Lactobacillus rhamnosus and its postbiotic for maintaining bone health and improving local inflammation of joints and application thereof

Abstract

The present application belongs to the technical field of microbial fermentation, and particularly relates to Lactobacillus rhamnosus for maintaining bone health and improving local inflammation of joints, postbiotic and application thereof. The present application prepares a postbiotic product by fermenting and culturing probiotic NKU ML1-2. The results of examples prove that the postbiotic drug can enhance the antioxidant capacity of chondrocytes and osteoblasts, promote the synthesis of anti-inflammatory factors and cartilage matrix (type II collagen and proteoglycan), has the potential to reduce local oxidative stress and inflammatory response of joints, relieve joint inflammation, and delay cartilage destruction. The postbiotic drug prepared by the present application has high safety and good stability, and can be applied to the preparation of drugs for preventing and / or treating osteoarthritis; the postbiotic drug can also be used to promote bone health, enhance the antioxidant capacity of osteoblasts and / or chondrocytes, promote osteoblast activity and / or promote calcium absorption and fixation, and provides a new solution for joint health management.

Description

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a type of Lactobacillus rhamnosus that maintains bone health and improves local joint inflammation, along with its postbiotics and applications. Background Technology

[0002] Chronic osteoarthritis is a common degenerative change in joints, characterized by progressive destruction of articular cartilage, subchondral bone sclerosis, osteophyte formation, and synovitis. Persistent chronic inflammation in the joint is a core factor driving the occurrence and development of osteoarthritis. Inflammatory factors can promote the production of large amounts of matrix metalloproteinases and proteoglycans by chondrocytes, thereby accelerating the degradation of the extracellular matrix (such as type II collagen and proteoglycans), ultimately leading to cartilage structural damage and loss of function.

[0003] Currently, clinical treatments for osteoarthritis mainly include nonsteroidal anti-inflammatory drugs, intra-articular injections of corticosteroids or hyaluronic acid, etc. Although these methods can relieve symptoms to some extent, they often only treat the symptoms and not the root cause, and long-term use may cause gastrointestinal, cardiovascular and kidney side effects.

[0004] In recent years, the link between gut microbiota and systemic inflammation has received increasing attention, known as the "gut-joint axis" hypothesis. Probiotics, as live microorganisms, have been shown to indirectly influence the immune status of distal organs by regulating gut microbiota balance. However, live probiotics have limitations such as high requirements for storage stability, sensitivity to the gastrointestinal environment, and the potential to carry antibiotic resistance genes. Compared to live probiotics, metabiotics have significant advantages, including higher safety, better stability, longer shelf life, a clear mechanism of action, and ease of standardized production.

[0005] Therefore, there is an urgent need for a probiotic post-biotic powder that can improve the local immune microenvironment of joints, safely and effectively inhibit inflammation, and protect cartilage. Summary of the Invention

[0006] The purpose of this invention is to provide a probiotic postbiotic product. The postbiotic product provided by this invention can enhance the antioxidant capacity of cartilage and osteoblasts, while promoting the synthesis of anti-inflammatory factors and cartilage matrix (type II collagen, proteoglycans). It has the potential to reduce local oxidative stress and inflammatory response in joints, relieve joint inflammation, and delay cartilage destruction.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a strain of Lactobacillus rhamnosus NKU ML1-2, which maintains bone health and improves local joint inflammation. The Latin name is Lactobacillus rhamnosus. The Lactobacillus rhamnosus NKU ML1-2 is deposited at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC No: 66837 and deposit date of August 13, 2025.

[0009] The present invention also provides a metabiotic composition, wherein the metabiotic composition uses the fermentation metabolites of the above-mentioned Lactobacillus rhamnosus as active ingredients.

[0010] Preferably, the composition further includes a pharmaceutically or food-grade carrier.

[0011] Preferably, the dosage form of the composition is powder, granules, capsules, tablets, or oral liquid.

[0012] The present invention also provides a method for preparing the postbiotic composition as described in any of the above claims. The method involves resuscitating the Lactobacillus rhamnosus NKU ML1-2 described above, subculturing it 2-3 times, and then plotting it on MRS solid medium. A single colony is inoculated into MRS liquid medium and cultured in an incubator at 35-38°C for 24 hours. After the culture is completed, the colony is inactivated in an 80°C water bath for 10 minutes and centrifuged at 8000 rpm. The supernatant is collected to prepare lyophilized powder, which is the probiotic postbiotic composition.

