Use of lactobacillus plantarum hi188 in promoting bone health

Abstract

This invention relates to the application of *Lactobacillus plantarum* Hi188 in promoting bone health. The invention provides *Lactobacillus plantarum* Hi188 or its metagener, or combinations thereof, for increasing bone density, improving bone mineral density, promoting bone development, increasing bone mineral content, and / or promoting tibial growth, or for the preparation of foods for increasing bone density, improving bone mineral density, promoting bone development, increasing bone mineral content, and / or promoting tibial growth; wherein the composition comprises: (A) *Lactobacillus plantarum* Hi188 or its metagener, and (B) *Bifidobacterium lactis* Ca360 or its metagener.

Description

Technical Field

[0001] This invention belongs to the fields of microecology, food and life sciences, and more specifically, this invention relates to the application of *Lactobacillus plantarum* Hi188 in promoting bone health. Background Technology

[0002] Bone mineral density is the result of a dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. In malnutrition, this delicate balance is disrupted. On the one hand, the body faces a direct deficiency of key building blocks such as calcium, phosphorus, vitamin D, and high-quality protein. On the other hand, nutritional deficiencies induce systemic chronic low-grade inflammation and endocrine disorders, leading to elevated levels of inflammatory factors (such as TNF-α and IL-6). These levels not only directly activate osteoclasts to promote bone resorption but also simultaneously inhibit osteoblast function and muscle protein synthesis. Furthermore, malnutrition is inevitably accompanied by severe gut microbiota dysbiosis and impaired intestinal barrier function, resulting in the translocation of endotoxins into the bloodstream, further exacerbating systemic inflammation and creating a vicious cycle of "gut disorder - systemic inflammation - bone and muscle loss."

[0003] Malnutrition is not simply about "eating too little"; it's a complex systemic disease involving the immune, metabolic, and endocrine systems. Malnutrition (especially long-term or severe malnutrition) is closely related to decreased bone density and reduced bone mineral content. Bone loss is the most direct effect of malnutrition, and the recovery of bone density after nutritional restoration often lags behind the recovery of body weight. Malnutrition affects bones primarily at two levels: "nutrient supply" and "growth and development."

[0004] Traditional bone health research primarily focuses on the effects of calcium, vitamin D, endocrine hormones, and mechanical stress on bones. Traditional views emphasize calcium and vitamin D supplementation, but some analyses show that calcium supplementation alone may increase the risk of cardiovascular events, and the dosage and effectiveness of vitamin D supplementation also vary considerably (Holick 2009. Clinic RevBone Miner Metab). While hormone therapy can significantly improve bone density, its safety has long been controversial, potentially increasing the risk of venous thromboembolism, stroke, and heart disease, especially in women with pre-existing cardiovascular disease. Furthermore, long-term estrogen use may increase the risk of breast cancer and endometrial cancer. Exercise is a natural remedy for bone health; some strength training exercises (such as squats and weightlifting) are effective ways to stimulate bone growth, but they come with the risk of joint wear and soft tissue injury. Aerobic exercise (such as running) can effectively stimulate bone density, but it puts significant stress on the knee and ankle joints, potentially leading to arthritis in the long term. In recent years, the concept of the "gut-bone axis" has emerged, revealing a complex network of bidirectional communication between the gut and skeleton through the microbiome, immune system, and metabolites. The theory derived from the "gut-bone axis" provides a new perspective for intervening in bone health, especially bone problems caused by malnutrition. As the core organ for nutrient absorption and immune regulation, the gut microbiota homeostasis remotely regulates bone metabolism through multiple pathways, including regulating immune inflammation, affecting nutrient absorption efficiency, producing bioactive metabolites, and regulating endocrine function.

[0005] However, current interventions for bone mineral density related to malnutrition are mainly limited to simple nutritional supplements (such as calcium, vitamin D, and protein powder) and medications used in severe cases. The efficiency of nutritional supplementation is highly dependent on individual absorption capacity, and its effectiveness is often limited in the context of impaired intestinal function associated with malnutrition. Drug therapy suffers from high costs, significant side effects, and long intervention periods (requiring 3-5 years or longer), and it is difficult to achieve a true improvement in bone mineral density. Taking osteoporosis medications as an example, osteoporosis drug treatment is mainly divided into anti-resorption agents, bone formation agents, and bone metabolism modulators, and clinically, it generally faces challenges such as poor long-term adherence, adverse reactions, and the reversibility of efficacy. Bisphosphonates and denosumab are the two most commonly used first-line drugs in clinical practice. Alendronate sodium, in particular, has abundant long-term safety data, but requires strict fasting and an upright posture, leading to poor long-term adherence. Long-term use (>5 years) increases the risk of ONJ (onset japonic anterior joint fracture) and atypical femoral fractures. More importantly, most existing intervention strategies fail to address the root cause of initiating intestinal flora imbalance triggered by malnutrition. From the perspective of microecological intervention, patent CN202510547002.0 discloses the application of *Bifidobacterium adolescentis* and *Bifidobacterium longum* strains in improving osteoporosis, whose mechanism of action is to enhance the physiological activity of vitamin D and maintain bone health. However, current research indicates that the effect of probiotics on improving bone health is limited and specific. Most studies focus on osteoporosis caused by ovariectomy or on assisting growth promotion during the growth and development period. No literature has yet explored the role of probiotics in promoting bone health under the specific systemic metabolic disorder model of malnutrition. Therefore, developing a microecological preparation that can target the gut-bone axis, is safe and effective, and can fundamentally break the vicious cycle of malnutrition has become an urgent problem to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide the application of *Lactobacillus plantarum* Hi188 in promoting bone health.

[0007] In a first aspect, the present invention provides *Lactobacillus plantarum* Hi188 or its metagener, or a combination thereof, for the use of increasing bone density, improving bone mineral density, promoting bone development, increasing bone mineral content and / or promoting tibial growth, or in the preparation of foods for increasing bone density, improving bone mineral density, promoting bone development, increasing bone mineral content and / or promoting tibial growth, wherein the composition comprises: (A) *Lactobacillus plantarum* Hi188 or its metagener, and (B) *Bifidobacterium lactis* Ca360 or its metagener; wherein *Lactobacillus plantarum* Hi188 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30250; wherein *Bifidobacterium lactis* Ca360 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32403.

[0008] In one or more embodiments, the *Lactobacillus plantarum* Hi188 comprises: *Lactobacillus plantarum* Hi188, its fermentation broth, dilution, fermentation precipitate, and / or lyophilized powder.

[0009] In one or more embodiments, the metagenes of *Lactobacillus plantarum* Hi188 include: inactivated fermentation broth, supernatant, filtrate, inactivated dilution, inactivated fermentation precipitate, and / or inactivated lyophilized powder of *Lactobacillus plantarum* Hi188.

[0010] In one or more embodiments, the Bifidobacterium lactis Ca360 comprises: Bifidobacterium lactis Ca360, its fermentation broth, dilution, fermentation precipitate, and / or lyophilized powder.

