Lactococcus lactis with remarkable bone strengthening activity and application thereof

Abstract

The invention belongs to the technical field of microorganisms and provides lactococcus lactis with remarkable bone strengthening activity and application of the lactococcus lactis 3-1, the lactococcus lactis 3-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.34671, and the preservation date is May 26, 2025. Fermentation supernate of the strain can produce butyric acid, can promote calcium nodule formation and up-regulate expression of osteogenic differentiation genes RUNX2, BGLAP and ALP, and has stable fermentation performance and gastric acid and cholate tolerance. By optimizing the process, the method is successfully applied to preparation of the sheep cheese. Animal experiments show that the strain and the sheep cheese fermented by the strain can improve osteoporosis symptoms of tail suspension rats, promote bone trabecula remodeling and mineralization, improve bone mineral density and bone biomechanical properties, regulate osteogenesis / osteoclast activity and increase the content of butyric acid in cecum contents, and have application potential in the field of osteoporosis prevention and treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a Lactococcus lactis with significant bone health activity and application thereof. BACKGROUND

[0002] Osteoporosis is a metabolic bone disease characterized by decreased bone mass, damaged bone microstructure, and increased risk of fracture. Current clinical treatment mainly relies on bisphosphonates, RANKL inhibitors, and hormone replacement therapy. However, long-term use of these drugs can cause serious side effects such as mandibular osteonecrosis, gastrointestinal damage, and cardiovascular risk. Therefore, developing safe and sustainable intervention strategies has become a research hotspot.

[0003] Recent studies have found that gut microbiota regulates bone metabolism through the "gut-bone axis", and short-chain fatty acids, especially butyric acid, play a key role. Butyric acid can promote bone health by inhibiting osteoclast differentiation and reducing bone resorption, promoting osteoblast mineralization by activating GPR43 receptors, and enhancing intestinal calcium absorption efficiency. In addition, probiotics can increase butyric acid production by regulating gut microbiota, thereby improving bone density.

[0004] However, existing probiotic strains generally have low butyric acid production and insufficient gastrointestinal survival rate, and functional strains with high-efficiency fermentation characteristics and clear bone health functions are still in the blank. Therefore, developing lactic acid bacteria that can tolerate gastrointestinal environment, produce high butyric acid, and directly promote bone formation, and applying them to functional fermented foods, is of great significance for dietary intervention of osteoporosis. SUMMARY

[0005] In view of the problems in the prior art, the present application aims to provide a Lactococcus lactis with significant bone health activity and application thereof. The strain has outstanding fermentation performance and osteogenic activity, providing a new strategy for the microecological treatment of osteoporosis.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The present application provides a Lactococcus lactis 3-1 with significant bone health activity in the first aspect. The Lactococcus lactis 3-1 is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Beichen West Road, Haidian District, Beijing, China, and has a preservation number of CGMCC No. 34671 and a preservation date of May 26, 2025.

[0008] The present application provides a microbial inoculant with significant bone health activity in the second aspect, wherein the inoculant contains the Lactococcus lactis 3-1 of the first aspect.

[0009] Further, the Lactococcus lactis 3-1 in the bacterial agent is a live bacterium or an inactivated bacterium.

[0010] Further, in the bacterial agent, the number of live Lactococcus lactis 3-1 is not less than 1×10 10 CFU / g.

[0011] The third aspect of the present application provides a preparation containing the Lactococcus lactis 3-1 fermentation broth of the first aspect.

[0012] The fourth aspect of the present application provides the use of the Lactococcus lactis 3-1 of the first aspect or the preparation of the second aspect in the preparation of a product for promoting bone health.

[0013] Further, the product is one or more of the following:

[0014] (1) The product can promote bone growth and development;

[0015] (2) The product can improve osteoporosis symptoms;

[0016] (3) The product can promote the remodeling and mineralization of bone trabeculae;

[0017] (4) The product can alleviate the sparseness of bone trabeculae;

[0018] (5) The product can enhance the bone structure.

[0019] Further, the product promotes bone health by up-regulating the expression levels of key genes RUNX2, BGLAP and ALP in osteogenic differentiation.

