Lactobacillus plantarum and application thereof in promoting bone health and regulating intestinal flora

Abstract

The invention discloses lactobacillus plantarum and application thereof in promoting bone health and regulating intestinal flora. The name of the lactobacillus plantarum is lactobacillus plantarum GXS77, the preservation number of the lactobacillus plantarum is CGMCC No.35899, and the lactobacillus plantarum is preserved in China General Microbiological Culture Collection Center (CGMCC) in No. 3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing in 2025. The yield of acetic acid and the yield of butyric acid of the lactobacillus plantarum GXS77 obtained through screening are increased by 18% and 16% respectively compared with the yield of acetic acid and the yield of butyric acid of common lactobacillus plantarum, the peak calcium absorption capacity of Caco-2 cells can be increased by 12.3%, osteoblasts are differentiated, and then bone health is promoted; meanwhile, the lactobacillus plantarum GXS77 has good acid resistance, cholate resistance and a good cell adhesion effect. Therefore, the strain can be used for developing fermented food, health-care products and medicines with the effects of promoting bone health, regulating intestinal flora and the like.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering, and specifically relates to a strain of Lactobacillus plantarum and its application in promoting bone health and regulating gut microbiota. Background Technology

[0002] Related studies indicate that bone loss, a precursor to osteoporosis, has a global prevalence of approximately 40%, while the global prevalence of osteoporosis itself has reached nearly 20%. Although fracture rates have stabilized in some European countries, the prevalence continues to rise in regions like North America due to factors such as population aging and inadequate treatment. Fracture cases are rising sharply in Asia and Latin America, and it is projected that by 2050, more than half of all hip fractures worldwide will occur in Asia. Furthermore, women have a significantly higher risk of developing osteoporosis than men. In individuals over 50, the diagnosis rate for women is approximately 4.5 times that of men. Globally, about one-third of women will experience an osteoporotic fracture in their lifetime, compared to one-fifth for men (Wang F., Wei W., Liu P.-J., Effects of probiotic supplementation on bone health in postmenopausal women: a systematic review and meta-analysis , Frontiers in Endocrinology (2024, 15:1487998). Clearly, with the enormous medical costs of hundreds of billions of RMB each year, osteoporosis and the resulting fragility fractures have become one of the most common metabolic bone diseases and a global public health issue that urgently needs to be addressed.

[0003] Osteoporosis is a systemic bone disease characterized by low bone mass and destruction of bone microstructure due to an imbalance in bone metabolism. The internationally accepted diagnostic standard is bone mineral density (BMD) measured using dual-energy X-ray absorptiometry (DXA). A T-score of -2.5 or lower is sufficient for diagnosis. The dynamic balance between bone resorption (dominated by osteoclasts) and bone formation (dominated by osteoblasts) in the body is disrupted, leading to a situation where the rate of bone resorption exceeds the rate of bone formation, resulting in porous and fragile bones. Osteoporosis often develops without obvious early symptoms, earning it the nickname "silent killer." Its most serious consequence is fragility fractures, typically occurring in the vertebrae, hip, and wrist. Fractures are often accompanied by severe pain, decreased height, and kyphosis (hunchback), and can induce complications such as prolonged bed rest, lung infections, and thrombosis. Severe cases (especially after hip fractures) have extremely high mortality and disability rates, causing immense suffering, severely impacting the ability to live independently, and even significantly shortening life expectancy.

[0004] Osteoporosis is a long-term degenerative bone disease, its development influenced by a variety of factors including genetics, nutrition, lifestyle, and hormone levels. It is characterized by insidious onset and complex etiologies. "Prevention first, combined with treatment, and comprehensive management" is the clinical consensus in the field of bone health. Currently, clinical interventions mainly involve long-term use of drugs that inhibit bone resorption or promote bone formation to increase bone mass and reduce fracture risk. However, long-term use of some existing drugs may be accompanied by potential side effects and safety risks. Therefore, developing novel functional intervention programs with high safety, few side effects, and suitability for long-term bone health maintenance, such as using probiotics to regulate bone metabolism, has become a current research hotspot. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a strain of *Lactobacillus plantarum*.

[0006] A second objective of this invention is to provide a series of intracellular enzymes of Lactobacillus plantarum.

