtain from food to maintain normal physiological functions. Common examples may be selected from one or more of vitamin A, B vitamins, vitamin C, vitamin D, vitamin E, or vitamin K.

[0039] In this invention, the term "mineral" refers to an inorganic element essential to the human body, which often exists in the form of metal salts, such as calcium salts, phosphates, potassium salts, sodium salts, magnesium salts, iron salts, zinc salts, copper salts, iodates, or selenates.

[0040] In this invention, the term "probiotics" refers to live microorganisms that, when administered in adequate amounts, confer health benefits to the host, such as restoring or improving the composition of the gut microbiota. They are often provided as dietary supplements containing potentially beneficial bacteria or yeasts and are widely used in foods, including dairy products and probiotic-fortified foods.

[0041] In this invention, the term "anti-inflammatory" refers to the ability of probiotics to restore and maintain a healthy, balanced immune state by regulating the host's (such as human) immune system and microbial community, characterized by effectively controlling unnecessary, excessive, or harmful inflammatory responses.

[0042] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0043] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0044] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0045] The nucleotide sequence described in this application is as follows:

[0046] SEQ ID No.1

[0047]

[0048] Example 1: Lactobacillus paracasei MG-B200 can promote alkaline phosphatase secretion.

[0049] Lactobacillus paracasei MG-B200 was isolated from the saliva of healthy Chinese individuals and cryopreserved in glycerol tubes at -80°C. Colonies were obtained by inoculating the bacteria onto the surface of MRS+L-cysteine ​​solid agar plates and anaerobic incubating at 37°C. Single colonies were then picked and inoculated into MRS+L-cysteine ​​liquid agar plates and anaerobically incubated at 37°C to obtain activated strains.

[0050] The specific preparation of the culture medium is as follows:

[0051] MRS+L-cysteine ​​liquid culture medium: Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 1g Tween 80, and 0.5g L-cysteine, dissolve in 1L distilled water, and sterilize at high temperature for 15min.

[0052] MRS+L-cysteine ​​solid medium: Weigh 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 1g Tween 80, 0.5g L-cysteine, and 20g agar, dissolve in 1L distilled water, and sterilize at high temperature for 15min.

[0053] Lactobacillus paracasei MG-B200 was inoculated into MRS+L-cysteine ​​liquid medium and then cultured at 37 °C to ensure sufficient growth of the strain. After culture, the bacterial culture was centrifuged and the bacterial cells were collected.

[0054] Mouse embryonic osteoblast precursor cells (MC3T3-E1) were revived and cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies at 37°C with 5% CO2. The medium was changed every 2 days. When confluence reached 80%–90%, the cells were passaged: washed with sterile PBS, digested with 0.25% Trypsin-EDTA, and neutralized with DMEM complete medium. The cells were then transferred to sterile centrifuge tubes, centrifuged at 1000×g for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium. The cell culture was then aliquoted into new flasks and cultured again. Experiments were conducted after 3 passages.

[0055] MC3T3-E1 cells were loaded at 2.5 × 10⁻⁶. 4Inoculate 24-well plates at a density of CFU / ml and incubate for 24 h until adherent. Discard the original culture medium and add 1 ml of induction culture medium containing 1 μl of the test bacterial sludge for further differentiation. Repeat the incubation process three times per sample. Incubate at 37°C and 5% CO2 for 5 days to induce differentiation. Collect the supernatant, centrifuge, and determine ALP activity according to the Beyotime ALP kit procedure.

[0056] The results are as follows Figure 1 As shown, using Lactobacillus paracasei 8700:2 (a commercial strain of Probiotics) as a control strain, Lactobacillus paracasei MG-B200 significantly increased the activity of alkaline phosphatase, that is, Lactobacillus paracasei MG-B200 promoted the secretion of alkaline phosphatase by MC3T3-E1.

[0057] Example 2: Lactobacillus paracasei MG-B200 has anti-inflammatory effects.

