Lactococcus lactis subsp. Lactis capable of strengthening bones and improving osteoporosis, metagen and application of lactococcus lactis subsp. Lactis

Abstract

The invention relates to the technical field of microorganisms, in particular to lactococcus lactis subsp. Lactis capable of strengthening bones and improving osteoporosis, a metagen and application of the lactococcus lactis subsp. Lactis. The preservation number of the lactococcus lactis subsp. Lactis BS99 is CGMCC (China General Microbiological Culture Collection Center) No.33932. The lactococcus lactis subsp. Lactis BS99 disclosed by the invention is a natural strain, and a fermentation supernatant of the lactococcus lactis subsp. Lactis BS99 has relatively high butyric acid production capacity and mouse osteoblast differentiation promoting calcium nodule production capacity, can resist gastric acid and cholate, and has good intestinal colonization capacity. Besides, rat test results show that the strain can improve osteoporosis symptoms caused by tail suspension, promote remodeling and mineralization of bone trabecula, relieve sparsity of the bone trabecula and improve the structure and function of bones, and has important application value in the fields of food, health care products and the like for assisting bone strengthening, promoting bone development and growth, improving osteoporosis and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a strain of Lactococcus lactis subspecies lactis capable of strengthening bones and improving osteoporosis, a postbiotic and an application thereof. Background Art

[0002] Osteoporosis is a systemic, metabolic bone disease. As people age, they gradually lose bone mass, their bone microstructure deteriorates, and their bone strength decreases, leading to osteoporosis.

[0003] Butyrate is a short-chain fatty acid that has been shown to regulate bone mass and prevent bone loss. It also activates immune system cells in the gut and bone marrow, called T cells. These T cells secrete a protein called Wnt10b, which is crucial for bone development and preventing bone loss.

[0004] Research reports indicate that the gut and bone have a complex connection. Gut microbes and their derived molecules may participate in bone metabolism regulation at various levels through the gut-bone axis, positively impacting bone health. Probiotics are defined as live microorganisms that, when consumed in sufficient amounts, can confer health benefits on the host. Probiotics can modulate the host's intestinal flora, altering the synthesis and release of relevant metabolites, promoting optimal metabolism, and creating a healthy intestinal environment. Lactic acid bacteria enhance intestinal calcium absorption by maintaining a balanced intestinal flora. A healthy intestinal environment facilitates mineral solubility and transport, thereby increasing calcium bioavailability. Numerous studies, both domestic and international, have confirmed that some probiotic strains have positive effects on improving human bone health. The short-linked fatty acids, phytase, and bioactive peptides produced by probiotic strains can promote bone mineral absorption. The immunomodulatory and antioxidant effects of probiotics can inhibit osteoclast differentiation. Specific probiotic strains can also reduce the expression of systemic proinflammatory cytokines and increase bone density. Therefore, studies have proposed probiotic interventions as adjunctive therapies to promote bone development and improve osteoporosis.

[0005] Most importantly, current treatments for osteoporosis include medication, surgery, rehabilitation therapy combined with lifestyle adjustments, dietary interventions, and exercise. These treatments are slow to produce results, and medication and surgery are relatively expensive, leaving many patients without effective treatment. To address this issue, research into probiotic therapy is poised to benefit osteoporosis patients.

[0006] The Chinese invention patent with publication number CN118064332A discloses a strain of Lactococcus lactis subsp. lactis JYLL-72 that promotes bone growth, its applications and preparations, and screens a strain of Lactococcus lactis subsp. lactis that can ferment and produce vitamin K2, which can stimulate intestinal cells to secrete insulin-like growth factor-1 (IGF-1) to mediate the host's growth and development process.

[0007] In summary, how to develop a probiotic that can improve human bone health is a technical difficulty that researchers in this field are committed to solving. Summary of the Invention

[0008] In order to solve the deficiencies of the prior art mentioned in the above background, the present invention provides a strain of Lactococcus lactis subsp. lactis BS99 having the functions of strengthening bones and improving osteoporosis and its application.

[0009] The present invention provides a Lactococcus lactis subspecies lactis BS99, whose Latin name is: Lactococcus lactis subsp. Lacti , deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: March 24, 2025, deposit number: CGMCC No.33932.