[0007] A third objective of this invention is to provide a fermented product containing Lactobacillus plantarum.

[0008] The fourth objective of this invention is to provide the application of the aforementioned *Lactobacillus plantarum*, *Lactobacillus plantarum* intracellular enzymes, and fermented products containing *Lactobacillus plantarum* in products that promote calcium absorption, osteoblast differentiation, improve osteoporosis, promote bone health, improve intestinal microbiota structure, inhibit the growth and reproduction of pathogenic bacteria, maintain the balance of normal intestinal microbiota, and increase the content of acetic acid and / or butyric acid in the intestine.

[0009] The objective of this invention is achieved through the following technical solution: This invention provides a strain of *Lactobacillus plantarum*, named *Lactobacillus plantarum* GXS77 (… Lactiplantibacillusplantarum GXS77, with accession number CGMCC No. 35899, was deposited on September 12, 2025, at the China General Microbiological Culture Collection Center (CMGCC), No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0010] Furthermore, the 16S rDNA gene sequence of Lactobacillus plantarum GXS77 is shown in SEQ ID NO.1.

[0011] Furthermore, Lactobacillus plantarum GXS77 exhibits relatively high yields of acetic acid and butyric acid, representing increases of 18% and 16% respectively compared to typical Lactobacillus plantarum.

[0012] Furthermore, Lactobacillus plantarum GXS77 exhibits good tolerance, high intestinal adhesion, and antibacterial activity, which can inhibit the growth and reproduction of pathogenic bacteria and help maintain the balance of normal intestinal microbiota.

[0013] Furthermore, the pathogenic bacteria with antibacterial activity include at least one of Escherichia coli, Staphylococcus aureus, Listeria, and Salmonella.

[0014] Furthermore, the method for culturing Lactobacillus plantarum GXS77 involves the following steps: inoculating Lactobacillus plantarum GXS77 into a culture medium and culturing it at 28℃~37℃.

[0015] Furthermore, the culture medium contains 10.0-12.0 g of casein digest or peptone per liter, 8.0-12.0 g of beef meal, 3.5-4.5 g of yeast extract, 2.0-2.2 g of diammonium hydrogen citrate or triammonium hydrogen citrate, 5.0-5.5 g of sodium acetate, 0.1-0.3 g of magnesium sulfate, 0.03-0.05 g of manganese sulfate, 2.0-2.3 g of dipotassium hydrogen phosphate, 20.0-22.0 g of glucose, and 1.0-1.2 g of Tween-80, with a pH of 3.0-6.8. The incubation time is 14-36 hours.

[0016] Furthermore, the biological characteristics of *Lactobacillus plantarum* GXS77 are as follows: Lactiplantibacillusplantarum It has the following biological characteristics: (1) Growth characteristics: After culturing in MRS liquid medium for 72 h, the growth of the colony conformed to the S-shaped curve. The strain began to enter the logarithmic growth phase and produced a large amount of acid after 4 h. It then grew rapidly until about 22 h and entered the stationary phase. The pH value also tended to level off and finally stabilized at about 3.7.

[0017] (2) Bacterial characteristics: After being cultured at 37°C for 48 h on MRS agar medium, the strain grew well. The colonies were milky white and round, with a diameter of 0.5–1.0 mm. The colonies had raised edges, were opaque, and had smooth surfaces. The strain was Gram-positive. Under a microscope, the cells were mostly short rods and did not form spores.

[0018] This invention also provides intracellular enzymes from *Lactobacillus plantarum* that participate in the synthesis of short-chain fatty acids such as acetic acid and butyric acid, including acetylkinase ack, phosphorylacetyltransferase pta, phosphoryltransferase gene ptb, 4-aminobutyrate aminotransferase puuE, and glutamate decarboxylase gadA.

[0019] This invention also provides the application of the above-mentioned Lactobacillus plantarum in promoting bone health.

[0020] Furthermore, Lactobacillus plantarum GXS77 is used in the preparation of products that promote calcium absorption, facilitate osteoblast differentiation, and alleviate osteoporosis. Furthermore, the products include microbial preparations, health foods, or pharmaceuticals.