[0058] Remove the cell cryovials from the liquid nitrogen container and thaw them quickly in a 37°C water bath. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and transfer the cells to a T25 cell culture flask. Incubate in a 37°C incubator with 5% CO2 until the cells adhere. After 2-3 passages, seed the cells into 96-well plates at a concentration of 10^5 CFU per well and incubate overnight until adherence.

[0059] The activated bacterial suspension was centrifuged at 4000 r / min for 10 minutes, the supernatant was discarded, and the cells were washed once with PBS and resuspended in DMEM medium to an OD of 1 (10^9 CFU / mL). 15 µL of each bacterial strain was added to 150 µL of DMEM medium containing 0.5 µg / mL LPS, mixed well, and then 100 µL was transferred to a 96-well plate. Each sample was tested in triplicate. After co-incubating with cells for 18 hours, the supernatant was collected to detect NO and TNF-α. The Beyotime reagent kit (S0021S) was used to detect NO according to the kit's instructions, and the Wuhan Feien reagent kit (EM0183) was used to detect TNF-α according to the kit's instructions.

[0060] The results are as follows Figure 2 As shown, using Lactobacillus paracasei 8700:2 (a commercial strain of Propofibrillar) as a control strain, Lactobacillus paracasei MG-B200 significantly inhibited the expression of pro-inflammatory factors NO and TNF-α, indicating that Lactobacillus paracasei MG-B200 has a good anti-inflammatory effect.

[0061] Example 3: Lactobacillus paracasei MG-B200 improves osteoporosis

[0062] Preparation of bacterial powder: Select a single colony of purified Lactobacillus paracasei MG-B200, inoculate it into MRS medium, and culture it anaerobically at 37℃; transfer the activated bacterial solution to MRS medium, expand the culture under the same conditions, and then transfer it to Lactobacillus paracasei special medium for further culture. Collect the bacterial solution, centrifuge and collect the bacterial cells to prepare bacterial powder and record the viable number.

[0063] Fifty-two rats were acclimatized for 7 days and then randomly divided into four groups of 13 rats each. The sample intervention group was administered *Lactobacillus paracasei* MG-B200 via gavage for 2 weeks. The model group, alendronate sodium tablet group, and sample intervention group underwent bilateral ovariectomy. For two weeks post-surgery, the bacterial powder was dissolved in sterile ultrapure water and vigorously shaken to ensure uniform dispersion. Water bottles were changed daily from 9:00 to 9:30 AM. Rats in all groups had free access to water. The viability of probiotics in the drinking water was monitored at 0 h, 8 h, and 24 h, and the water intake of each rat in each cage was recorded over 24 hours. Two weeks post-surgery, after the rat wounds healed, rats in each group continued to be administered *Lactobacillus paracasei* MG-B200 via gavage. 9 Lactobacillus paracasei MG-B200 was administered by gavage for 6 weeks, and samples were collected at the end of the experiment.

[0064] Physiological indicators and behavioral monitoring:

[0065] General observation of rats

[0066] During the experiment, the rats' activity level and mental state were observed every morning. Any abnormalities were recorded in detail in the "Rat Project Abnormality Record Form".

[0067] Weight and height measurement

[0068] The rats were weighed twice a week, and their body length (from the tip of the nose to the base of the tail) was measured once at the beginning and once at the end of the experiment.

[0069] Femoral bone collection and testing

[0070] After blood collection, the skin of the rat's leg was cut open, and the femurs and tibias on both sides of the rat were completely dissected. The muscle tissue and ligaments attached to the femurs and tibias were removed, and the wet weight and length of the femur were measured and photographed. The left femur of 6 rats in each group was randomly selected for Micro-CT to detect bone morphological parameters, including bone mineral density (BMD), bone volume (BV), total bone volume (TV), bone volume fraction (BV / TV), average trabecular number (Tb.N), average trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp).

[0071] 2. Colon tissue marker detection

[0072] Colon tissue was harvested from rats in each group. The contents were initially washed away, but the intestinal wall was not scraped. The colon tissue was placed on ice and further rinsed with pre-chilled PBS buffer, then the tissue was minced. An appropriate amount of lysis buffer was added at a weight-to-volume ratio (generally 1 g of tissue to 9 mL of lysis buffer), and a protease inhibitor was added to the lysis buffer. The tissue was homogenized on ice using a tissue homogenizer. The homogenate was centrifuged at 5000 x g for 5 min at 4°C, and the supernatant was used to detect the levels of IL-6 and TNF-α.