[0011] In one or more embodiments, the metabiotic of Bifidobacterium lactis Ca360 includes: inactivated fermentation broth, supernatant, filtrate, inactivated dilution, inactivated fermentation precipitate and / or inactivated lyophilized powder of Bifidobacterium lactis Ca360.

[0012] In one or more embodiments, the composition of *Lactobacillus plantarum* Hi188 and *Bifidobacterium lactis* Ca360 may be a composition of *Lactobacillus plantarum* Hi188 and *Bifidobacterium lactis* Ca360, a composition of *Lactobacillus plantarum* Hi188 post-biotic and *Bifidobacterium lactis* Ca360 post-biotic, or a composition of *Lactobacillus plantarum* Hi188 post-biotic and *Bifidobacterium lactis* Ca360 post-biotic.

[0013] In one or more embodiments, the food includes food ingredients, semi-finished food products, finished food products, processed food, and unprocessed food.

[0014] In one or more embodiments, the food also includes food science acceptable materials.

[0015] In one or more embodiments, the food-grade acceptable materials include: food ingredients or their processed products, nutritional additives, ingredients that can be added to food, excipients and / or auxiliary materials.

[0016] In one or more embodiments, the food raw material or its processed product includes raw milk, whey protein powder, whey powder, the nutritional additives include one or more of lactose, fructooligosaccharides, galactooligosaccharides, dietary fiber, prebiotics, protein, lipids, minerals, vitamins, nucleotides, choline, and taurine, and / or the ingredients that may be added to the food include one or more of jujube, hawthorn, wolfberry, longan, lily, poria cocos, and dried tangerine peel, and / or the excipients or adjuvants include one or more of calcium carbonate, calcium phosphate, sugars, cellulose derivatives, gelatin, vegetable oil, and polyethylene glycol.

[0017] In one or more embodiments, the food includes ordinary food and special food.

[0018] In one or more embodiments, the special foods include infant foods, foods for special medical purposes, and health foods.

[0019] In one or more embodiments, the food is infant formula.

[0020] In one or more embodiments, the food further includes one or more of the following: raw milk, whey protein powder, whey powder, α-lactalbumin, β-lactalbumin, lactoferrin, milk fat globule membrane protein, lactose, galactose, oligosaccharides, dietary fiber, prebiotics, calcium, iron, phosphorus, zinc, vitamins, and nervonic acid.

[0021] In a second aspect, the present invention provides the use of *Lactobacillus plantarum* Hi188 or its metagener, or a combination thereof, in the preparation of a medicament for increasing bone density, improving bone mineral density, promoting bone development, increasing bone mineral content, promoting tibial growth, treating or preventing osteoporosis and / or treating or preventing skeletal dysplasia, wherein the composition comprises: (A) *Lactobacillus plantarum* Hi188 or its metagener, and (B) *Bifidobacterium lactis* Ca360 or its metagener; wherein *Lactobacillus plantarum* Hi188 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30250; wherein *Bifidobacterium lactis* Ca360 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32403.

[0022] In one or more embodiments, the *Lactobacillus plantarum* Hi188 or its post-genetic derivative is as defined in any embodiment of the present invention.

[0023] In one or more embodiments, the Bifidobacterium lactis Ca360 or its postbiotic is as defined in any embodiment of the present invention.

[0024] In one or more embodiments, the composition is as defined in any embodiment of the present invention.

[0025] In one or more embodiments, the osteoporosis includes decreased bone mass, bone microstructure damage, increased bone fragility, decreased osteoblast activity, decreased bone formation, increased osteoclast activity, and increased bone resorption.

[0026] In one or more embodiments, the skeletal dysplasia includes slow skeletal development, incomplete skeletal development, decreased bone density, and decreased bone mineral content.

[0027] In one or more embodiments, the skeletal dysplasia is skeletal dysplasia caused by malnutrition.

[0028] A third aspect of the present invention provides the use of a medicament in the preparation of a reagent for increasing bone density, improving bone mineral density, promoting bone development, increasing bone mineral content, promoting tibial growth, treating or preventing osteoporosis and / or treating or preventing skeletal dysplasia, said medicament comprising an effective amount of *Lactobacillus plantarum* Hi188 or its metagener as described in any embodiment of the present invention, or an effective amount of the composition as described in any embodiment of the present invention, and a pharmaceutically acceptable carrier.

[0029] In one or more embodiments, the osteoporosis includes decreased bone mass, bone microstructure damage, increased bone fragility, decreased osteoblast activity, decreased bone formation, increased osteoclast activity, and increased bone resorption.

[0030] In one or more embodiments, the skeletal dysplasia includes slow skeletal development, incomplete skeletal development, decreased bone density, and decreased bone mineral content.

[0031] In one or more embodiments, the skeletal dysplasia is skeletal dysplasia caused by malnutrition.

[0032] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0033] Figure 1 Effects of different treatments on zebrafish skeletal development (typical figure). D indicates inactivation. Scale bar = 100 μm.

[0034] Figure 2 Effects of different treatments on osteoporotic zebrafish (typical figure). D indicates inactivation. Scale bar = 100 μm.

[0035] Figure 3 A schematic diagram of a research protocol for studying the effects of Lactobacillus plantarum Hi188 on the skeletal health of malnourished cynomolgus monkeys.

[0036] Figure 4 Hi188 significantly increased bone mineral content in malnourished cynomolgus monkeys. d0, d30, d60, d90, d120, d150, and d180 represent days 0, 30, 60, 90, 120, 150, and 180 of the intervention, respectively. Control represents the control group, and Hi188 represents the Hi188 group.

[0037] Figure 5 Hi188 significantly increased the rate of change in bone mineral content in malnourished cynomolgus monkeys. d0, d30, d60, d90, d120, d150, and d180 represent days 0, 30, 60, 90, 120, 150, and 180 of the intervention, respectively. Control represents the control group, and Hi188 represents the Hi188 group.

[0038] Figure 6 Hi188 significantly increased bone mineral density in malnourished cynomolgus monkeys. Control group represents the control group, and Hi188 group represents the Hi188 group.

[0039] Figure 7 Hi188 significantly increased the rate of change in bone mineral density in malnourished cynomolgus monkeys. d0, d30, d60, d90, d120, d150, and d180 represent days 0, 30, 60, 90, 120, 150, and 180 of the intervention, respectively. Control represents the control group, and Hi188 represents the Hi188 group.

[0040] Figure 8 The images show photographs and tibial length statistics of Hi188 significantly increasing tibial growth in malnourished cynomolgus monkeys. The left image is a photograph of the tibia after dissection of malnourished cynomolgus monkeys in the control group, the middle image is a photograph of the tibia after dissection of malnourished cynomolgus monkeys in the Hi188 group, and the right image is a statistical chart of tibial length after dissection of malnourished cynomolgus monkeys in the control group and the Hi188 group. Detailed Implementation

[0041] Through in-depth research, the inventors used zebrafish and cynomolgus monkeys as animal models to study the role of *Lactobacillus plantarum* Hi188 in promoting bone health. The results showed that *Lactobacillus plantarum* Hi188 can promote bone development, increase bone mineral content, improve bone density, prevent or treat osteoporosis, and significantly improve malnutrition-induced bone dysplasia, such as bone mineral content, bone density levels, and tibial growth, thereby promoting bone development.