[0021] Furthermore, *Lactobacillus plantarum* GXS77 promotes calcium absorption by secreting acetic acid and butyric acid. After secreting acetic acid and butyric acid in the intestine, *Lactobacillus plantarum* GXS77 enhances calcium absorption through two main pathways: First, acetic acid and butyric acid lower the local pH of the intestinal lumen, converting some insoluble calcium salts into free calcium ions, thereby increasing the absorbable proportion. Second, short-chain fatty acids can regulate calcium transport-related proteins in the intestinal epithelium, including promoting the expression of claudin-2, claudin-12, and the calcium-binding protein Calbindin-D9k, making calcium transport via the paracellular and transmembrane pathways more efficient. Butyric acid also maintains the state of epithelial cells, improves villous absorption capacity, and further promotes calcium absorption; by promoting calcium absorption, it promotes osteoblast differentiation; and by promoting osteoblast differentiation, it promotes bone health.

[0022] This invention also provides the application of the above-mentioned Lactobacillus plantarum in regulating intestinal flora.

[0023] The present invention also provides a fermented product, which is produced by fermentation using the above-mentioned Lactobacillus plantarum.

[0024] The present invention also provides the application of at least one of the above-mentioned Lactobacillus plantarum, intracellular enzymes and fermented products in the preparation of products that increase the content of acetic acid and / or butyric acid in the intestine, promote calcium absorption, promote osteoblast differentiation, improve osteoporosis, promote bone health, improve the structure of intestinal microbiota, inhibit the growth and reproduction of pathogenic bacteria, and maintain the balance of normal intestinal microbiota.

[0025] The present invention has the following advantages and effects compared with the prior art: The *Lactobacillus plantarum* in this invention was first screened from fermented kimchi. It was further screened through tests of acid resistance, bile salt resistance, gastrointestinal environment resistance, and intestinal adhesion ability to obtain probiotics. This strain has probiotic characteristics such as resistance to the gastrointestinal environment and high adhesion. It has broad application prospects in the development and utilization of probiotic products that promote calcium absorption and osteoblast differentiation. Long-term use can improve the intestinal microbiota structure and prevent and alleviate osteoporosis. Attached Figure Description

[0026] Figure 1 It is Lactobacillus plantarum Lactiplantibacillusplantarum ) Electrophoretic identification image of 16S rDNA of GXS77.

[0027] Figure 2This is a growth curve of Lactobacillus plantarum GXS77.

[0028] Figure 3 This is a graph showing the acid-producing capacity of Lactobacillus plantarum GXS77.

[0029] Figure 4 This is a graph showing the acid resistance of Lactobacillus plantarum GXS77.

[0030] Figure 5 This is a diagram showing the cell adhesion results of Lactobacillus plantarum GXS77.

[0031] Figure 6 This is a graph showing the effect of Lactobacillus plantarum GXS77 on promoting calcium absorption.

[0032] Figure 7 This is a graph showing the survival rate of Lactobacillus plantarum GXS77 under different concentrations of bile salts.

[0033] Figure 8 This is a diagram showing the adhesion of Lactobacillus plantarum GXS77 to Caco-2 cells. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0035] Example 1: Screening for lactic acid bacteria that alleviate osteoporosis (I) Isolation and purification of lactic acid bacteria Take 0.1 mL of pickled vegetable juice (from pickled vegetables sold in local markets in Hubei province) and mix thoroughly with 0.9 mL of sterile saline (0.9% g / mL NaCl solution) to dilute tenfold. After further appropriate decimal dilutions of the pickled vegetable juice using the same method, take 0.1 mL of each diluted solution (10⁻¹⁰). 2 10 3 10 4 10 5 10 6 and 10 7The bacterial culture was poured onto an empty agar plate, and MRS agar medium containing 0.1% g / ml bromocresol purple was added (the formula is as follows: per liter, it contains 10.0g casein digest, 11.5g beef extract, 3.5g yeast extract, 2.1g triammonium citrate, 5.4g sodium acetate, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate tetrahydrate, 2.0g dipotassium hydrogen phosphate, 21.7g glucose, 4g agar powder, 10mg bromocresol purple, 1.12g Tween-80, pH 5.7±0.2). The medium was moved clockwise on a clean bench to mix thoroughly. After the MRS agar medium solidified, it was incubated at 37℃ for 48h.