[0010] The present invention also provides a Lactococcus lactis subsp. lactis BS99 bacterial agent, the components of which include the Lactococcus lactis subsp. lactis BS99 described above.

[0011] The present invention also provides a Lactococcus lactis subsp. lactis BS99 probiotic product, the components of which include the Lactococcus lactis subsp. lactis BS99 described above.

[0012] In one embodiment, the viable count of Lactococcus lactis subspecies BS99 in the probiotic product is not less than 1×10 10 CFU / g or 1×10 10 CFU / mL.

[0013] The present invention also provides a Lactococcus lactis subsp. lactis BS99 functional product, which is a functional food or health product, and its components include small molecule active functional peptides and the Lactococcus lactis subsp. lactis BS99 described above.

[0014] In one embodiment, the viable cell count of Lactococcus lactis subspecies BS99 in the functional product is not less than 1×10 10 CFU / g or 1×10 10 CFU / mL.

[0015] Furthermore, the small molecule active functional peptide is one or more combinations of collagen peptide, sturgeon tendon peptide and sturgeon cartilage peptide.

[0016] In one embodiment, the functional food includes one or more combinations of functional foods that assist in bone health, promote bone development and growth, improve osteoporosis symptoms, promote trabecular remodeling and mineralization, alleviate trabecular sparsity, and enhance bone structure, and acid-resistant and bile-resistant foods.

[0017] The present invention also provides a fermentation liquid, wherein the Lactococcus lactis subsp. lactis BS99 as described above is inoculated into a culture liquid, cultured at 30-40° C. for 24-48 hours, the culture liquid is separated into solid and liquid, and the liquid is collected to obtain a fermentation liquid; Wherein, the fermentation broth is a fermentation broth containing butyric acid.

[0018] It should be noted that the culture medium may be other culture medium suitable for the growth of Lactococcus lactis subsp. lactis BS99, including but not limited to MRS liquid culture medium.

[0019] The present invention also provides a traditional Chinese medicine composition, the components of which include the Lactococcus lactis subsp. lactis BS99 described above.

[0020] In one embodiment, the traditional Chinese medicine composition further comprises one or more of Astragalus, Schisandra, Lycium barbarum, Radix Trichosanthis, Rhizoma Anemarrhenae, Radix Puerariae, Radix Angelicae Sinensis, and Salvia miltiorrhiza.

[0021] Furthermore, the Chinese medicine composition includes, by weight, 40-100 parts of Lactococcus lactis subspecies BS99 agent, 30-100 parts of Astragalus, 20-100 parts of Schisandra chinensis, 50-100 parts of Lycium barbarum, 30-100 parts of Radix Trichosanthis, 25-100 parts of Rhizoma Anemarrhenae, 20-100 parts of Radix Puerariae, 60-100 parts of Radix Angelicae Sinensis, and 30-100 parts of Salvia miltiorrhiza.

[0022] The present invention also provides a postbiotic product, which is prepared by inoculating Lactococcus lactis subspecies BS99 into a culture solution containing skim milk, and then fermenting, centrifuging, inactivating, and freeze-drying. The Lactococcus lactis subspecies lactis BS99 adopts the Lactococcus lactis subspecies lactis BS99 described above.

[0023] In one embodiment, the amount of Lactococcus lactis subsp. lactis BS99 in the postbiotic product is not less than 1×10 10 CFU / g.

[0024] The present invention also provides a Lactococcus lactis subspecies BS99 proecological preparation, the components of which include one or more of Lactococcus lactis subspecies BS99, fermentation broth of Lactococcus lactis subspecies BS99, fermentation broth precipitate of Lactococcus lactis subspecies BS99, bacterial agent of Lactococcus lactis subspecies BS99, and freeze-dried bacterial powder of Lactococcus lactis subspecies BS99; The Lactococcus lactis subspecies lactis BS99 adopts the Lactococcus lactis subspecies lactis BS99 described above.

[0025] The present invention also provides a use of the above-mentioned probiotic preparation in a bone-strengthening product for improving osteoporosis and / or strengthening bone structure.