[0073] 16S sequencing of ileocecal contents

[0074] At the endpoint, 6 rats were randomly selected from each group, and the contents of the ileocecal junction were collected in sterile cryovials. The operation was rapid and contamination was avoided as much as possible during the process. All samples were flash-frozen in liquid nitrogen after collection and sent for external testing for 16S sequencing of gut microbiota.

[0075] Data processing and statistical methods

[0076] The experimental data are expressed as mean ± standard error (Mean ± SEM). All data were processed and statistically analyzed using GraphPad Prism 8.0. ANOVA was used to compare the means of multiple groups, and p < 0.05 was considered statistically significant.

[0077] Experimental results and explanations:

[0078] Clinical observation results

[0079] Throughout the experiment, before modeling, the activity level, food intake, hair growth, and mental state of the rats in each group were normal. After modeling, the animals experienced a temporary decrease in activity level and food intake. After a two-week recovery period, the surgical incisions in the rats' abdomens healed well, and hair growth, activity level, food intake, and mental state all returned to normal levels.

[0080] Weight test results

[0081] The results of rat weight monitoring during the experiment are as follows: Figure 3 As shown, the body weight of rats in all groups showed a continuous increasing trend. Compared with the sham-operated group, there was no significant difference in body weight in the model group, indicating that the modeling operation itself did not significantly affect the normal growth of the animals. Compared with the model group, the body weight of rats in the sample group did not change significantly.

[0082] Detection results of IL-6 in colon tissue

[0083] In OVX-induced osteoporosis animal models, estrogen deficiency leads to impaired intestinal barrier function and immune dysregulation, prompting excessive production of pro-inflammatory cytokines such as IL-6 in tissues like the colon. These factors not only exacerbate local inflammation but also enter the bloodstream, systemically stimulating osteoclast differentiation, inhibiting osteoblast function, and directly driving increased bone resorption. Therefore, detecting the levels of key inflammatory factors in the colon can assess whether sample intervention exerts a bone-protective effect by regulating intestinal immune homeostasis and reducing the pro-inflammatory environment. The results of colon tissue inflammatory factor detection are as follows: Figure 4 As shown, compared with the sham-operated group, the level of IL-6 in the colonic tissue of rats in the model group was significantly increased, while the level of TNF-α showed an increasing trend, with no significant difference. After sample intervention, compared with the model group, *Lactobacillus paracasei* MG-B200 could significantly reduce the level of IL-6 in the colonic tissue, and the level of TNF-α also showed a decreasing trend.

[0084] Gut microbiota abundance results

[0085] The results of the analysis of the relative abundance of beneficial bacteria (such as AKK bacteria, Lactobacillus, Adlerella, etc.) and harmful bacteria (such as Desulfovibrio, Escherichia coli, Escherichia-Shigella, etc.) in the rat gut microbiota are as follows: Figure 5 As shown, compared with the sham-operated group, the abundance of beneficial bacteria and the abundance of harmful bacteria in the intestines of rats in the model group were significantly increased and decreased, respectively. After intervention with different samples, compared with the model group, the Lactobacillus paracasei MG-B200 group significantly increased the abundance of beneficial bacteria and showed a decreasing trend in the abundance of harmful bacteria.

[0086] Micro-CT bone morphology results of femoral tissue

[0087] Bone mineral density (BMD) refers to the mineral density of bone, reflecting its strength and hardness, and is often used to assess the degree of osteoporosis and fracture risk. Bone volume fraction (BBS) refers to the proportion of bone tissue volume to the total tissue volume; a higher BBS indicates a larger bone volume. Trabeculae are extensions of the cortex within cancellous bone, forming an irregular three-dimensional network structure. The number and thickness of trabeculae are positively correlated with the hardness of the rat femur. Trabecular separation refers to the degree of connection between trabeculae in the bone; a higher value indicates a greater distance between trabeculae and a less tighter bone connection. Micro-CT results are shown below. Figure 6 As shown, compared with the sham-operated group, the bone mineral density, bone volume fraction, number of trabeculae, and thickness of the femur in the model group rats were significantly decreased, while the trabecular separation was significantly increased. Compared with the model group, the *Lactobacillus paracasei* MG-B200 group significantly increased the bone mineral density, bone volume fraction, number of trabeculae, and thickness of the rat femur and significantly decreased the trabecular separation.