[0042] the term

[0043] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as would be understood by those skilled in the art. Furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as would be understood by those skilled in the art.

[0044] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0045] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0046] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0047] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0048] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0049] The term "skeletal development" encompasses the formation, development, and resorption of bone tissue. During skeletal development, osteoblasts first secrete collagen fibers and bone matrix, forming osteoid. After osteoid formation, hydroxyapatite is deposited to complete matrix ossification, becoming bone tissue. Subsequently, new osteoblasts continue to form osteoid on the surface of the formed bone tissue, depositing calcium salts and transforming into bone, thus achieving continuous bone growth.

[0050] The term "bone mineral density" refers to the amount of bone contained per unit volume (volume density) or per unit area (areal density). Osteoblasts are the main functional cells in bone formation, and they play a crucial role in maintaining and increasing bone mineral density.

[0051] The term "bone mineral content" usually refers to the mineral content within bones. Bone minerals mainly include substances such as calcium, phosphorus, and magnesium. Maintaining normal bone mineral content can ensure bone strength and prevent osteoporosis.

[0052] The term "bone mineral density" refers to the mass of minerals (primarily calcium and phosphorus) contained in a unit volume or area of ​​bone, and can be used to assess bone strength and health.

[0053] The term "osteoporosis" is a systemic bone disease characterized by decreased bone mass and destruction of bone microstructure, leading to increased bone fragility and a higher risk of fractures. Decreased osteoblast activity results in reduced bone formation, while relatively increased osteoclast activity leads to increased bone resorption, ultimately causing decreased bone mass and osteoporosis.

[0054] The term "skeletal dysplasia" refers to abnormalities that occur during skeletal development, such as slow skeletal growth, incomplete skeletal development, decreased bone density, reduced bone mineral content, or decreased bone mineral density. Osteoblasts are responsible for the synthesis and mineralization of the bone matrix during skeletal development; impaired function of osteoblasts leads to skeletal dysplasia. In some implementations, the skeletal dysplasia referred to is skeletal dysplasia caused by malnutrition.

[0055] The terms "subject," "host," or "individual" refer to any animal of interest. In some embodiments, the subject includes humans and non-human animals. In some embodiments, the non-human animals include fish (e.g., zebrafish), monkeys (e.g., cynomolgus monkeys), orangutans, mice, rats, rabbits, pigs, cattle, sheep (e.g., sheep, goats), dogs, cats, deer, horses, donkeys, etc.

[0056] The term "metasec" refers to products of non-living microorganisms and / or their components that are beneficial to the health of the host. Metabolic agents include processed inactivated bacteria and metabolites or lysates produced by live bacteria during their growth (e.g., short-chain fatty acids, enzymes, peptides, polysaccharides, etc.).

[0057] Lactobacillus plantarum Hi188 or its metagener

[0058] In this invention, *Lactobacillus plantarum* Hi188 refers to the Latin name... Lactiplantibacillus plantarum The strain, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30250, has been disclosed in CN118240712B. Its isolation and identification are based on CN118240712B. The relevant content of CN118240712B concerning Lactobacillus plantarum Hi188 is quoted in full here.

[0059] In this invention, *Lactobacillus plantarum* Hi188 refers to *Lactobacillus plantarum* Hi188 in its live bacterial form. It should be understood that *Lactobacillus plantarum* Hi188 can exist in various forms, such as, but not limited to, fermentation broth, diluted solution, fermentation precipitate, and / or lyophilized powder (also known as bacterial powder). Exemplarily, fermentation processes of *Lactobacillus plantarum* well-known in the art can be used to obtain the fermentation broth of *Lactobacillus plantarum* Hi188. The fermentation broth produced by *Lactobacillus plantarum* Hi188 fermentation can be processed through dilution and other steps to obtain its diluted solution. The fermentation broth or diluted solution of *Lactobacillus plantarum* Hi188 can be further processed through precipitation and / or lyophilization steps to obtain the fermentation precipitate and / or lyophilized powder of *Lactobacillus plantarum* Hi188.

[0060] This invention also provides a metagene of *Lactobacillus plantarum* Hi188 with accession number CGMCC No. 30250. The metagene can be inactivated *Lactobacillus plantarum* Hi188. For example, during processing, the supernatant or filtrate of the fermentation broth produced by *Lactobacillus plantarum* Hi188 fermentation can be obtained by centrifugation, filtration, and / or ultrafiltration. Alternatively, the fermentation broth, dilution, fermentation precipitate, or lyophilized powder of *Lactobacillus plantarum* Hi188 can be inactivated to obtain the metagene of *Lactobacillus plantarum* Hi188. Therefore, the metagene of *Lactobacillus plantarum* Hi188 can be an inactivated fermentation broth, supernatant, filtrate, inactivated dilution, inactivated fermentation precipitate, and / or inactivated lyophilized powder (also called sterilized powder) of *Lactobacillus plantarum* Hi188.

[0061] food

[0062] This invention provides the application of *Lactobacillus plantarum* Hi188 or its metagener, as described in any embodiment of the invention, in the preparation of foods that increase bone density, improve bone mineral density, promote bone development, increase bone mineral content, promote tibial growth, and / or improve osteoporosis. This invention also provides a food comprising *Lactobacillus plantarum* Hi188 or its metagener, as described in any embodiment of the invention.

[0063] The term "food" refers to articles or substances that can be ingested by an individual, including food ingredients, semi-finished food products, finished food products, processed food products, and unprocessed food products. In this invention, the term "individual" refers to any animal of interest. In some embodiments, the individual is a mammal, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats. The *Lactobacillus plantarum* Hi188 or its metagenes described herein can be formulated into a food product using standard techniques well known to those skilled in the art. For example, *Lactobacillus plantarum* Hi188 or its metagenes can be added directly to a food-grade acceptable material, or it can be used to prepare an intermediate composition (e.g., a food additive or premix) suitable for subsequent addition to a food-grade acceptable material.

[0064] The term "food-acceptable material" refers to food ingredients or their processed products (e.g., raw milk, whey protein powder, whey powder, etc.), nutritional additives used in food (e.g., lactose, fructooligosaccharides, galactooligosaccharides, dietary fiber, prebiotics, proteins, lipids, minerals, vitamins, nucleotides, choline, taurine), ingredients that can be added to food (e.g., jujubes, hawthorn, goji berries), excipients or adjuvants (e.g., calcium carbonate, calcium phosphate, various sugars, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol), etc. The food products of this invention can be in any form suitable for oral administration, such as powders, tablets, capsules, granules, solutions, suspensions, etc.