[0036] During their metabolism, lactic acid bacteria produce acidic organic matter such as lactic acid. The bromocresol purple indicator on the agar plate changes color from purple to yellow as the lactic acid bacteria produce these organic acids. Single colonies surrounded by a yellow ring are picked, streaked onto fresh MRS solid medium for dilution, and incubated overnight at 37°C with the plate inverted. This process is repeated for secondary purification of the single colonies. Single colonies are then inoculated into fresh MRS liquid medium, sealed with sealing film, and incubated at 37°C and 220 rpm for 24 hours. The enriched and purified lactic acid bacteria are then collected.

[0037] Add glycerol to a final concentration of 25% (v / v) (prepare a glycerol solution with physiological saline at a volume ratio of 1:1 to a final concentration of 50% (v / v). When using, mix 50% (v / v) glycerol with bacterial solution at a volume ratio of 1:1 to achieve a final glycerol concentration of 25% (v / v)). After thorough mixing, freeze at -80°C for testing.

[0038] (II) Evaluation of the effect of lactic acid bacteria on promoting calcium absorption 1. Screening and identification of the high butyrate production capacity of Lactobacillus plantarum GXS77 To screen for high-butyric acid-producing *Lactobacillus plantarum*, this invention establishes a precise quantitative method based on the DMAB colorimetric assay. First, a series of butyric acid standard working solutions (0.0 mM to 1.0 mM) were precisely prepared and reacted with 1% (g / ml) DMAB solution in a 96-well plate for 30 minutes. The absorbance was measured at 425 nm, successfully constructing a linear butyric acid standard curve ($A = kC + b$). Subsequently, the candidate strain was anaerobically cultured in MRS medium at 37°C for 24 hours. The fermentation supernatant was then collected, and the colorimetric reaction and absorbance were measured under the same conditions. If the absorbance value of the sample was too high, appropriate dilution was performed to ensure the reading fell within the linear range. Finally, the mean effective absorbance of the sample was substituted into the regression equation to calculate the concentration, and multiplied by the dilution factor, yielding a final butyric acid content of 0.14 g / L in the fermentation broth. Based on these quantitative results, the butyric acid yield of Lactobacillus plantarum was increased by 16% compared to the general yield of 0.12 g / L. The strain with the highest butyric acid yield, Lactobacillus plantarum GXS77, was successfully screened and identified, laying the foundation for subsequent functional verification.

[0039] 2. Verification and evaluation of the acetic acid production capacity of Lactobacillus plantarum GXS77 To further confirm the short-chain fatty acid production characteristics of strain GXS77, this invention employs a method based on ferric ions (Fe3+). 3+ The acetic acid production capacity of *Lactobacillus plantarum* GXS77 was quantitatively evaluated using a complexochromic method. This method constructed a standard curve of sodium acetate from 0 to 100 mM and detected the reddish-brown complex formed by acetic acid and FeCl3 at a wavelength of 370 nm. For the strain samples, the fermentation broth underwent rigorous pretreatment including high-speed centrifugation, protein precipitation with 0.2 M hydrochloric acid, and a 5-fold dilution. The broth was then reacted with the chromogenic reagent and stop solution, and the absorbance was measured. The net absorbance value of the sample was substituted into the linear regression equation and multiplied by the overall dilution factor (10-fold) to accurately calculate the acetic acid concentration in the original fermentation broth as 10.95 g / L, representing an 18% increase compared to the acetic acid production of 9.29 g / L in typical *Lactobacillus plantarum* strains. This result, combined with butyrate screening results, further confirms that *Lactobacillus plantarum* GXS77 possesses excellent short-chain fatty acid secretion capacity and is an ideal functional candidate strain.