[0026] Based on the above characteristics, the Lactococcus lactis subspecies lactis provided by the present invention has the following beneficial effects: The Lactococcus lactis subspecies lactis of the present invention is a natural strain with high food safety; its fermentation supernatant has a high butyrate production capacity and the ability to promote differentiation and calcium nodule production in mouse osteoblasts. It is also resistant to gastric acid and bile salts and has good intestinal colonization ability. In addition, the strain can improve the symptoms of osteoporosis caused by tail suspension in rats, promote the remodeling and mineralization of trabecular bone, alleviate the sparseness of trabecular bone, and improve the structure and function of bones. It has important application value in the fields of food, health products and medicines such as assisting bone health, promoting bone development and growth, and improving osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the standard curve for butyric acid in Example 1 of the present invention; Figure 2 This is the peak diagram of butyric acid detected by HPLC in the fermentation supernatant of the strain in Example 1 of the present invention; Figure 3 The butyrate content of the fermentation supernatant of the strain in Example 1 of the present invention; Figure 4 This is the microscopic imaging of osteoblasts MC3T3-E1 stained with Alizarin red treated with the supernatant of the strain in Example 2 of the present invention; Figure 5 This is an analysis of the significance of the differences in butyrate production by four bacterial strains that can promote the differentiation of mouse osteoblasts MC3T3-E1 according to Example 2 of the present invention; Figure 6 This is a Micro-CT scan of the femur of a rat in the animal experiment of Example 4 of the present invention; Figure 7 This is the femoral bone density analysis of rats in the animal experiment of Example 4 of the present invention; Figure 8 This is the analysis of femoral bone volume fraction in rats in the animal experiment of Example 4 of the present invention; Figure 9This is the analysis of rat femoral trabeculae in the animal experiment of Example 4 of the present invention. DETAILED DESCRIPTION

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0030] The present invention also provides the following operation examples and embodiments: The present invention provides a strain of Lactococcus lactis subsp. lactis BS99, whose Latin name is: Lactococcus lactis subsp. Lacti , deposited in the General Microbiology Center of China Culture Collection Administration, deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: March 24, 2025, deposit number: CGMCC No.33932.

[0031] Source: Lactococcus lactis subspecies BS99 was isolated and screened from yak milk samples collected from Lakang Village, Alang Township, Linzhou County, Lhasa City, Tibet Autonomous Region.

[0032] Colony morphology: The colony morphology of Lactococcus lactis subspecies BS99 is round, milky white, with a smooth and raised surface, neat edges, and opaque.

[0033] The specific process of isolating and screening Lactococcus lactis subsp. lactis from yak milk in Lakang Village, Alang Township, Linzhou County, Lhasa City, Tibet Autonomous Region is as follows: Yak milk samples were collected from Lakang Village, Alang Township, Linzhou County, Lhasa City, Tibet Autonomous Region. Using the spread plate method, 20 g of yak milk sample was placed in a sterile homogenizing bag, marked, and 20 mL of 0.85% saline was added and patted thoroughly to mix.

[0034] Then, 1 mL of the sample was drawn for 10-fold gradient dilution. 3 , 10 4 , 10 5 Spread 1 mL of the sample onto an MRS plate containing 2.5% CaCO₃ and incubate inverted at 37°C for 48 hours. Select well-growing colonies with large calcium-dissolving zones and repeatedly isolate and purify them by streaking until single colonies are obtained. Isolate the strains, number them, and store them in glycerol tubes at -80°C in a bacterial library.

[0035] Example 1 Screening of strains with high butyrate production by HPLC 1. Determination of butyric acid standard curve HPLC conditions were as follows: Column: Agilent TC-C18; 250 x 4.6 nm; Partial size: 5 μm. Mobile phase: Organic phase A: 0.05% trifluoroacetic acid in methanol; Aqueous phase B: 0.05% trifluoroacetic acid in water. Elution conditions are shown in Table 1, with column temperature at 35°C, injection volume of 10 μL, and detection wavelength at 210 nm. The butyric acid gradient dilution and corresponding peaks are shown in Table 2.

[0036] Table 1 Elution conditions

[0037] Table 2 Butyric acid gradient dilution

[0038] The experiment showed that the average peak elution time of the gradient diluted butyric acid standard was approximately between 12 and 13 minutes. The standard curve was drawn based on the peak area of ​​butyric acid. Figure 1 As shown, Y=579332900*X+234397, where R 2 =0.9996.