[0065] In some embodiments, the food is a general food or a special food. In some embodiments, the special food is infant food, such as infant formula. The term "infant" as used in this invention includes infants and toddlers, generally referring to individuals aged 0-36 months. The term "infant" refers to individuals aged 0-12 months. The term "toddler" refers to individuals aged 12-36 months. As an example, the food may be selected from dairy products, confectionery, beverages, bread, and biscuits; for example, the food may be selected from milk powder or fermented foods. In some embodiments, the food is infant formula. Exemplarily, when the food is milk powder, in addition to *Lactobacillus plantarum* Hi188 or its metabiotic as described in this invention, the milk powder may also include proteins such as α-lactalbumin, β-lactalbumin, lactoferrin, and milk fat globule membrane protein; carbohydrates such as lactose, galactose, and oligosaccharides; lipids; minerals such as calcium, iron, phosphorus, and zinc; vitamins; nervonic acid, etc. The food may be produced using preparation methods commonly used in the art. In some embodiments, the special food is a food for special medical purposes or a health food (also known as a functional food). The terms "health food" or "functional food" refer to items or substances that can be ingested by an individual and have health benefits (such as increasing bone density, improving bone mineral density, promoting bone development, increasing bone mineral content, promoting tibial growth, and / or improving osteoporosis).

[0066] It should be understood that when added to the food, the content of *Lactobacillus plantarum* Hi188 or its postbiotic meets the requirements for strain content in the art. In some embodiments, the viable count of *Lactobacillus plantarum* Hi188 in the food may be 10-1. 6 CFU / mL(g) or higher, for example, 10 6 ~10 12 CFU / mL (g).

[0067] In some embodiments, the food may also contain Bifidobacterium lactis Ca360 or its post-biotic. In some embodiments, the food contains (A) Lactobacillus plantarum Hi188 or its post-biotic, and (B) Bifidobacterium lactis Ca360 or its post-biotic. In one or more embodiments, the food contains Lactobacillus plantarum Hi188 and Bifidobacterium lactis Ca360, or contains Lactobacillus plantarum Hi188 post-biotic and Bifidobacterium lactis Ca360, or contains Lactobacillus plantarum Hi188 post-biotic and Bifidobacterium lactis Ca360 post-biotic, or contains Lactobacillus plantarum Hi188 post-biotic and Bifidobacterium lactis Ca360 post-biotic.

[0068] In one or more embodiments, the *Bifidobacterium lactis* Ca360 comprises: *Bifidobacterium lactis* Ca360, its fermentation broth, dilution, fermentation precipitate, and / or lyophilized powder. In one or more embodiments, the metabiotic of *Bifidobacterium lactis* Ca360 comprises: inactivated fermentation broth of *Bifidobacterium lactis* Ca360, supernatant, filtrate, inactivated dilution, inactivated fermentation precipitate, and / or inactivated lyophilized powder.

[0069] drug

[0070] The present invention also provides a medicine comprising *Lactobacillus plantarum* Hi188 or its metagener. The medicine may also include a pharmaceutically acceptable carrier.

[0071] In this document, "pharmaceutically acceptable carriers" refer to carriers that do not significantly irritate the organism and do not impair the bioactivity and properties of the reagents in the pharmaceutical composition, such as, but not limited to: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, or liposomes. In some embodiments, pharmaceutically acceptable carriers may be inert substances added to the pharmaceutical composition to further facilitate the administration of the reagents, such as calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, etc.

[0072] The medicament of this invention can be formulated into any suitable dosage form for administration by oral or other routes. Suitable dosage forms for oral administration include, but are not limited to: sterile powder, tablet, troche, lozenge, pellet, capsule, dispersible powder or granule, solution, suspension, drop, emulsion, syrup, elixir, or slurry. The medicament of this invention can also be stored in sterile instruments suitable for injection or infusion. The *Lactobacillus plantarum* Hi188 or its metagener described in the medicament is typically present in an effective amount (e.g., a therapeutic effective amount, a preventative effective amount). An effective amount is a dosage sufficient to improve or in some way alleviate symptoms associated with a disease, such as effectively improving or eliminating one or more symptoms, and can be determined based on the subject's age, sex, physical condition, etc. The dosage may cure the disease, but administration is usually intended to improve the symptoms of the disease. Repeated administration is generally required to achieve the desired symptom improvement.

[0073] In specific embodiments of this invention, zebrafish and cynomolgus monkeys were used as experimental animals to propose a dosing regimen for *Lactobacillus plantarum* Hi188 or its post-biotic to improve bone density, increase bone mineral density, promote skeletal development, increase bone mineral content, promote tibial growth, and / or treat or prevent osteoporosis or skeletal dysplasia. When zebrafish are used as experimental animals, the concentration of *Lactobacillus plantarum* Hi188 can be 10... 4 CFU / mL. When using cynomolgus monkeys as experimental animals, the concentration of *Lactobacillus plantarum* Hi188 can be 10. 11 CFU / mL. It should be understood that converting the dosage from zebrafish or cynomolgus monkeys to human dosage is readily achievable by those skilled in the art. When necessary, the *Lactobacillus plantarum* Hi188 or its post-biotic may also be administered in combination with other active ingredients or drugs, such as *Bifidobacterium lactis* Ca360 or its post-biotic.

[0074] In some embodiments, the drug contains (A) *Lactobacillus plantarum* Hi188 or its post-generant, and (B) *Bifidobacterium lactis* Ca360 or its post-generant. In one or more embodiments, the drug contains *Lactobacillus plantarum* Hi188 and *Bifidobacterium lactis* Ca360, or contains *Lactobacillus plantarum* Hi188 post-generant and *Bifidobacterium lactis* Ca360, or contains *Lactobacillus plantarum* Hi188 post-generant and *Bifidobacterium lactis* Ca360 post-generant.

[0075] In one or more embodiments, the *Bifidobacterium lactis* Ca360 comprises: *Bifidobacterium lactis* Ca360, its fermentation broth, dilution, fermentation precipitate, and / or lyophilized powder. In one or more embodiments, the metabiotic of *Bifidobacterium lactis* Ca360 comprises: inactivated fermentation broth of *Bifidobacterium lactis* Ca360, supernatant, filtrate, inactivated dilution, inactivated fermentation precipitate, and / or inactivated lyophilized powder.

[0076] Applications / Methods of Lactobacillus plantarum Hi188

[0077] This invention provides the use of *Lactobacillus plantarum* Hi188 or its metagener, or a combination thereof, in the preparation of medicaments for increasing bone density, improving bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, improving bone mineral content), improving bone mineral content, promoting tibial growth, treating or preventing osteoporosis (e.g., but not limited to osteopenia, bone microarchitectural damage, increased bone fragility, decreased osteoblast activity, decreased bone formation, increased osteoclast activity, increased bone resorption), and / or treating or preventing skeletal dysplasia (e.g., but not limited to slow bone development, skeletal hypoplasia, decreased bone density, decreased bone mineral content); wherein the composition contains (A) *Lactobacillus plantarum* Hi188 or its metagener, and (B) *Bifidobacterium lactis* Ca360 or its metagener.