[0040] 3. Verification of the effect of Lactobacillus plantarum GXS77 in significantly promoting calcium absorption in Caco-2 cells To verify the promoting effect of *Lactobacillus plantarum* GXS77 on intestinal calcium absorption, this invention established a calcium influx model of Caco-2 cells based on the Fluo-4 AM fluorescent probe. A monolayer of differentiated mature Caco-2 cells was then compared with 1×10⁻⁶ cells. 8After co-incubating with a CFU / mL GXS77 bacterial suspension for 24 hours, the cells were loaded with a Fluo-4 AM probe. The fluorescence intensity dynamics caused by changes in intracellular calcium ion concentration after the addition of CaCl2 were monitored in real time using a multifunctional microplate reader (excitation wavelength 488 nm, emission wavelength 516 nm). The results showed that, compared with the untreated control group, the experimental group treated with *Lactobacillus plantarum* GXS77 exhibited significantly enhanced calcium uptake. Data showed that the peak relative fluorescence intensity of the control group was 1.63, while the peak intensity of the GXS77-treated group increased to 1.83, an increase of 12.3%. t-test statistical analysis showed that the difference in peak intensity between the two groups was highly statistically significant (p < 0.01, n = 9). This result strongly demonstrates that the *Lactobacillus plantarum* GXS77 screened in this invention can effectively promote calcium uptake by intestinal epithelial cells. Real-time kinetic curves of intracellular calcium uptake were obtained. The results are as follows... Figure 6 As shown.

[0041] Example 2: Identification and Characteristics of Lactic Acid Bacteria (I) Molecular biological identification of Lactobacillus plantarum GXS77, including the following steps: (1) 16S rDNA sequencing and amplification: GXS77 colonies were picked from MRS solid plates, 20 μL of lysis buffer was added, and the plates were incubated at 100°C for 15 min, then centrifuged at 12000 r / min for 5 min. 1 μL of the supernatant was used as the DNA template. PCR amplification was performed using universal primers 27F / 1492R. The components of the PCR system are shown in Table 1. Table 1

[0042] The primer sequences are as follows: 27F sequence: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.2); 1492R sequence: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO. 3).

[0043] The PCR procedure is shown in Table 2: Table 2

[0044] (2) Identification of PCR amplification products: This invention uses agarose gel electrophoresis to detect DNA molecular weight. Specifically, 0.3 g of agar powder is mixed with 30 mL of 1×Bis-Tris buffer solution, heated in a microwave oven until the agar powder is completely dissolved, 3 μL of Goldview nucleic acid dye is added, and the mixture is poured into a mold for shaping. The agarose gel is placed in a Bio-Rad horizontal electrophoresis apparatus, and 3 μL of marker (100–2000 bp) and PCR amplification product are added to each well sequentially. After running at 125 V for 14 min, the DNA bands are observed using a Bio-Rad gel imaging system. The results are as follows: Figure 1 Ultimately, it was determined that the target fragment was 1504 bp in length.

[0045] (3) 16S rDNA sequencing: The validated PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained 16S rDNA sequences were BLASTed on NCBI and compared with the sequencing information of known strains for homology to identify the strains. Multiple sequence matching analysis was performed on the sequences of the strains screened in this invention and the sequences of reference strains retrieved from the NCBI database using MEGA 5.0. The strains screened in this invention were identified as... Lactiplantibacillus (Chinese name: Lactobacillus), after three generations of whole-genome analysis and GCF_014131735.1 Lactiplantibacillusplantarum The ANI value is 99.09, which is presumed to be... Lactiplantibacillusplantarum (Chinese name: *Lactobacillus plantarum*). Ultimately, this invention named this strain *Lactobacillus plantarum*. Lactiplantibacillusplantarum GXS77.

[0046] The 16S rDNA gene sequence of Lactobacillus plantarum GXS77 is as follows (SEQ ID NO.1): (4) Whole genome sequencing: The extracted whole genome was sent to Shanghai Paisenno Biotechnology Co., Ltd. for combined second-generation and third-generation sequencing. The obtained sequences were compared with the KEGG database. It was found that Lactobacillus plantarum GXS77 has intracellular enzymes involved in the synthesis of short-chain fatty acids such as acetic acid and butyric acid, as described in Table 3.

[0047] Table 3

[0048] (II) Observation of colony morphology characteristics of Lactobacillus plantarum GXS77 Lactobacillus plantarum GXS is a Gram-positive, non-spore-forming bacterium. Its optimal growth temperature is 37℃, and its optimal growth pH is 6-7. It enters the stationary phase after 12 hours of shake-incubation. On MRS agar plates, the colonies are milky white, round, with neat edges, opaque, and smooth, with a diameter of 0.5–1.0 mm.