[0020] Strain preservation information:

[0021] Lactococcus lactis 3-1, preservation agency: China General Microbiological Culture Collection Center (CGMCC); address: No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing, Chaoyang District, Beichen West Road No. 1, postcode: 100101; preservation date: May 26, 2025; preservation number: CGMCC No. 34671; classification and naming: Lactococcus lactis.

[0022] The present application has the following beneficial effects compared with the prior art:

[0023] The present application screens a strain of Lactococcus lactis 3-1 with significant bone health activity, which is identified as Lactococcus lactis with preservation number CGMCC No. 34671; classification and naming: Lactococcus lactis. The strain has many significant beneficial effects and shows great application potential in the field of bone health:

[0024] 1. High butyric acid production and bone differentiation promoting ability

[0025] The fermentation supernatant of the strain has significant butyric acid production ability, which can be confirmed by HPLC detection. At the same time, it can significantly promote the differentiation of osteoblasts, and the alizarin red staining experiment shows that the formation of calcium nodules is obviously increased, and the RT-qPCR analysis confirms that it can up-regulate the expression levels of key genes RUNX2, BGLAP and ALP of osteogenic differentiation, and directly promote the growth and mineralization of bone.

[0026] 2. Excellent fermentation performance and gastrointestinal tolerance

[0027] The strain growth curve and acid production characteristics determination show that it has stable fermentation performance, and is suitable for the preparation of fermented foods such as goat cheese. The gastrointestinal tolerance experiment shows that the strain can tolerate gastric acid (pH 2.5-4) and bile salt environment, has high survival rate in simulated intestinal environment, has good intestinal colonization potential, and can effectively play the probiotic function.

[0028] 3. Significant improvement of osteoporosis verified by animal experiment

[0029] In the tail-suspended rat osteoporosis model, the strain and its fermented goat cheese can significantly improve the trabecular bone structure remodeling and mineralization, and increase the bone density through micro-CT analysis; histological analysis confirms that it regulates the activity of osteoblasts and osteoclasts; three-point mechanical test shows that the bone biomechanical properties are enhanced; GC-MS detection confirms that it can increase the content of butyric acid in cecal contents, which verifies the bone health function from multiple dimensions.

[0030] 4. Natural safety and multi-functional application value

[0031] The strain is isolated from natural fermented food and has high food safety, which can be applied to probiotic products, functional foods (such as auxiliary bone health food, acid and bile salt resistant food), bacterial agents and microecological preparations, etc. Its fermentation products (such as goat cheese) have excellent taste and bone health activity, which provides high-quality strain resources and scientific basis for the development of innovative fermented foods with bone health function. BRIEF DESCRIPTION OF DRAWINGS

[0032] The present application will be further described below in combination with the drawings and examples:

[0033] Figure 1 The standard curve of butyric acid in Example 2 of the present application;

[0034] Figure 2 The butyric acid HPLC detection peak chart of the fermentation supernatant of the strain in Example 2 of the present application;

[0035] Figure 3 The butyric acid content of the fermentation supernatant of the strain in Example 2 of the present application;

[0036] Figure 4 A: blank, B: sodium butyrate, C: MRS, D: A508-1, E: A201-1-1, F: BS99, G: 274-1, H: 126-2, I: 13-1, J: 10-4-1, K: 3-1, L: 1-1;

[0037] Figure 5 Example 4: Basic characteristics of the strains and simulated gastrointestinal tolerance experiment

[0038] Figure 6 Example 5: Expression analysis of osteogenic genes

[0039] Figure 7 Example 6: Radar chart of the taste of Lactococcus lactis 3-1 fermented goat cheese

[0040] Figure 8 Example 7: Rat bone apparent analysis of animal experiment

[0041] Figure 9 Example 7: Rat femur micro-CT analysis of animal experiment

[0042] Figure 10 Example 7: Rat femur HE and TRAP staining section analysis of animal experiment DETAILED DESCRIPTION

[0043] The examples are used to better illustrate the present application, but are not intended to limit the present application to only the examples. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above disclosure, which still fall within the scope of the present application.

[0044] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and values are provided as approximate descriptions of the ranges and values. The endpoints of the ranges of values, the endpoints of the ranges of values and individual values, and the individual values can each be combined with one another to form new ranges of values, which are also contemplated as being within the scope of the present disclosure.