[0078] The present invention also provides the use of the aforementioned *Lactobacillus plantarum* Hi188 or its metagenes, or combinations thereof, in increasing (or improving) bone density, increasing bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, increasing bone mineral content), increasing bone mineral content, promoting tibial growth and / or, or in the preparation of foods for increasing (or improving) bone density, increasing bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, increasing bone mineral content), and / or increasing bone mineral content; wherein the composition contains (A) *Lactobacillus plantarum* Hi188 or its metagenes, and (B) *Bifidobacterium lactis* Ca360 or its metagenes.

[0079] The present invention provides a method for increasing (or improving) bone density, improving bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, improving bone mineral content), increasing bone mineral content and / or promoting tibial growth for non-therapeutic purposes, the method comprising using an effective amount of *Lactobacillus plantarum* Hi188 or its postgenerate, or a combination thereof; wherein the composition contains (A) *Lactobacillus plantarum* Hi188 or its postgenerate, and (B) *Bifidobacterium lactis* Ca360 or its postgenerate.

[0080] The present invention also provides the use of the food in increasing (or improving) bone density, increasing bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, increasing bone mineral content), increasing bone mineral content and / or promoting tibial growth for non-therapeutic purposes.

[0081] The present invention also provides the use of the said drug in the preparation of agents for increasing (or improving) bone density, increasing bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, increasing bone mineral content), increasing bone mineral content, promoting tibial growth, treating or preventing osteoporosis (e.g., but not limited to bone loss, bone microstructure damage, increased bone fragility, decreased osteoblast activity, decreased bone formation, increased osteoclast activity, increased bone resorption) and / or treating or preventing skeletal dysplasia (e.g., but not limited to slow bone development, skeletal hypoplasia, decreased bone density, decreased bone mineral content).

[0082] In this invention, "promoting bone development" includes, but is not limited to, promoting osteoblast formation, increasing bone mineral content, and increasing bone mineral density. In some embodiments, "promoting bone development" is non-therapeutic and non-diagnostic.

[0083] The present invention also provides the use of the said drug in the preparation of agents for increasing bone density, improving bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, increasing bone mineral content), increasing bone mineral content, promoting tibial growth, treating or preventing osteoporosis (e.g., but not limited to bone loss, bone microstructure damage, increased bone fragility, decreased osteoblast activity, decreased bone formation, increased osteoclast activity, increased bone resorption) and / or treating or preventing skeletal dysplasia (e.g., but not limited to slow bone development, skeletal hypoplasia, decreased bone density, decreased bone mineral content).

[0084] The present invention also provides a method for increasing bone density, improving bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation, improving bone mineral content), improving bone mineral content, promoting tibial growth, treating or preventing osteoporosis (e.g., but not limited to bone loss, bone microstructure damage, increased bone fragility, decreased osteoblast activity, decreased bone formation, increased osteoclast activity, increased bone resorption) and / or treating or preventing skeletal dysplasia (e.g., but not limited to slow bone development, incomplete bone development, decreased bone density, decreased bone mineral content), the method comprising: administering *Lactobacillus plantarum* Hi188 or its metagener, or a combination thereof, to an individual in need, or administering the drug of the present invention to an individual in need; wherein the composition contains (A) *Lactobacillus plantarum* Hi188 or its metagener, and (B) *Bifidobacterium lactis* Ca360 or its metagener.

[0085] The present invention also provides a method for increasing bone density, improving bone mineral density, promoting bone development (e.g., but not limited to promoting osteoblast formation and increasing bone mineral content), increasing bone mineral content, and / or promoting tibial growth, the method comprising: giving the food of the present invention to an individual in need.

[0086] In this invention, the methods described are typically non-therapeutic and non-diagnostic.

[0087] In this invention, "giving" can refer to introducing, providing, or delivering a substance to an individual through any suitable means to achieve its intended function.

[0088] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0089] In this invention, *Lactobacillus plantarum* Hi188 refers to the Latin name... Lactiplantibacillus plantarum The strain, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30250, has been disclosed in CN118240712B. The relevant content of CN118240712B concerning Lactobacillus plantarum Hi188 is quoted in full here.

[0090] In this invention, the Bifidobacterium lactis Ca360 is a strain with accession number CGMCC No. 32403. Bifidobacterium lactis Ca360 has been disclosed in CN120330110B, and the relevant content of Bifidobacterium lactis Ca360 in CN120330110B is quoted in full here.

[0091] In this invention, the sodium alendronate was purchased from Savio Industrial SrL (white tablets, batch number X020681).

[0092] Example 1: Lactobacillus plantarum Hi188 promotes bone development in zebrafish.

[0093] Zebrafish have become an important model widely used in recent years for establishing disease models and drug discovery. The study of their skeleton has a physiological and genetic basis: zebrafish skeletal development is remarkably similar to that of other vertebrates, with skeletons composed of both cartilage and bone. At the cellular level, zebrafish osteoblasts and osteoclasts possess functions similar to those in mammals, with osteoclasts playing a role in bone resorption during early embryonic development. Anatomically, the morphology and developmental process of zebrafish juvenile and adult skeletons are largely well understood, showing a high degree of consistency with mammalian skeletal development, involving both intramembranous and endochondral ossification mechanisms. Alizarin red is a dye that specifically binds to calcified bone structures. The fluorescence intensity of zebrafish skeletons, collected under a fluorescence microscope, reflects the amount of calcified bone, and bone density is closely related to the calcium content in the bone. Using skull fluorescence intensity as an indicator, if a sample has a bone-promoting effect, the skull fluorescence intensity will be enhanced.

[0094] In this embodiment, wild-type AB strain zebrafish with a 3-day pf growth rate (dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish per well and a volume of 3 mL per well. The zebrafish from different wells were randomly divided into a normal control group (no treatment), a positive control group (administered water-soluble Swisse "Calcium & Vitamin D", final concentration 500 μg / mL (milky white tablets, purchased from Swisse)), and a commercial probiotic group (administered water-soluble Bifidobacterium lactis Ca360, final concentration 10). 8 CFU / mL), commercial probiotic inactivated group (administered water-soluble inactivated Bifidobacterium lactis Ca360 (inactivated at 75℃ for 30 min, final concentration of 10 CFU / mL), 8 The study group consisted of two subgroups: a live Hi188 group (treated with water-soluble Hi188 bacterial powder, with a final concentration of 10 CFU / mL) and an experimental group. 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 CFU / mL), Hi188 inactivated bacteria group (Hi188-D group, administered water-soluble inactivated Hi188 bacterial powder (inactivated at 75℃ for 30 min), final concentration of 10 ...). 8 The group receiving Hi188 live bacteria + Ca360 live bacteria (administered water-soluble Hi188 and Ca360, with a live bacteria ratio of 1:1, and a final Hi188 concentration of 5 × 10⁻⁶ CFU / mL) and the group receiving Hi188 live bacteria + Ca360 live bacteria (administered water-soluble Hi188 and Ca360, with a live bacteria ratio of 1:1, and the final concentration of Hi188 being 5 7 CFU / mL, final Ca360 concentration is 5×10 7(CFU / mL). Except for the normal control group which received no treatment, samples were administered the solution from fish tanks. After treatment at 28℃ for 72 h, samples were collected and stained with 50.0 μg / mL Alizarin Red (Shanghai Maclean Biotechnology Co., Ltd.). 24 h later, 10 zebrafish from each experimental group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity of the zebrafish skull was analyzed, and the statistical analysis results of this index were used to evaluate the sample's efficacy in promoting bone development. Statistical results are expressed as mean ± SEM. Statistical analysis was performed using SPSS 27.0 software; p < 0.05 indicated statistical significance.