[0013] The present invention also provides the use of the above-described post-biotic composition in the preparation of a medicine for promoting bone health, increasing bone density and / or supplementing calcium to protect bones.

[0014] Preferably, the promotion of bone health includes enhancing the antioxidant capacity of osteoblasts and / or chondrocytes, promoting osteoblast activity, and / or promoting calcium absorption and fixation.

[0015] The present invention also provides the use of the above-described post-biotic composition in the preparation of a medicament for the prevention and / or adjunctive treatment of osteoarthritis.

[0016] Preferably, the prevention and / or adjunctive treatment of osteoarthritis includes inhibiting local inflammatory responses in the joint, promoting the synthesis of type II collagen and proteoglycans by chondrocytes, and / or delaying cartilage destruction.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The post-biotic product of this invention is prepared by fermentation culture of Lactobacillus rhamnosus NKU ML1-2. Experiments have shown that this post-biotic product can enhance the antioxidant capacity of cartilage and osteoblasts, while promoting the synthesis of anti-inflammatory factors and cartilage matrix (type II collagen, proteoglycans). It has the potential to reduce local oxidative stress and inflammatory response in joints, relieve joint inflammation, and delay cartilage destruction.

[0019] The post-adrenergic drugs prepared by this invention have high safety and good stability, and can be used to prepare drugs for the prevention and / or treatment of osteoarthritis. The post-adrenergic drugs can also be used to promote bone health, enhance the antioxidant capacity of osteoblasts and / or chondrocytes, promote osteoblast activity and / or promote calcium absorption and fixation, providing a new solution for joint health management. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the Gram staining results for NKU ML1-2.

[0022] Figure 2 This is a schematic diagram of the results of the antibiotic resistance test for NKU ML1-2.

[0023] Figure 3 This is a schematic diagram of the hemolysis test results of NKU ML1-2.

[0024] Figure 4 This is a schematic diagram showing the experimental results of nitrate reductase and amino acid deacidase activity in NKU ML1-2.

[0025] Figure 5 The graph shows the experimental results of acid and alkali resistance of NKU ML1-2.

[0026] Figure 6 The image shows the results of the CCK-8 cell activity assay, where M represents the model group, T represents the intervention group, and D-Gal represents D-galactose.

[0027] Figure 7 This is a schematic diagram of the analysis of type I and type II collagen in mice.

[0028] Figure 8 This is a diagram showing the analysis of Y-frame protein 9 in the mouse sex-determining region.

[0029] Figure 9 This is a graph showing the analysis of osteopontin in mice.

[0030] Figure 10 This is a graph showing the changes in the content of markers of bone formation and osteogenic activity.

[0031] Figure 11 This is a graph showing the analysis of biomarkers for oxidative stress levels.

[0032] Preservation certificate information

[0033] Preservation name: Lacticaseibacillus rhamnosus NKU ML1-2;

[0034] Taxonomical name: Lacticaseibacillus rhamnosus

[0035] Preservation institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC);

[0036] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0037] Accession number: GDMCC No: 66837;

[0038] Deposit date: August 13, 2025. Detailed Implementation

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0041] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0042] Example 1: Preparation of a probiotic postbiotic composition

[0043] The preparation method is as follows:

[0044] Revive the preserved strain NKU ML1-2, subculture it 2-3 times, and then plate it on MRS solid medium. Next, inoculate a single colony onto MRS liquid medium and incubate it at 35-38℃ for 24 hours. After incubation, inactivate the bacteria in an 80℃ water bath for 10 minutes, centrifuge at 8000 rpm, and collect the supernatant to prepare lyophilized powder, which is the probiotic postbiotic composition.

[0045] Example 2: Validation of the strain

[0046] To evaluate the safety of the probiotic NKU ML1-2 selected in Example 1, six experiments were conducted sequentially: Gram staining test, antibiotic resistance test, hemolytic activity test, nitrate reductase activity test, amino acid decarboxylase activity test, and acid and bile salt tolerance test. The results of each experiment corresponded to... Figures 1-5 .