[0045] The present application will be described in detail below through examples. It should be understood that the following examples are only used to exemplarily further explain and illustrate the content of the present application, and are not used to limit the present application.

[0046] Example 1: Isolation and identification of strains

[0047] The Lactococcus lactis lactis 3-1 described in the present application is isolated from yak milk in Lhasa City, Tibet Autonomous Region, and the specific process for specific isolation and screening is as follows:

[0048] After transferring 5 mL of yak milk (Lhasa City, Tibet) sample into a 25 mL sterile Erlenmeyer flask, adding 10 mL of sterile water for mechanical stirring, taking 1 mL and diluting 10 times in 0.9% NaCl solution, and taking 50 μL and coating on MRS medium (peptone 10.0 g, beef powder 5.0 g, glucose 20.0 g, yeast powder 4.0 g, sodium acetate 5.0 g, potassium phosphate 2.0 g, magnesium sulfate 0.2 g, triammonium citrate 2.0 g, manganese sulfate 0.05 g, Tween 80 1 mL, add distilled water to 1 L, agar powder 15.0 g), incubate at 37°C for 48 h. After incubation, single colonies are selected and transferred to MRS medium for purification culture, and 62 strains are obtained.

[0049] Example 2: HPLC method for screening strains with high butyric acid production

[0050] In this example, the butyric acid production capacity of the 62 strains isolated in Example 1 is detected, and strains with high butyric acid production are expected to be obtained. The specific method is as follows:

[0051] (1) Determination of butyric acid standard curve

[0052] The HPLC conditions are as follows: chromatographic column: Agilent TC-C18; 250x4.6 nm; partical size 5 μm. Mobile phase: A organic phase 0.05% trifluoroacetic acid methanol solution; B aqueous phase 0.05% trifluoroacetic acid aqueous solution. Elution conditions are shown in Table 1, wherein the column temperature is 35°C; the injection volume is 10 μL; the detection wavelength is 210 nm. Butyric acid gradient dilution and corresponding peak are shown in Table 2.

[0053] Table 1 Elution conditions

[0054]

[0055] Table 2 Butyric acid gradient dilution

[0056]

[0057] The average peak time of the gradient-diluted butyric acid standard is about 12-13 min. The standard curve made from the peak area of butyric acid is shown in Figure 1 Y = 579332900 * X + 234397, R 2 = 0.9996.

[0058] (2) Determination of butyric acid content of strains

[0059] Experiments from low temperature preservation of 62 strains of target strains selected for activation culture. First, the frozen strains were inoculated into MRS solid medium surface, 37°C for 48 hours to form single colonies. Select typical colonies into 5 mL of MRS liquid medium for 12 hours of preliminary expansion, and then inoculated into 50 mL of fresh medium for 48 hours of secondary culture. After the bacteria grew to the stationary phase, 10 mL of culture medium was centrifuged at 14000 r / min for 10 minutes to separate the bacteria and fermentation supernatant. The collected supernatant was filtered through a 0.22 μm filter to remove bacteria. The butyric acid content in the supernatant was quantitatively analyzed by high performance liquid chromatography. The test results showed that the HPLC detection peak of the strain was as shown in Figure 2 According to the regression equation, the butyric acid concentration was converted from the peak area, and the butyric acid content in the fermentation supernatant of the strain was as shown in Figure 3 .

[0060] Through high performance liquid chromatography analysis, 9 strains of high butyric acid-producing strains were selected from 62 strains of edible lactic acid bacteria, including Lactobacillus plantarum 10-4-1 and 126-2, Lactobacillus paracasei 274-1, Lactococcus lactis BS99, A201-1-1 and 3-1, Lactobacillus fermentum 1-1 and 13-1, and Streptococcus thermophilus A508-1.

[0061] Example 3: Alizarin red staining analysis of alizarin red staining of high butyric acid-producing strain fermentation supernatant promoting osteogenesis

[0062] This example further detects the osteogenic effect of the 9 strains selected in Example 2, and the specific method includes:

[0063] (1) Strain supernatant treatment of osteoblasts MC3T3-E1

[0064] First, the frozen strains were inoculated into MRS solid medium surface, 37°C for 48 hours to form single colonies. Select typical colonies into 5 mL of MRS liquid medium for 12 hours of preliminary expansion, and then inoculated into 50 mL of fresh medium for 48 hours of secondary culture. After the bacteria grew to the stationary phase, 10 mL of culture medium was centrifuged at 14000 r / min for 10 minutes to separate the bacteria and fermentation supernatant. The collected supernatant was filtered through a 0.22 μm filter to remove bacteria.