[0095] Experimental results: As shown in Table 1, the skull bone mineral density of zebrafish in both the Hi188 live bacteria group and the Hi188 inactivated bacteria (Hi188-D) group was significantly higher than that in the normal control group (p<0.0001). Both the Hi188 live bacteria and Hi188 inactivated bacteria groups had a density of 10... 8 At CFU / mL, the levels were 45% and 38% higher than the normal control group, respectively, indicating that both live and inactivated Hi188 bacteria can promote skeletal development in zebrafish.

[0096] Compared to the commercial probiotic Ca360, the skull bone mineral density of zebrafish in the Hi188 live bacteria group was significantly higher than that in the commercial probiotic (Ca360) group. p The bone density of zebrafish in the Hi188 inactivated bacteria group (Hi188-D) was significantly higher than that in the commercial probiotic inactivated bacteria group (Ca360-D), indicating that both live and inactivated Hi188 bacteria were more effective than commercial probiotic Ca360 in promoting zebrafish skeletal development. Both the Hi188 and Ca360 groups were significantly lower than the Ca+VD group (p<0.0001), but there was no significant difference between the Hi188+Ca360 group and the Swiss group (Ca+VD3). p =0.7042), and increased by 100.42% and 106.59% respectively compared with the normal control group.

[0097] Furthermore, Hi188 promotes bone development with a dose-response effect, when the dose is 10... 5 At a concentration of CFU / mL, it begins to show a tendency to promote bone development; at a dose of 10... 6 At CFU / mL, it was significantly higher than that of the normal control group ( p <0.05).

[0098] Table 1. Effects of different treatments on skull bone mineral density in zebrafish

[0099] Note:" "This indicates that compared with the normal control group, p < 0.05;" "This indicates that compared with the normal control group, p < 0.01;" "" indicates that compared with the normal control group, p < 0.001.

[0100] Example 2: Lactobacillus plantarum Hi188 improves osteoporosis in zebrafish

[0101] Osteoporosis is a common skeletal disease, a systemic metabolic bone disease caused by various factors leading to bone loss and reduction, damage to bone tissue microstructure, increased bone fragility, and decreased bone density, resulting in patients' susceptibility to fractures. Clinical manifestations typically include lower back pain or generalized bone pain, spinal deformities, and fractures. In severe cases, it can compress the heart and lungs, causing circulatory and respiratory system dysfunction. The skeletal development process of zebrafish is remarkably similar to that of other vertebrates; their skeletons are composed of cartilage and bone. Cartilage is formed by chondrocytes, while bone is composed of osteoblasts and osteoclasts.

[0102] Glucocorticoids such as dexamethasone directly damage bone tissue cells and their regulatory factors, reducing bone formation and increasing bone resorption. This leads to impaired bone remodeling, decreased ability of the body to restore bone microstructure, increased bone fragility, and ultimately, fractures and osteonecrosis. Simultaneously, glucocorticoids inhibit the absorption of calcium and phosphorus in the gastrointestinal tract, lowering blood calcium levels and causing secondary increases in parathyroid hormone, thereby inhibiting osteoblast activity, activating osteoclasts, and promoting bone turnover. Furthermore, long-term use of glucocorticoids affects the hypothalamic-pituitary-gonadal axis, reducing the secretion of growth hormone and other hormones, resulting in impaired bone matrix synthesis, inhibiting bone formation, and increasing skeletal muscle protein degradation, thus exacerbating osteoporosis. Bone mineralization matrix deposition is an important indicator of bone formation and is usually evaluated using alizarin red staining. Alizarin red is a dye that can adhere to calcium salts and is widely used to observe and measure bone mineralization. The fluorescence intensity collected by fluorescence microscopy after staining zebrafish bones reflects the calcified bone content, and bone density is closely related to the calcium content in bones. Therefore, inducing zebrafish with glucocorticoids will reduce the number of bones, weaken bone density, and decrease the intensity of bone fluorescence after alizarin red staining.

[0103] Wild-type AB strain zebrafish with a 3-day pf (dpf) count were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well and a volume of 3 mL per well. The zebrafish from different wells were randomly divided into a normal control group (treated with normal fish tank water), a positive control group 1 (treated with alendronate sodium dissolved in fish tank water, final concentration 5 μg / mL), a positive control group 2 (treated with Swisse "Calcium & Vitamin D" tablets, milky white, purchased from Swisse, final concentration 500 μg / mL, dissolved in fish tank water), and a commercial probiotic group (treated with Bifidobacterium lactis Ca360 dissolved in fish tank water, final concentration 10 μg / mL). 8 CFU / mL), commercial probiotic inactivated group (inactivated Bifidobacterium lactis Ca360 dissolved in fish tank water (inactivated at 75℃ for 30 min), final concentration of 10 8 The study group consisted of two groups: a live bacteria group (Hi188 group, with a final concentration of 10 CFU / mL) and an experimental group. The experimental group was further divided into a Hi188 live bacteria group (Hi188 group, with a final concentration of 10 CFU / mL). 3 , 10 4 , 10 5 , 10 6 , 10 7 and 10 8 CFU / mL), Hi188 inactivated bacteria group (Hi188-D group, Hi188 bacterial powder inactivated at 75℃ for 30 min, final concentration of 10). 8 CFU / mL) and Hi188 live bacteria + Ca360 live bacteria group (Hi188 + Ca360, final Hi188 concentration was 5×10⁻⁶ ...). 7 CFU / mL, final Ca360 concentration is 5×10 7 (CFU / mL). Drug administration and model establishment were performed simultaneously: the test samples were administered water-soluble solutions. Except for the normal control group, all experimental groups received 2.00 μM dexamethasone water-soluble solutions to establish a zebrafish osteoporosis model. After treatment at 28℃ for 96 h, samples were collected and stained with 50.0 μg / mL alizarin red for 24 h. Ten zebrafish from each group were randomly selected and photographed under a fluorescence microscope. Data were analyzed and collected using NIS-Elements D3.20 advanced image processing software. The fluorescence intensity of the zebrafish skull was analyzed, and the statistical analysis results of this index were used to evaluate the anti-osteoporosis efficacy of the samples. Statistical results are expressed as mean ± SEM. Statistical analysis was performed using SPSS 27.0 software; p < 0.05 indicated statistical significance.