[0047] 2.1 Gram staining experiment

[0048] By observing the cell wall structure of the strain through staining, the strain type can be preliminarily determined. Prepare a clean glass slide, take 1-2 loops of bacterial solution and spread it. After the slide is air-dried, place the dried slide with the smear side facing up and quickly heat-fix it by passing it through an alcohol lamp flame 2-3 times, and then perform staining.

[0049] First, add crystal violet solution to cover the entire smear area and let it stand for 1 minute; then gently rinse with running water to remove excess crystal violet; finally, add iodine solution to cover the smear area and let it stand for 1 minute.

[0050] Rinse off the iodine solution with running water; then proceed with destaining. Hold the slide at an angle and add 95% ethanol using a dropper, allowing the ethanol to flow from the top of the smear across the entire area until the flowing ethanol is almost colorless. Immediately rinse with a gentle stream of water to stop the destaining process. Add safranin counterstain to cover the smear area and let it stand for 30 seconds. Rinse off the counterstain with a gentle stream of water. Finally, observe the results under a microscope. Figure 1 As shown.

[0051] according to Figure 1 The results showed that the colonies on solid culture medium were small, smooth, milky white, with neat and raised edges and a soft texture.

[0052] Under an optical microscope, the bacterial cells showed Gram-positive staining. When cultured in liquid medium, the medium was turbid, with a sterile membrane and bacterial ring on the surface, flocculent precipitate at the bottom, and a brownish-yellow color. This directly confirms that *Lactobacillus rhamnosus* NKU ML1-2 belongs to the genus *Lactobacillus*, subspecies *Rhamnosus*, and is a facultative anaerobic, acid-resistant, non-spore-forming Gram-positive probiotic.

[0053] 2.2 Antibiotic resistance test

[0054] To detect the resistance spectrum of this strain to commonly used clinical antibiotics, a bacterial suspension was first prepared by inoculating a single colony of NKU ML1-2 onto MRS liquid medium. The suspension was then evenly spread onto a solid medium using a spreader. After drying, the medium was sterilized by flaming with alcohol using sterile forceps. After cooling, antibiotic discs were carefully placed on the agar surface and gently pressed down with forceps to ensure complete contact. The medium was then incubated at 37°C for 24 hours. The experimental results were then recorded. Figure 2 As shown.

[0055] The results are as follows Figure 2 As shown, NKU ML1-2 has some resistance to antibiotics, but this inherent resistance is not passed on to other pathogens at the genetic level, so there is no risk of developing drug-resistant bacteria.

[0056] 2.3 Hemolytic activity test

[0057] To detect whether the strain produces hemolysin, Columbia blood agar plates were prepared before the experiment. Colonies of the NKU ML1-2 strain to be tested were then picked up with an inoculation loop and streaked on the surface of the Columbia blood agar plate to obtain single, well-isolated colonies. The plates were then incubated at 37°C for 24 hours, and the hemolytic characteristics of individual colonies were observed. Results... Figure 3 As shown.

[0058] The results are as follows Figure 3 As shown, NKU ML1-2 did not exhibit a hemolytic region when grown on Columbia blood agar plates, and therefore can be classified as γ-hemolysis with no hemolytic activity or no toxicity, while the positive control Staphylococcus aureus ATCC25923 showed β-hemolysis.

[0059] 2.4 Nitrate reductase activity experiment

[0060] To test whether the strain can reduce nitrate to nitrite in the culture medium, nitrate medium, 0.8% p-aminobenzenesulfonic acid (dissolved in 5M acetic acid), and 0.5% α-naphthylamine (dissolved in 5M acetic acid) were first prepared. The nitrate medium contained potassium nitrate (KNO3) as a substrate, and MRS broth + 0.1% KNO3. Two nitrate broth test tubes were used, and NKUML1-2 was inoculated extensively into one tube using an inoculation loop (the experimental tube). The other tube was left uninoculated as a negative control. The tubes were incubated at 37℃ for 24 hours. After incubation, the experimental tube was first observed for growth (whether the liquid became cloudy). Then, 2-3 drops of the test reagent were added to both the experimental tube and the uninoculated control tube to observe the color change and confirm the experimental results. Figure 4 As shown.