[0065] MC3T3-E1 mouse pre-osteoblast cell line was used for in vitro mineralization experiment. Cells were routinely cultured in a-MEM complete medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. When the cells reached 80% confluence in a 10 cm culture dish, they were gently washed twice with PBS, then digested with 0.25% trypsin (containing EDTA) at 37°C for 2 min. After confirming that the cells were detached under a microscope, an equal volume of complete medium was added to terminate the digestion. The cell suspension was inoculated in a 6-well plate (2 mL / well) at the same density, and the cells were cultured for 10 h to allow them to adhere to the wall. The experimental group was added with 1:20 diluted bacterial fermentation supernatant (v / v), and the positive control group was added with 0.5 mM sodium butyrate solution. Fresh medium containing treatment factors was replaced every 48 h during the intervention. After 7 days of continuous culture, alizarin red staining was performed to evaluate the formation of cell mineralization nodules. All operations were completed under sterile conditions.

[0066] (2) Cell alizarin red staining

[0067] On the seventh day of culture and passage, the cells were removed, the culture medium was discarded, 1 mL of PBS was added to wash the cells twice, 1 mL of fixing solution was added to each well, and the cells were fixed for 15 min. After the cells were fixed, the fixing solution was discarded, 1 mL of alizarin red staining solution was added to each well for 2 h, then the staining solution was discarded, 1 mL of ddH2O was added to wash twice, and the alizarin red staining of osteoblasts MC3T3-E1 was observed under a microscope as shown in Figure 4 .

[0068] The osteogenic activity analysis showed that Streptococcus thermophilus A508-1, Lactococcus lactis BS99, Lactobacillus plantarum 126-2, Lactobacillus scottomini 13-1 and Lactococcus lactis 3-1 significantly promoted the mineralization differentiation of MC3T3-E1 osteoblasts, showing a significant increase in alizarin red staining calcium nodules. Among them, Lactococcus lactis 3-1 not only showed the highest butyric acid production, but also showed the strongest osteogenic activity.

[0069] Example 4: Basic characteristics of strains and simulated gastrointestinal tolerance experiment

[0070] Activated *Lactococcus lactis* 3-1 was cultured in liquid MRS medium at 37°C for 24 hours. Every 2 hours, 2 mL of fermentation broth was sampled to measure OD600 and pH to monitor growth and acid production. In a simulated gastrointestinal tolerance test, the strain was harvested after centrifugation at 8,000g for 10 minutes at 4°C, washed three times with sterile PBS, and resuspended in PBS containing 3 g / L pepsin at pH 2.5, 3, and 4. The OD600 was adjusted to 1.0, and the strain was cultured at 37°C. Samples (2 mL) were taken at 0 and 3 hours, diluted proportionally, and cultured on MRS agar plates. Colonies were counted after 36–48 hours of culture at 37°C. To simulate gastrointestinal digestion, the strain was cultured in PBS containing 3 g / L pepsin (pH 2.5) for 3 hours, then transferred to simulated intestinal fluid (1 g / L trypsin, pH 8.0) and cultured at 37°C for 4 hours. See the graphs below. Figure 5 The formula for calculating the survival rate of the strain is shown in (1):

[0071]

[0072] N1 — viable bacterial count after strain treatment, cfu / mL;

[0073] N0 – Initial viable count of the strain, cfu / mL.