[0049] (III) Growth curve and acid production curve After the bacteria were activated and expanded, they were inoculated into MRS liquid medium at an inoculum of 2% (v / v) and incubated at 37°C. Samples were taken every 2 hours to measure the absorbance at 600 nm and the pH value of the culture medium. The results were repeated 3 times, and then growth curves and acid production curves were plotted.

[0050] Growth curves as follows Figure 2 As shown: the growth of the bacteria follows an S-shaped curve, entering the logarithmic growth phase after 4 hours, followed by rapid growth until reaching the stationary phase around 18-22 hours. Acid production capacity is as follows... Figure 3 As shown: after 4-6 hours, a large amount of acid is produced, and the acid value drops rapidly to 3.5-4.0. Then the acid production curve tends to flatten out and finally stabilizes at around 3.7.

[0051] Based on the colony morphology and molecular biological identification results of *Lactobacillus plantarum* GXS77, *Lactobacillus plantarum* (GXS77) was classified as a bacterial strain. Lactiplantibacillusplantarum GXS77 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35899 and deposit date of September 12, 2025.

[0052] Example 3: Probiotic Properties of Lactobacillus plantarum GXS77 (a) Acid resistance test The pH of MRS liquid medium was adjusted to analyze the acid tolerance of *Lactobacillus plantarum* GXS77. The pH of the MRS liquid medium was adjusted to 3.0, 4.0, and 5.0 using 0.1M HCl solution, with the MRS liquid medium at its natural pH serving as a control. Each experiment was repeated three times. The bacterial suspension was inoculated at a 2% (v / v) inoculation rate into MRS culture media at different pH values ​​and incubated at 37°C. Samples were taken after 24 hours, and the OD values ​​in the different media were measured, using uninoculated MRS liquid medium as a reference. 600 The acid tolerance of the strain was calculated by performing three parallel samples and taking the average value. Where: the acid tolerance survival rate of the strain = (OD value of the experimental group) / ... 600 nm OD of the natural pH control group 600 nm (Value) × 100%.

[0053] Survival rates after 2 hours of treatment under different pH conditions are as follows: Figure 4 As shown, the acid-resistant survival rate decreases continuously with decreasing pH value, and the strain can still grow at a pH value of 3.0.

[0054] Table 4 shows the survival rate of Lactobacillus plantarum GXS77 in simulated gastrointestinal environments. The survival rate was 83.18 ± 0.42 in simulated gastric fluid at pH 3.0 and 60.26 ± 0.57 in simulated small intestinal fluid at pH 6.8.

[0055] Table 4. Survival results of Lactobacillus plantarum GXS77 in simulated gastrointestinal environment

[0056] (ii) Bile salt tolerance test The tolerance of *Lactobacillus plantarum* GXS77 to bile salts was determined by analyzing its growth in a bile-containing culture medium. (MRS) Porcine bile salts were added to THIO liquid medium (MRS liquid medium with 0.2 g / 100 mL sodium thioglycolate) at concentrations of 0.03 g / 100 mL, 0.06 g / 100 mL, 0.1 g / 100 mL, and 0.3 g / 100 mL, and the pH was adjusted to 7.2. MRS liquid medium without bile salts was used as a control.

[0057] The bacterial suspension was added at an inoculum rate of 2% (v / v) to MRS liquid medium containing different bile salt concentrations. Each experiment was repeated three times. After incubation at 37°C for 24 hours, the medium was shaken well, and the turbidity (absorbance OD) of different strains grown in different concentrations of bile salts was measured. 600 To analyze its tolerance level, the following formula is used: Bile salt tolerance (%) = (OD value of bile salt-containing medium / OD value of bile salt-free medium) × 100%.

[0058] Survival rates of Lactobacillus plantarum GXS77 under different concentrations of bile salts were as follows: Figure 7 As shown, within the bile salt concentration range of 0–0.1% (g / mL), the bile salt tolerance of Lactobacillus plantarum GXS77 decreases rapidly with increasing bile salt concentration. When the bile salt concentration rises to 0.1–0.3% (g / mL), the bile salt tolerance of Lactobacillus plantarum GXS77 is almost zero.