[0061] The results are as follows Figure 4As shown, nitrates can be converted into nitrites by nitro reductases. Nitrites can react with α-naphthylamine solution and p-aminobenzylsulfonic acid solution to produce a red diazo compound.

[0062] NKU ML1-2 did not show a color reaction, indicating that it does not contain active nitrate reductase.

[0063] 2.5 Amino acid decarboxylase activity experiment

[0064] To test whether the strain can decompose basic amino acids such as lysine and ornithine to produce biogenic amines, Moeller's decarboxylase broth was first prepared. A set of decarboxylase culture medium test tubes was taken, including: control tubes (containing no amino acids) and experimental tubes containing specific amino acids (lysine and ornithine). A small number of colonies were picked from the plate using an inoculation needle and inoculated to the bottom of the test tube, then streaked up the tube wall. A layer of liquid paraffin, approximately 3-5 mm thick, was slowly added to each inoculated test tube to cover the surface of the culture medium. An anaerobic environment was created to induce the production of decarboxylase. The test tubes were placed in a 37°C incubator and incubated for 24 hours. The color change of the culture medium was observed, such as... Figure 4 As shown.

[0065] The results are as follows Figure 4 As shown, bacteria with specific amino acid decarboxylases can ferment glucose to produce acid (causing the culture medium to turn from purple to yellow). Under acidic and anaerobic conditions, the decarboxylase system is activated, removing the carboxyl groups from the amino acids in the culture medium to generate highly alkaline amines. The accumulation of these amines neutralizes the acid produced earlier, causing the pH of the culture medium to rise again, thus turning it back to purple.

[0066] NKU ML1-2 did not show a color reaction in the amino acid decarboxylase activity test, therefore it does not contain active amino acid decarboxylase.

[0067] 2.6 Acid and bile salt resistance tests

[0068] The concentration of bile salts in the gastrointestinal tract varies from 0.03% to 0.3%. This invention selects bile salt concentrations of 0.05% and 0.1% to evaluate the bile salt tolerance of strain NKU ML1-2. First, MRS broth was prepared, along with 1M HCl and bile salts. For the acid tolerance test, the pH of the culture medium was adjusted to 3 and 4 using HCl. NKU ML1-2 colonies were then inoculated and cultured at 37°C. Culture samples were taken at 0 and 3 hours for dilution, spread, and counting. For the bile salt tolerance test, 0.05% and 0.1% bile salts were added according to the volume of the culture medium, respectively. NKU ML1-2 colonies were inoculated and cultured at 37°C. Culture samples were taken at 0 and 3 hours for dilution, spread, and counting. The experimental results were then analyzed as follows: Figure 5 As shown.

[0069] The results are as follows Figure 5 As shown, compared with 0 h, the number of viable bacteria in the strain treated with 0.05% bile salt for 3 h decreased by two orders of magnitude; compared with 0 h, the number of viable bacteria in the strain treated with 0.1% bile salt for 3 h also decreased by two orders of magnitude.

[0070] NKU ML1-2 needs to pass through the acidic environment of the stomach and colonize the intestines after oral administration to exert its efficacy. When NKU ML1-2 is treated at pH 3 and pH 4, the number of viable bacteria in strains treated at pH 3 and pH 4 for 3 hours decreased by two orders of magnitude compared to 0 hours.

[0071] The above experimental results show that the test strains can grow in MRS medium containing 0.05% and 0.1% bile salts, as well as in MRS medium at pH 3 and pH 4, with little difference in the number of viable cells.

[0072] This demonstrates moderate tolerance to bile salts and acids, and the strain can survive in a simulated gastrointestinal environment.

[0073] Example 3 CCK-8 Viability Detection

[0074] The metagenic composition prepared in Example 1 of this invention was selected, and osteoblasts MC3T3-E1 and chondrocytes ATDC-5 were selected as research subjects, and two cell-specific culture media were used.

[0075] Pathological model creation:

[0076] Inflammation model: Cells were treated with IL-6 (interleukin-6, a typical inflammatory factor) to simulate the local inflammatory microenvironment of the joint and induce inflammation-related pathological changes in the cells;

[0077] Aging Model: By intervening in cells with D-galactose to disrupt normal cellular metabolic processes, a cellular aging model is constructed to simulate the functional decline of the skeletal-joint system with age.