[0074] Depend on Figure 5 The growth curves showed that *Lactococcus lactis* 3-1 entered the logarithmic phase after 2.5 hours and reached the stationary phase after 12 hours, indicating rapid growth under optimal conditions. This strain continuously produced acid during fermentation, leading to a stable decrease in pH, demonstrating its strong acid-producing capacity. This capacity not only regulates the fermentation pH but also creates ideal conditions for goat cheese fermentation. Furthermore, the acid production was closely related to the growth rate, highlighting the synergistic effect of the strain during fermentation. In the acid tolerance experiment, *Lactococcus lactis* 3-1 achieved growth rates of 102.62% and 100.67% at pH 3 and pH 4, respectively. These results indicate that the strain has strong tolerance to the acidic conditions of gastric juice and maintains good growth even under low pH conditions. Subsequently, when the strain was transferred to simulated intestinal fluid at pH 8 for 4 hours, its growth rates were 91.52% and 103.02%, respectively. These results highlight the strain's ability to maintain high growth levels in the alkaline environment of a simulated intestine, demonstrating its excellent gastrointestinal adaptability. It is noteworthy that the growth rate of this strain reached 103.02% at a pH of 4, indicating that it has a strong ability to recover and adapt after exposure to an acidic gastric environment.

[0075] Example 5: Analysis of osteogenic gene expression in Lactococcus lactis 3-1

[0076] This example is directed to osteogenic gene expression analysis of Lactococcus lactis 3-1, the specific method is as follows:

[0077] The supernatant of 12-hour LAB culture was used to induce osteoblast MC3T3-E1 cells, and then total RNA of Lactococcus lactis 3-1 group and control group was extracted by universal RNA extraction kit. Then the cDNA was treated with DNase I, and reverse transcription was performed using Evo M-MLVRT mixed kit. RT-qPCR was performed on four genes of GAPDH, RUNX2, BGLAP and ALP using quantitative PCR instrument and SYBR Green Premix Pro Taq HS qPCR reagent kit. The fold change of gene expression was calculated by 2 -ΔΔCt method. As shown in Figure 6 , the gene primer sequences used in RT-qpcr are shown in Table 3.

[0078] Table 3 Gene primer sequences

[0079]

[0080] After seven days of treatment with Lactococcus lactis 3-1 supernatant, the expression levels of osteogenic genes RUNX2, BGLAP and ALP in MC3T3-E1 cells were significantly higher than those in the control group (p<0.0001). Notably, the expression of RUNX2 and BGLAP was even higher than that of the positive control group, indicating that the supernatant of Lactococcus lactis 3-1 strain may enhance the expression of osteogenic-related genes. These findings further confirm the potential of Lactococcus lactis 3-1 strain in promoting bone metabolism and osteogenesis.

[0081] Example 6: Optimization of Lactococcus lactis 3-1 Fermented Goat Cheese Conditions

[0082] It was found through experiments that the taste of goat cheese fermented by Lactococcus lactis 3-1 is closely related to the proportion of fat contained in goat milk. Full-fat goat cheese has a richer, more concentrated and smooth taste, but has a goat odor and is not easy to shape. Defatted goat cheese has no goat odor and is easy to shape, but the taste is dry, solid and rough. Therefore, in order to make the overall quality of goat cheese fermented by Lactococcus lactis 3-1 optimal, it is necessary to explore the optimal fat ratio of goat cheese, so in the following experimental process, this example is divided into five defatted ratios: 100% defatted, 85% defatted ratio, 70% defatted ratio, 62.5% defatted ratio and 55% defatted ratio for comparison.

[0083] The specific fermentation method is as follows:

[0084] I. Sterilization

[0085] Put the bottles, centrifuge bottles, milk pots, gauze wrapped with tin foil and molds in advance in the sterilization pot, adjust to 121℃, sterilize for 15 minutes, and store in the incubator.

[0086] II. Expansion of the strain

[0087] In advance, transfer the strain to be used for making cheese to a shaking tube, shake well after 12 hours, pour into 300mL MRS medium, and place in a 37℃ incubator for 12 hours.

[0088] III. Preservation of the strain

[0089] In the super-clean bench, mix the bacterial solution and pour it into centrifuge bottles, weigh to ensure an error of 0.5g, adjust the centrifuge to 8000r, and centrifuge for 8 minutes. Discard the supernatant. Rinse the bacteria with 30mL ddH2O, transfer to a 50mL centrifuge tube, weigh to ensure an error of 0.02g, balance, centrifuge at 8000r for 8 minutes, discard the supernatant, pour into 5mL milk, shake well, and store in a -20℃ refrigerator.

[0090] IV. Inoculation

[0091] Take the bacteria out of the refrigerator and thaw; fill the bottles with 800mL of milk, adjust the sterilization pot to 72℃, sterilize for 4 minutes, and then reduce the temperature to 30℃. Add 0.05% bacteria to the milk, shake well, pour into the milk pot, and continue heating at 30℃. Measure the pH of the milk every hour and record the results.