[0088] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A type of Lactobacillus paracasei MG-B200 that can alleviate osteoporosis, characterized in that, The preservation number of Lacticaseibacillus paracasei MG-B200 is CGMCC No. 38263.

2. The *Lactaseibacillus paracasei* MG-B200 according to claim 1, characterized in that, The Lacticaseibacillus paracasei MG-B200 contains a DNA fragment with the nucleotide sequence shown in SEQ ID NO.

1.

3. The *Lactaseibacillus paracasei* MG-B200 according to claim 1, characterized in that, The Lacticaseibacillus paracasei MG-B200 has one or more of the following functions: 1) Increase alkaline phosphatase levels; 2) Reduce the level of inflammatory factors; 3) Increase bone density, bone volume fraction, or the number and thickness of trabeculae.

4. A microbial agent, characterized in that, The microbial agent contains Lacticaseibacillus paracasei MG-B200 as described in any one of claims 1-3.

5. The microbial agent according to claim 4, characterized in that, The microbial agent also contains one or more of the following: Lactobacillus plantarum, Akkermansia, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus helveticus, Lactobacillus bulgaricus, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium breve, Bacillus subtilis, Streptococcus thermophilus, Prevotella acidogenae, Clostridium plasminogene, or Propionibacterium acidogenae.

6. The microbial agent according to claim 4, characterized in that, The microbial agent also contains one or more of the following: prebiotics, vitamins, minerals, amino acids, polypeptides, emulsifiers, thickeners, sweeteners, flavorings, stabilizers, or preservatives.

7. The microbial agent according to claim 4, characterized in that, The concentration of Lactobacillus paracasei MG-B200 in the bacterial agent is at least 1 × 10⁻⁶. 8 CFU / g, at least 2×10 8 CFU / g, at least 3×10 8 CFU / g, at least 4×10 8 CFU / g, at least 5×10 8 CFU / g, at least 6×10 8 CFU / g, at least 7×10 8 CFU / g, at least 8×10 8 CFU / g, at least 9×10 8 CFU / g, 1×10 9 CFU / g, at least 2×10 9 CFU / g, at least 3×10 9 CFU / g, at least 4×10 9 CFU / g, at least 5×10 9 CFU / g, at least 6×10 9 CFU / g, at least 7×10 9 CFU / g, at least 8×10 9 CFU / g, at least 9×10 9 CFU / g, at least 1×10 10 CFU / g, at least 2×10 10 CFU / g, at least 3×10 10 CFU / g, at least 4×10 10 CFU / g, at least 5×10 10 CFU / g, at least 6×10 10 CFU / g, at least 7×10 10 CFU / g, at least 8×10 10 CFU / g, at least 9×10 10 CFU / g, or at least 1×10 11 CFU / g or higher.

8. The microbial agent according to claim 4, characterized in that, The microbial agent is selected from one or more of the following: compressed candy, tablets, powders, granules, powders, oral liquids, drops, soft candy, capsules, jellies, solid beverages, or fermented dairy products.

9. Use of Lacticaseibacillus paracasei MG-B200 as described in any one of claims 1-3 or the bacterial agent as described in any one of claims 4-8 in the preparation of products for the prevention, treatment or improvement of bone disease symptoms.

10. The use according to claim 9, characterized in that, The bone disease is selected from one or more of osteoporosis, osteomalacia, rheumatoid arthritis, degenerative arthritis, osteogenesis imperfecta, osteopenia, bone atrophy, rickets, or fracture; preferably, the product is a food, health product, or medicine; preferably, the product also contains excipients acceptable for food, health product, or medicine.

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