[0104] Table 2

[0105] Note: Positive control group 1 was given alendronate sodium dissolved in water for fish farming, with a final concentration of 5 μg / mL; positive control group 2 was given Swisse "Calcium & Vitamin D" dissolved in water for fish farming, milky white tablets, purchased from Swisse, with a final concentration of 500 μg / mL; # indicates that compared with positive control group 1, # indicates p<0.05, ## indicates p<0.01, ### indicates p<0.001; This indicates that compared to the Ca360-D group, '&' indicates p < 0.01; '&' indicates group (10) with Hi188. 8 Compared to (CFU / mL), & indicates p<0.05. This indicates that compared to the Hi188+Ca360 group, This indicates that p < 0.05. This indicates that p < 0.01. p<0.001.

[0106] The results are shown in Table 2 and Figure 2 As shown, the skull bone density of zebrafish in the model control group was significantly lower than that in the normal control group. p The result was <0.0001, indicating that the osteoporosis model was successfully established. Both the Hi188 live bacteria group and the Hi188 inactivated bacteria (Hi188-D) group showed varying degrees of increased skull bone mineral density, significantly higher than the model control group (p<0.01). The concentration of Hi188 live bacteria or Hi188 inactivated bacteria was 10... 8 At CFU / mL, the levels were 54.67% and 24.49% higher than the model group, respectively (p<0.0001), indicating that both live and inactivated probiotics Hi188 can significantly improve bone mineral density in osteoporotic zebrafish.

[0107] The probiotic Hi188 exhibits a dose-dependent effect in improving osteoporosis, with increased skull fluorescence intensity as the dose increases. At a dose of 10... 4 Starting from CFU / mL, the levels were significantly higher than in the model group (p<0.001), and there was no significant difference between the two groups and the positive control group given Swisse (Ca+VD) (p>0.05).

[0108] Hi188 live bacteria (10) 8 The skull bone mineral density in the (CFU / mL) group was 54.67% higher than that in the model control group, which was superior to the commercial probiotic (Ca360) group. Hi188 inactivated bacteria (10 8 The skull bone mineral density of the group with CFU / mL was 36.53% higher than that of the model control group, which was superior to that of the commercial probiotic inactivated (Ca360-D) group. This indicates that both live and inactivated Hi188 bacteria are more effective than commercial probiotic Ca360 in improving bone mineral density and treating osteoporosis.

[0109] When Hi188 live bacteria were combined with commercial probiotics (Ca360) (Hi188+Ca360 group), the skull bone mineral density increased by 75.97% compared with the model group, which was significantly higher than that of the Swisse (Ca+VD) positive control group (p<0.05), and was comparable to the effect of alendronate sodium (bone mineral density increased by 73.99%), indicating that the combination of the two was more effective.

[0110] At the same time, and Hi188-D group ( p <0.0001) and Ca360-D group ( p The values ​​of <0.001) were significantly higher than those of the model control group.

[0111] Example 3: Lactobacillus plantarum Hi188 can promote bone health in cynomolgus monkeys.

[0112] 1. Research Methods

[0113] 1.1 Screening of malnourished cynomolgus macaques

[0114] Using the Body Condition Score (BCS), a semi-quantitative indicator for assessing the nutritional status of cynomolgus monkeys, as the initial assessment method, male cynomolgus monkeys with developmental delays and malnutrition were selected for this study. Referring to Xu et al. (2024), the Weight-for-age Z-score (WAZ) was calculated, with a Z-score <0 for age and weight used as the diagnostic criterion for malnourished cynomolgus monkeys.

[0115] The WAZ value is calculated based on weight, body length, and age distribution, using the following formula:

[0116] 1.2 Probiotic gavage intervention

[0117] After a week of acclimatization feeding, malnourished cynomolgus monkeys were randomly divided into a control group and a Hi188 group, with 6 monkeys in each group. Baseline values ​​of age, WAZ value, weight, body length, bone mineral content, and bone mineral density were recorded for all cynomolgus monkeys.

[0118] The Hi188 group was administered 2 mL / animal via gavage with *Lactobacillus plantarum* Hi188 + PBS (total viable count 1×10⁻⁶) 0.5–1.0 h before breakfast daily. 11 The control group received 2 mL / animal of maltodextrin + PBS (without live bacteria) 0.5-1.0 h before breakfast daily. Figure 3As shown, all animals underwent a continuous intervention for 180 days, and bone mineral content (BMC) and bone mineral density (BMD) were measured on days 0, 30, 60, 90, 120, 150, and 180 of the intervention. Dual-energy X-ray absorptiometry (DEXA) was used to measure the BMC and BMD of the cynomolgus monkeys. After the intervention ended on day 180, the cynomolgus monkeys were fasted overnight and anesthetized by intramuscular injection of ketamine hydrochloride (10 mg / kg), followed by deep anesthesia via intravenous injection of sodium pentobarbital (100 mg / kg). After veterinarians assessed the monkeys' respiration and heartbeat and confirmed the absence of neurological reflexes, dissection was performed to measure bone length.

[0119] 1.3 Data Statistics and Analysis

[0120] GraphPad Prism version 10.1.2 was used to analyze statistical data and generate graphs. Experimental results are expressed as mean ± standard error (Mean ± SEM). Differences between groups were tested and analyzed using a mixed-effects model and the Turkey test. , p < 0.05 indicates a statistically significant difference; , p < 0.01 indicates a statistically significant difference; , p < 0.001 indicates that it is statistically highly significant; , p < 0.0001 indicates that it is statistically significant.

[0121] 2. Results Analysis

[0122] 2.1 Baseline Value

[0123] The baseline values ​​for age, WAZ, weight, body length, bone mineral content, and bone mineral density of all cynomolgus monkeys are shown in Table 3. It can be seen that all cynomolgus monkeys used in this study were adult males and were malnourished. There were no significant differences in age, nutritional status (WAZ), weight, body length, bone mineral content, and bone mineral density at enrollment. p >0.05).

[0124] Table 3. Baseline values ​​for all cynomolgus monkeys

[0125] According to Wei Zhumei et al. (Experimental Animals and Comparative Medicine, 2022, 42(5). DOI:10.12300 / j.issn.1674-5817.2022.043), the body mass index (BMD) of normal cynomolgus monkeys increased year by year from 1 to 8 years of age, and then remained at a plateau. The body mass index (BMC) of healthy cynomolgus monkeys aged 5-6 years and 7-8 years were 304.03±17.35g and 382.21±13.29g, respectively; the BMD values ​​were 0.53±0.05 g / cm³, respectively. 2 and 0.57±0.05 g / cm 2 Therefore, the cynomolgus monkey used in this embodiment is a clearly malnourished cynomolgus monkey.

[0126] 2.2 Hi188 significantly increased bone mineral content in cynomolgus monkeys.