[0092] V. Coagulation

[0093] When the pH drops below 6.2, stop heating and remove the milk pot to cool down. Prepare 0.015% rennet, dilute with 0.9% NaCl to 8mL; and 0.02% CaCl2, dilute with ddH2O to 8mL, shake well, and wait for 30 minutes for activation. Add the activated CaCl2 and rennet to the milk pot in sequence, stir well, and transfer the milk pot to a 37℃ incubator and wait for 30 minutes.

[0094] At this time, the milk has coagulated and the supernatant has separated out. Cut it into small pieces with a knife and stir with a blender for 15 minutes.

[0095] VI. Hot water treatment

[0096] Stir the milk at 40℃ for 10-30 minutes, and stop heating when the pH reaches 5.3-5.7.

[0097] VII. Molding

[0098] Pour the milk into the gauze-wrapped mold, squeeze out the whey, and press for 12 hours to form the cheese.

[0099] Take photos throughout the process and measure the pH, as shown in Table 4.

[0100] The optimal fermented goat cheese was selected according to the hardness, mouthfeel, color, flavor, etc. of the cheese, and a radar chart was prepared as follows Figure 7 .

[0101] Table 4 pH record of Lactococcus lactis 3-1 fermented goat cheese

[0102]

[0103] Finally, according to the mouthfeel radar chart of Lactococcus lactis 3-1 fermented goat cheese with different defatted ratios, it can be concluded that the mouthfeel of Lactococcus lactis 3-1 fermented goat cheese is best at a defatted ratio of 70%, and the fermentation is optimal.

[0104] Example 7: Rat experiment study using Lactococcus lactis 3-1 fermented goat cheese

[0105] In this example, a rat model of disuse osteoporosis was used to simulate the model of aging and disuse osteoporosis. Among them, 8-week-old male Sprague-Dawley rats (180-200 grams) were raised under specific pathogen-free conditions, with a temperature of 23±2℃, a humidity of 50-70%, and a light / dark cycle of 12 hours / 12 hours. After one week of adaptation, the rats were randomly assigned to five groups (eight rats per group): free living group, tail suspension group, tail suspension and Lactococcus lactis 3-1 gavage group, tail suspension and Lactococcus lactis 3-1 fermented goat cheese gavage group, and tail suspension and Lactobacillus plantarum 10-4-1 fermented goat cheese gavage group (Note: 10-4-1 was not conducive to the growth of osteoblasts in the previous cell experiment, so it was used as a fermented goat cheese group without therapeutic effect as a control). The tail suspension method is to suspend the rat in a tail suspension box at an angle of 30° with the ground, while allowing the rat to move freely. The Lactococcus lactis group was orally administered 2 milliliters of Lactococcus lactis 3-1 (10 9 CFU / mL) in normal saline per day. The gavage cheese group was given 1 gram of fermented goat cheese containing Lactococcus lactis 3-1 or 10-4-1 (10 9 CFU / g) in 2 milliliters of normal saline per day, while the model group received an equal amount of normal saline. All rats had food and water every day. After 4 weeks, the rats were weighed, anesthetized with intraperitoneal injection of 20% urethane (1 mL / 100 g body weight), and the femur and tibia were collected for analysis.

[0106] (1) Bone apparent analysis

[0107] First, the weight of the rat femur was measured with a precision analytical balance. The length, diameter, and maximum width of the femur were measured with a digital caliper. A CMT4304 tester was used to perform a three-point bending test on the rat tibia. The test parameters were set as follows: speed 0.5 mm / min; span 16 mm; support radius 5 mm. The ultimate force and breaking force of the tibia were recorded during the test as followsFigure 8 As shown.

[0108] The therapeutic effect of Lactococcus lactis 3-1 fermented goat cheese on osteoporosis was evaluated by assessing the morphological changes in the femur of different experimental groups. By measuring the length, midshaft diameter, weight, and widest diameter of the femur, it was found that the values of the osteoporosis group were significantly lower than those of the normal group, indicating that bone morphology had severely deteriorated. In contrast, the 3-1 group, 3-1 fermented goat cheese group, and 10-4-1 fermented goat cheese group all showed significant improvements. Specifically, the fermented goat cheese group showed the most significant improvements, with a 7.05% increase in femur length, a 25.52% increase in midshaft diameter, a 34.25% increase in femur weight, and an 11.52% increase in the widest diameter. These results indicate that Lactococcus lactis 3-1 fermented goat cheese has the most significant therapeutic effect on osteoporosis.