[0059] (III) Adhesion effect of Lactobacillus plantarum GXS77 on Caco-2 cells 1. Culture of Caco-2 cells 2 mL of Caco-2 cell (human cloned colon adenocarcinoma cells, from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection) suspension was transferred to 25T cell culture flasks, and 2-4 mL of 20% FBS-DMEM medium (DMEM medium with 20% (v / v) fetal bovine serum FBS added) was added. The cell culture flasks were shaken using the cross-shaking method to distribute the cells evenly, and the flasks were placed at 37℃ and 5% (v / v) CO2 for adherent culture. The medium was changed every other day.

[0060] 2. Plate count method to test the adhesion effect of Lactobacillus plantarum GXS77 When the cell density in the culture flask reaches 80%~90%, digest and collect the cells, then place them into a 12-well cell culture plate using a cell spreader, and add 1×10⁻⁶ cells per well. 5 Add cells / well to a cell culture plate and incubate overnight at 37°C in a 5% (v / v) CO2 incubator. Add 1 mL of Caco-2 cells to the wells containing the Caco-2 cells to adjust the concentration to 1×10⁻⁶. 8 The bacterial suspension at CFU / mL was incubated with DPBS buffer as a blank control for 2 hours. After incubation, the suspension was discarded, and the cells were gently washed 2-3 times with DPBS buffer to remove unadhered *Lactobacillus plantarum* GXS77 cells. Adhered cells were then gently scraped off with the tip of a pipette to prepare a cell suspension. The number of adhered colonies was counted using the plating method, and the number of Caco-2 cells in the blank control group was counted using a hemocytometer. The adhesion effect of *Lactobacillus plantarum* GXS77 to Caco-2 cells was calculated using the formula: Cell adhesion rate (CFU / 100 cells) = N² × 100 / N₀; where N² represents the number of adhered *Lactobacillus plantarum* GXS77 cells, and N₀ represents the number of Caco-2 cells in the control group. Results are as follows: Figure 5 As shown, the Caco-2 adhesion rate of Lactobacillus plantarum GXS77 was 28.60 ± 2.05%.

[0061] 3. Gram staining method was used to observe the adhesion effect of Lactobacillus plantarum GXS77. When the cell density in the culture flask reaches 80%~90%, digest and collect the cells, then place them into a 12-well cell culture plate using a cell spreader, and add 1×10⁻⁶ cells per well. 5 Add cells / well to a cell culture plate and incubate overnight at 37°C in a 5% (v / v) CO2 incubator. After cell attachment, discard the original culture medium, wash 2-3 times with DPBS buffer, and add 1 mL of DPBS buffer to each well to adjust the concentration to 1×10⁻⁶ cells / well. 8 The bacterial suspension, diluted to CFU / mL, was incubated at 37°C in a 5% (v / v) CO2 incubator for 2 h. The suspension was then discarded, and the cells were washed 2-3 times with DPBS buffer to remove any unattached *Lactobacillus plantarum* GXS77 cells. The cell smears were carefully removed with forceps, fixed with 0.4% (g / mL) paraformaldehyde solution for 30 min, and allowed to air dry at room temperature before Gram staining. The adhesion of *Lactobacillus plantarum* GXS77 to Caco-2 cells was observed under a microscope, and photographs were taken. Results are as follows: Figure 8 As shown.

[0062] (iv) The effect of Lactobacillus plantarum GXS77 in inhibiting pathogenic bacteria This embodiment uses the Oxford cup agar diffusion method to determine the in vitro inhibitory effect of *Lactobacillus plantarum* GXS77 on four foodborne pathogens: *Listeria monocytogenes*, *Staphylococcus aureus*, and *Salmonella*. First, cell-free fermentation supernatant was prepared: the lactic acid bacteria suspension was centrifuged at 8000 rpm for 1 min, the supernatant was collected and filtered through a 0.22 μm microporous membrane for sterilization. Next, bacterial plates were prepared: 200 μL of indicator bacteria suspension was evenly spread on an LB agar plate, and three equidistant wells were prepared in a triangular pattern using an 8 mm diameter Oxford cup. 100 μL of the filtered, sterilized cell-free supernatant was injected into each well. After the liquid had completely penetrated the agar, the plate was incubated upright at 37°C overnight. After the experiment, the diameter of the transparent inhibition zone around each well was observed and measured. The final antibacterial activity was expressed as the measured diameter of the inhibition zone minus the diameter of the hole (8 mm). The strength of the antibacterial ability of the strain was determined based on the size of the inhibition zone.