[0127] like Figure 4-5 As shown, from day 30 of the intervention, the bone mineral content of the cynomolgus monkeys in the Hi188 group began to increase and showed an increasing trend; from day 60 of the intervention to the end of the intervention, the bone mineral content and the rate of change of bone mineral content in the Hi188 group were significantly higher than those in the control group (multiple time points). p < 0.05). Compared to baseline, on day 60 of the intervention, the mean bone mineral content in the Hi188 group and the control group was 83.39 ± 5.65 g and 66.43 ± 5.28 g, respectively. p <0.05, and the average change rate of bone mineral content in Hi188 was 23.22%, significantly higher than that in the control group ( p <0.05). At the end of the intervention, the average bone mineral content of the cynomolgus monkeys in the Hi188 group was 95.91±10.03g, while that in the control group was 64.30±6.79g, meaning that the Hi188 group increased by 31.47%, while the control group decreased by -2.79%. This indicates that Hi188 can significantly promote bone mineral deposition and form a stable bone mass advantage in the first 3-4 months of the intervention.

[0128] 2.3 Hi188 can significantly increase bone density in cynomolgus monkeys.

[0129] like Figure 6-7 As shown, from day 30 of the intervention, the bone mineral density of the cynomolgus monkeys in the Hi188 group began to increase significantly and showed an increasing trend. From day 60 of the intervention until the end of the intervention, the bone mineral density of the Hi188 group was significantly higher than that of the control group. p <0.05), and from day 150 of the intervention, the rate of change in bone mineral density in the Hi188 group was also significantly higher than that in the control group ( p <0.05). By the end of the intervention, the average bone mineral density of the cynomolgus monkeys in the Hi188 group was 0.238 ± 0.008 g / cm³. 2The control group had a concentration of 0.193 ± 0.009 g / cm³. 2 Specifically, the Hi188 group showed an increase of 12.40%, while the control group showed a decrease of -6.07%. This indicates that Hi188 can significantly promote bone mineral deposition and form a stable bone mass advantage in the first 3-4 months of intervention.

[0130] 2.4 Hi188 can significantly promote tibial growth in cynomolgus monkeys.

[0131] Tibial measurements were performed on cynomolgus monkeys, and the results of the absolute tibial length were obtained. Figure 8 The data showed that the tibia length in the Hi188 group was significantly longer than that in the control group. Analysis of the measured lengths revealed that after 180 days of intervention, the tibia length of the cynomolgus monkeys in the Hi188 group was significantly higher than that in the control group. p <0.05), suggesting that the probiotic Hi188 helps improve malnutrition-induced delayed bone development.

[0132] In summary, *Lactobacillus plantarum* Hi188 can significantly improve malnutrition-induced osteodystrophy, specifically by increasing bone mineral content and bone density levels, and promoting skeletal development in adult cynomolgus monkeys.

[0133] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. The use of *Lactobacillus plantarum* Hi188 or its metagener, or a combination thereof, in increasing bone density during normal bone development for non-therapeutic purposes, or in the preparation of foods intended to increase bone density during normal bone development; wherein the composition comprises: (A) Lactobacillus plantarum Hi188 or its postgenes, and (B) Bifidobacterium lactis Ca360 or its postgenes; The Lactobacillus plantarum Hi188 mentioned therein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30250; The Bifidobacterium lactis Ca360 mentioned therein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32403; The metagene of *Lactobacillus plantarum* Hi188 is an inactivated strain of *Lactobacillus plantarum* Hi188. The metagene of Bifidobacterium lactis Ca360 is an inactivated strain of Bifidobacterium lactis Ca360.

2. The application as described in claim 1, characterized in that, The food products include food ingredients, semi-finished food products, and finished food products.

3. The application as described in claim 1, characterized in that, The food products mentioned include processed food and unprocessed food.

4. The application as described in claim 2 or 3, characterized in that, The food also includes food science acceptable materials.

5. The application as described in claim 4, characterized in that, Food science-acceptable materials include: food ingredients or their processed products, and nutritional additives.

6. The application as described in claim 5, characterized in that, The food ingredients or their processed products include raw milk, whey protein powder, and whey powder, and the nutritional additives include one or more of lactose, dietary fiber, protein, lipids, minerals, vitamins, nucleotides, choline, and taurine.

7. The application as described in claim 5, characterized in that, The nutritional additives include prebiotics.

8. The application as described in claim 4, characterized in that, Food science acceptable materials include ingredients that can be added to food.

9. The application as described in claim 8, characterized in that, The ingredients that may be added to the food include one or more of the following: red dates, hawthorn, goji berries, longan, lily bulbs, poria cocos, and dried tangerine peel.

10. The application as described in claim 4, characterized in that, Food science acceptable materials include: additives.

11. The application as described in claim 10, characterized in that, The excipients include one or more of calcium carbonate, calcium phosphate, sugars, cellulose derivatives, gelatin, vegetable oil, and polyethylene glycol.

12. The application as described in claim 2 or 3, characterized in that, The food products mentioned include general food products and special food products.

13. The application as described in claim 12, characterized in that, The special foods mentioned include infant food, food for special medical purposes, and health food.

14. The application as described in claim 13, characterized in that, The food product in question is infant formula.

15. The application as described in claim 14, characterized in that, The food also includes one or more of the following: raw milk, whey protein powder, whey powder, α-lactalbumin, β-lactalbumin, lactoferrin, milk fat globule membrane protein, lactose, galactose, dietary fiber, calcium, iron, phosphorus, zinc, vitamins and nervonic acid.

16. The application as described in claim 14, characterized in that, The food also includes prebiotics.

17. Use of a composition in the preparation of a medicament for increasing bone mineral density in cases of osteoporosis; wherein the composition comprises: (A) Lactobacillus plantarum Hi188 or its postgenes, and (B) Bifidobacterium lactis Ca360 or its postgenes; The Lactobacillus plantarum Hi188 mentioned therein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30250; The Bifidobacterium lactis Ca360 mentioned therein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32403; The metagene of *Lactobacillus plantarum* Hi188 is an inactivated strain of *Lactobacillus plantarum* Hi188. The metagene of Bifidobacterium lactis Ca360 is an inactivated strain of Bifidobacterium lactis Ca360.

18. The application as described in claim 17, characterized in that, The osteoporosis described includes decreased bone mass, destruction of bone microstructure, increased bone fragility, decreased osteoblast activity, reduced bone formation, increased osteoclast activity, and increased bone resorption.

19. The use of a drug in the preparation of a reagent for increasing bone density in cases of osteoporosis, characterized in that, The drug comprises an effective amount of (A) *Lactobacillus plantarum* Hi188 or its postgeneratrium, and (B) *Bifidobacterium lactis* Ca360 or its postgeneratrium; and a pharmaceutically acceptable carrier; The Lactobacillus plantarum Hi188 mentioned therein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30250; The Bifidobacterium lactis Ca360 mentioned therein is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32403; The metagene of *Lactobacillus plantarum* Hi188 is an inactivated strain of *Lactobacillus plantarum* Hi188. The metagene of Bifidobacterium lactis Ca360 is an inactivated strain of Bifidobacterium lactis Ca360.

20. The application as described in claim 19, characterized in that, The osteoporosis mentioned includes decreased bone mass, destruction of bone microstructure, increased bone fragility, decreased osteoblast activity, decreased bone formation, increased osteoclast activity, and increased bone resorption.

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