[0109] The three-point bending test of the tibia was used to evaluate the effect of Lactococcus lactis 3-1 fermented goat cheese on bone strength. This mechanical test method provides precise measurement results, and the trends in fracture force and ultimate force closely reflect the improvement in bone strength. Compared to the control group, the mechanical properties of the tibia in the osteoporosis group were significantly lower. The 3-1 fermented goat cheese group showed the highest fracture force and ultimate force, significantly better than all other experimental groups (p<0.001), indicating that it has a superior effect on bone strength. Although the values of the 3-1 group were slightly lower than those of the 3-1 fermented goat cheese group, its performance was still significantly better than that of the 10-4-1 fermented goat cheese group (p<0.05).

[0110] (2) micro-CT analysis of femur density

[0111] Micro-computed tomography was used to evaluate the bone microstructure of the samples. Imaging was performed using a micro-computed tomography scanner with a resolution of 15 pm. The scanning parameters were configured as follows: the X-ray tube voltage was 70 kV, the current was 200 pA, and a 0.5 mm aluminum filter was used to minimize beam hardening artifacts. Each scan was performed with a 360° rotation, and the detector resolution was 2016 x 1344 to obtain high-quality images. The generated images were processed using NRecon (v.1.6.9.8), CTVox (v.2.6.0), and CTAnalyser (v.1.13.11.0) software for three-dimensional reconstruction and analysis. The analysis was performed as described in Figure 9 As shown.

[0112] Micro-CT scans showed that femurs of the normal group exhibited tightly packed trabeculae, high bone volume to trabecular volume ratio (BV / TV), and good bone mineral density (BMD). In contrast, femurs of the osteoporosis group exhibited sparse trabeculae, a 71.08% decrease in BV / TV, a 71.48% decrease in BMD, a 21.58% decrease in trabecular thickness (Tb.Th), a 50.79% increase in trabecular separation (Tb.Sp), a 66.33% decrease in trabecular number (Tb.N), a 67.09% increase in structure model index (SMI), indicating a significant deterioration in bone structure.

[0113] Femurs of the L. lactis 3-1 fermented goat cheese group showed significant improvements in key microstructural parameters. Specifically, BV / TV increased by 55.02%, Tb.Th increased by 88.46%, and Tb.Sp decreased by 96.29% compared to the osteoporosis group, indicating that L. lactis 3-1 fermented goat cheese has efficacy in restoring bone microstructure. While the L. plantarum 10-4-1 fermented goat cheese group also showed improvements, these effects were not as pronounced as the L. lactis 3-1 group. Notably, the BMD recovery rate was 38.58% for the 3-1 group, while the BMD recovery rate was 74.88% for the L. lactis 3-1 fermented goat cheese group, nearly doubling. Micro-CT analysis further confirmed that L. lactis 3-1 fermented goat cheese significantly improved bone microstructure in osteoporotic rats, particularly in terms of BV / TV, Tb.Th, and Tb.Sp, making it a promising osteoporosis treatment strategy.

[0114] (3) Rat HE and TRAP Stained Section Analysis

[0115] Rat femur samples were fixed in 4% paraformaldehyde for 48 hours and then decalcified in EDTA decalcifying solution for 3 weeks. The samples were then dehydrated, paraffin-embedded, and sectioned. The sections were deparaffinized with xylene, gradient dehydrated with ethanol, stained with hematoxylin and eosin for 5 minutes each, stained with hydrochloric acid ethanol for 30 seconds, re-stained with alkaline ammonia solution for 1 minute, stained with eosin for 2 minutes, and finally mounted. TRAP staining was performed according to the manufacturer's protocol: after fixation, samples were incubated in TRAP staining solution at 37°C for 30 minutes. Osteoclasts were stained red, and mature osteoblasts were identified by the presence of three or more nuclei. Tissue sections were scanned using a panoramic section scanner (3DHISTECH, PANNORAMIC DESK / MIDI / 250 / 1000), and image analysis was performed using CaseViewer 2.4 software, analyzing Figure 10 .