[0063] Table 5 shows the inhibitory effects of Lactobacillus plantarum GXS77 on pathogenic bacteria. The inhibition zone of Listeria monocytogenes was greater than 5 mm, the inhibition zone of Staphylococcus aureus was 0-2 mm, and the inhibition zone of Salmonella was greater than 5 mm.

[0064] Table 5. Effect of Lactobacillus plantarum GXS77 on inhibiting pathogenic bacteria.

[0065] Note: - indicates inhibition zone ≤ 0mm; + indicates inhibition zone 0~2mm; ++ indicates inhibition zone 2~5mm; +++ indicates inhibition zone > 5mm. The Lactobacillus plantarum provided by this invention ( Lactiplantibacillusplantarum GXS77 has been proven to promote calcium absorption and osteoblast differentiation. This strain meets the basic requirements for probiotics, exhibiting good tolerance to the gastrointestinal environment, maintaining a certain survival rate, and possessing strong intestinal adhesion. It plays a role in maintaining the balance of normal intestinal microbiota and can adhere well to intestinal epithelial cells, thus exerting its effects. It can be developed into microbial preparations, health foods, or pharmaceuticals. Long-term use can improve the intestinal microbiota structure and prevent and alleviate depressive symptoms.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, The name of the *Lactobacillus plantarum* is *Lactobacillus plantarum*. Lactiplantib acillusplantarum GXS77, with accession number CGMCC No. 35899, was deposited in 2025 at the China General Microbiological Culture Collection Center, No. 3, Beichen West Road, Chaoyang District, Beijing.

2. The *Lactobacillus plantarum* strain according to claim 1, characterized in that, The 16S rDNA gene sequence of the *Lactobacillus plantarum* GXS77 is shown in SEQ ID NO.

1.

3. The *Lactobacillus plantarum* strain according to claim 1, characterized in that, The *Lactobacillus plantarum* GXS77 has relatively high acetic acid and butyric acid production, increasing by 18% and 16% respectively compared to general *Lactobacillus plantarum*.

4. The *Lactobacillus plantarum* strain according to claim 1, characterized in that, The Lactobacillus plantarum GXS77 exhibits good tolerance, high intestinal adhesion and antibacterial activity, and can inhibit the growth and reproduction of pathogenic bacteria, thus helping to maintain the balance of normal intestinal microbiota.

5. The intracellular enzyme in *Lactobacillus plantarum* as described in claim 1, which participates in the synthesis of short-chain fatty acids such as acetic acid and butyric acid, is characterized in that... The intracellular enzymes include acetate kinase ack, phosphoacetyltransferase pta, phosphotransacetylbutyrate gene ptb, 4-aminobutyrate aminotransferase puuE, and glutamate decarboxylase gadA.

6. The application of *Lactobacillus plantarum* as described in any one of claims 1 to 4 in promoting bone health.

7. The application according to claim 6, characterized in that, The plant lactobacillus promotes calcium absorption by secreting acetic acid and butyric acid, thereby promoting osteoblast differentiation and ultimately promoting bone health.

8. The application of *Lactobacillus plantarum* as described in any one of claims 1 to 4 in regulating intestinal flora.

9. A fermented product, characterized in that, It is produced by fermentation using Lactobacillus plantarum as described in any one of claims 1 to 4.

10. The use of at least one of the following as described in any one of claims 1 to 4, the intracellular enzyme as described in claim 5, and the fermented product as described in claim 9 in the preparation of products that increase the content of acetic acid and / or butyric acid in the intestine, promote calcium absorption, promote osteoblast differentiation, improve osteoporosis, promote bone health, improve the structure of intestinal microbiota, inhibit the growth and reproduction of pathogenic bacteria, and maintain the balance of normal intestinal microbiota.

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