[0078] Three experimental groups were set up: the blank group, the model group, and the post-genetic intervention group.

[0079] During the experiment, cell supernatants were collected from each group, and core indicators related to bone health and joint inflammation were detected and analyzed.

[0080] 3.1 Cell viability and toxicity indicators

[0081] To evaluate the effect of NKU ML1-2 postbiotic on the viability of osteoblasts MC3T3-E1 and chondrocytes ATDC-5, CCK-8 viability was measured.

[0082] The results are as follows Figure 6 As shown, compared with the model group, the cell viability of both cell types showed an increasing trend after intervention with NKU ML1-2 post-biotics.

[0083] The cell viability of ATDC-5 chondrocytes in the IL-6 experiment, ATDC-5 chondrocytes in the D-galactose experiment, MC3T3-E1 osteocytes in the IL-6 experiment, and MC3T3-E1 osteocytes in the D-galactose experiment all showed an increasing trend to some extent under the intervention of post-genetic intervention.

[0084] In conclusion, the CCK-8 assay results indicate that supplementing cell culture with NKU ML1-2 postbiotics can, to some extent, promote the viability of osteoblasts MC3T3-E1 and chondrocytes ATDC-5. This provides preliminary experimental evidence that probiotic postbiotics can maintain bone health and improve local joint inflammation.

[0085] 3.2 Osteogenic Function Related Indicators

[0086] To investigate the effect of NKU ML1-2 postbiotic on improving cartilage synthesis and metabolic balance, this invention used mouse type I collagen (col-I), mouse type II collagen α1 (COL2A1), mouse sex-determining region Y-frame protein 9 (SOX-9), and mouse osteopontin (OPN) as metabolic biomarkers related to cartilage health. A control group (control), a model group (M), and an intervention group (T) were set up. By comparing the differences in the levels of the above biomarkers in the model group, intervention group, and control group, the patterns of their changes were analyzed, thereby clarifying whether NKU ML1-2 postbiotic has an improving effect on cartilage synthesis and metabolic balance.

[0087] The results are as follows Figure 7 As shown, compared with the control group, the content of col-I in the supernatant of the two cell cultures treated with IL-6 and D-galactose both showed an increasing trend, while the content of COL2A1 showed a decreasing trend, indicating that there was pathological repair or fibrosis of cartilage in the model group.

[0088] In the model group, the content of col-I decreased to some extent after intervention with NKU ML1-2 postbiotic, while the content of COL2A1 increased. This suggests that NKU ML1-2 postbiotic may alleviate joint inflammation, inhibit the dedifferentiation process of chondrocytes, protect and promote chondrocytes to maintain their normal phenotype of COL2A1, thereby delaying or preventing fibrotic lesions of cartilage.

[0089] like Figure 8As shown, mouse sex-determining region Y-box protein 9 (SOX-9) is a key transcription factor for chondrogenesis and maintenance. SOX-9 is continuously expressed in mature articular cartilage, which is crucial for maintaining the stable and mature state of chondrocytes and, to some extent, inhibits chondrocyte differentiation or hypertrophy. During the experiment, the SOX-9 level was significantly higher in the intervention group compared to the model group. Increased SOX-9 levels typically lead to increased synthesis of type II collagen, promoting stable maturation of chondrocytes.

[0090] like Figure 9 As shown, mouse osteopontin (OPN) is a multifunctional glycoprotein that is an active participant in bone metabolism and a key regulator of inflammatory and immune responses. High expression of OPN in the synovium is a significant factor contributing to synovitis, manifested as joint swelling, pain, and stiffness. OPN attracts and activates inflammatory cells, stimulating chondrocytes to produce large amounts of matrix metalloproteinases and proteoglycans. These enzymes degrade the core components of cartilage—type II collagen and proteoglycans.

[0091] Compared with the control group, the OPN content in the model group showed an increasing trend, while the OPN content in the intervention group was lower than that in the model group. This indicates that the NKU ML1-2 post-genetic inhibitor directly or indirectly inhibits the expression of OPN in the joint, which may ultimately disrupt the OPN-driven inflammatory circuit that leads to cartilage destruction, creating a more temperate microenvironment for cartilage.