[0116] In the osteoporosis group, osteoclasts were mainly located at the junction of the cartilage and the marrow cavity, and a significant increase was observed below the epiphyseal plate. The osteoclast activity was most evident in this group, with a very intense red staining. In contrast, the group of goat cheese fermented with Lactococcus lactis 3-1 showed the lowest osteoclast activity, with a significant decrease in TRAP staining compared to the other groups, showing a decrease in osteoclast activity of 66.84% (p < 0.0001), reflecting the most effective inhibition. Both groups of goat cheese fermented with Lactococcus lactis 3-1 and 10-4-1 showed a decrease in osteoclast activity, but to a lesser extent than the group of goat cheese fermented with Lactococcus lactis 3-1.

[0117] The trabecular area represents the spongy bone region, characterized by a network of trabeculae that are interconnected, contributing to the structural integrity and facilitating metabolic exchanges. Changes in the trabecular area reflect bone remodeling, with a decrease indicating bone loss and an increased risk of fractures. The osteoporosis group showed the smallest trabecular area, reflecting a clear degradation of the bone mass, while the normal group maintained a well-structured trabecular morphology. Both groups of goat cheese fermented with Lactococcus lactis 3-1 and Lactobacillus plantarum 10-4-1 showed improvements to varying degrees. However, the group of goat cheese fermented with Lactococcus lactis 3-1 was significantly better. TRAP staining showed that the osteoporosis group had significantly higher osteoclast activity. In contrast, the group of goat cheese fermented with Lactococcus lactis 3-1 showed the greatest inhibition of osteoclast activity, while the other treatment groups showed a decrease in osteoclast activity, but still at a high level.

[0118] In summary:

[0119] The Lactococcus lactis subsp. lactis of the present application is a natural strain with high food safety, and its fermentation supernatant has high butyric acid production capacity and mouse osteoblast differentiation-promoting calcium nodule production capacity, highlighting the therapeutic potential of goat cheese fermented with Lactococcus lactis 3-1 in the treatment of osteoporosis, demonstrating its significant impact on improving bone morphology, strength, and microstructure. The research results provide strong experimental evidence to support its potential as a substitute for traditional drug therapy. Probiotic fermented foods, such as goat cheese fermented with Lactococcus lactis 3-1, provide a biologically safe and sustainable approach to the treatment of osteoporosis, with higher biological safety and fewer side effects compared to traditional drugs.

[0120] Finally, it should be noted that the above merely serves to illustrate the technical solutions of the present application and is not limiting. Although the present application has been described in detail with reference to the preferred arrangement, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A Lactococcus lactis 3-1 strain having significant bone health activity, characterized in that, The Lactococcus lactis 3-1 is preserved in the China General Microbiological Culture Collection Center, the address of which is No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation number of which is CGMCC No. 34671, and the preservation date of which is May 26, 2025.

2. A microbial inoculum having significant bone health activity, characterized in that, The bacterial agent contains the Lactococcus lactis 3-1 according to claim 1.

3. The microbial inoculant of claim 2, wherein, The Lactococcus lactis 3-1 in the bacterial agent is a live bacterium or an inactivated bacterium.

4. The microbial inoculant of claim 3, wherein, In the bacterial agent, the viable cell number of the Lactococcus lactis 3-1 is not less than 1 x 10 10 CFU / g.

5. A preparation containing the fermentation liquor of the Lactococcus lactis 3-1 according to claim 1.

6. Use of the Lactococcus lactis 3-1 according to claim 1 or the preparation according to any one of claims 2 to 5 in the preparation of a product for promoting bone health.

7. Use according to claim 6, characterized in that, The product is one or more of the following: (1) The product can promote the growth of bone development; (2) The product can improve the symptoms of osteoporosis; (3) The product can promote the remodeling and mineralization of bone trabeculae; (4) The product can relieve the sparseness of bone trabeculae; (5) The product can enhance the bone structure.

8. Use according to claim 6, characterized in that, The product promotes bone health by up-regulating the expression levels of key genes RUNX2, BGLAP and ALP in osteogenic differentiation.

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