[0092] NKU ML1-2 post-biotic intervention significantly regulates core signaling pathways in osteogenic and cartilage metabolism, with effects such as... Figure 10 As shown, after metabiotic intervention, the expression level of Runt-related transcription factor 2 in mice was downregulated, indicating that metabiotics effectively inhibited a key driver of pathological hypertrophic differentiation in articular cartilage. Simultaneously, the activity of mouse alkaline phosphatase and the expression level of mouse osteocalcin both showed a synergistic increase. This result indicates that metabiotics, while inhibiting abnormal differentiation of articular cartilage, can maintain or even enhance normal osteogenic activity in the skeletal system, demonstrating its unique advantage in improving the pathological environment of osteoarthritis through multi-target coordinated action.

[0093] From the perspective of osteogenic activity, such as Figure 10 As shown, metatrophic agents effectively promoted osteoblast function, as evidenced by a significant increase in mouse alkaline phosphatase (ALP) activity and osteocalcin (BGP) expression levels, indicating enhanced bone formation. Metatrophic intervention significantly reduced the expression of Runt-related transcription factor 2 in mouse cartilage tissue, demonstrating its successful inhibition of the pathological hypertrophic differentiation program leading to cartilage degeneration.

[0094] In summary, NKU ML1-2 postbiotics exhibit a bidirectional regulatory effect: they both ensure the normal functioning of physiological osteogenic processes and inhibit pathological cartilage metabolism within the joint, providing key molecular biological evidence for improving the progression of osteoarthritis.

[0095] Example 4

[0096] To clarify the redox state of cells, including antioxidant defense capacity and the degree of oxidative damage, this invention detected and analyzed related indicators such as total superoxide dismutase (SOD), reduced glutathione (GSH), and malondialdehyde (MDA). Specific results are as follows: Figure 11 As shown.

[0097] The results showed that, compared with the model group, the total superoxide dismutase (SOD) activity and reduced glutathione (GSH) content in the supernatants of both cell cultures were increased to some extent in the intervention group, while the level of malondialdehyde (MDA), the end product of lipid peroxidation, was decreased.

[0098] These results demonstrate that post-biotics not only enhance the body's enzyme- and non-enzyme-based antioxidant defense systems but also effectively reduce free radical-mediated lipid peroxidation damage. Post-biotics can exert their beneficial effects by alleviating oxidative stress.

[0099] In conclusion, NKU ML1-2 postbiotics may reduce local oxidative stress and inflammation in joints by enhancing the body's antioxidant capacity, ultimately protecting chondrocytes and promoting the healthy synthesis of cartilage matrix, thereby maintaining bone health and improving local joint inflammation.

[0100] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus NKU ML1-2, characterized in that, The Lactobacillus rhamnosus NKU ML1-2 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66837, and the deposit date is August 13, 2025.

2. A post-genetic composition, characterized in that, The postbiotic composition uses the fermentation metabolites of Lactobacillus rhamnosus NKU ML1-2 as the active ingredient.

3. The post-genetic composition according to claim 2, characterized in that, The composition also includes a pharmaceutically or food-grade carrier.

4. The post-genetic composition according to claim 2, characterized in that, The composition is available in the form of powder, granules, capsules, tablets, or oral liquid.

5. A method for preparing the post-biotic composition according to any one of claims 2-4, characterized in that, The method involves reviving the Lactobacillus rhamnosus NKU ML1-2 described in claim 1, subculturing it 2-3 times, and then plotting it on MRS solid medium. A single colony is inoculated into MRS liquid medium and cultured in an incubator at 35-38℃ for 24 hours. After the culture is completed, the colony is inactivated in an 80℃ water bath for 10 minutes and centrifuged at 8000 rpm. The supernatant is collected to prepare lyophilized powder, which is the probiotic postbiotic composition.

6. The use of the post-biotic composition according to any one of claims 2-4 in the preparation of a food for maintaining bone and joint health.

7. The application according to claim 6, characterized in that, Maintaining bone and joint health includes alleviating chondrocyte damage, increasing osteoblast activity, and promoting bone metabolism and bone formation.

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