[0039] 2. Determination of butyric acid content in strains Eighty strains were removed from a -80°C bacterial library and inoculated onto MRS solid medium at 37°C for 48 hours. A single colony was transferred to a shake tube and incubated at 37°C for 12 hours. A 10% inoculum was then inoculated into another shake tube and incubated at 37°C for 48 hours. The grown bacteria were shaken to a uniform consistency, transferred to a 10 mL centrifuge tube, and centrifuged at 14,000 rpm for 10 minutes. The fermentation supernatant was filtered through a 0.22 μm filter to obtain a sterile supernatant. The butyric acid content of the fermentation supernatants of the 80 strains was determined by HPLC.

[0040] The test results showed that the strain HPLC detected peaks such as Figure 2 , compared the butyric acid peak area of ​​80 strains fermentation supernatant samples, screened out the 8 strains with higher butyric acid content, numbered 126-2, 10-4-1, BS99, 274-1, A201-1-1, 13-1, 1-1 and A508-1. According to the regression equation, the peak area was converted into butyric acid concentration, and the butyric acid content of the fermentation supernatant of the 8 strains was obtained as follows: Figure 3 .

[0041] Example 2 Analysis of the Effect of Fermentation Supernatants of Eight Strains with High Butyrate Content on the Differentiation Ability of Mouse Osteoblasts by Alizarin Red Staining 1. Treatment of osteoblasts MC3T3-E1 with strain supernatant The eight strains identified as high butyrate-producing strains were inoculated onto solid MRS medium and cultured at 37°C for 48 hours. A single colony was then selected and transferred to liquid MRS medium for 12 hours at 37°C. A 10% inoculum was then transferred to fresh liquid MRS medium and cultured at 37°C for 48 hours. The resulting cultures were shaken well, transferred to a 10mL centrifuge tube, and centrifuged at 14,000 rpm for 10 minutes. The fermentation supernatant was then filtered through a 0.22μm filter to obtain a sterile supernatant.

[0042] Osteoblastic MC3T3-E1 cells were first cultured in complete cell culture medium in a 10 cm dish in a 37°C, 5% CO2 incubator for 48 hours. Once cells had grown to 80% of the dish surface, the medium was aspirated and 2 mL of PBS was slowly added for washing. The cells were shaken and aspirated twice. Adherent cells were digested with 1 mL of trypsin for 2 minutes in a 37°C incubator. After microscopy, cells were removed and observed for spherical formation. Digestion was terminated by adding 2 mL of complete culture medium. Aliquots were transferred to a 6-well plate and each well was filled to 2 mL with complete culture medium. After 10 hours of growth in the incubator, cells were removed and the prepared bacterial supernatant was added to the plate at a ratio of 1:20. A positive control, sodium butyrate, was added at a ratio of 0.5 mM to the plate. The plate was incubated for 24 hours before the medium was changed. The medium was then changed every 48 hours. Alizarin red staining was performed on the seventh day of passage.

[0043] 2. Cell Alizarin Red Staining On the seventh day of culture, the cells were removed, the culture medium was discarded, and 1 mL of PBS was added to wash the cells twice. 1 mL of fixative was added to each well and the cells were left for 15 min. After the cells were fixed, the fixative was discarded and 1 mL of alizarin red staining solution was added to each well for staining for 2 h. The staining solution was then discarded and 1 mL of ddH20 was added to wash the cells twice. The osteoblasts MC3T3-E1 were observed under a microscope. Figure 4 shown.

[0044] Figure 4 In the figure, A: blank control, B: positive control sodium butyrate, C: A508-1, D: A201-1-1, E: BS99, F: 274-1, G: 126-2, H: 3-1, I: 10-4-1, J: 1-1.

[0045] Analysis of the above experimental results revealed that cells treated with the supernatants of strains 274-1 and 10-4-1 showed significant cell death and no calcium nodules, likely because these strains are not conducive to cell growth. No significant changes were observed with strains A201-1-1 and 1-1, while numerous calcium nodules were observed with strains A508-1, BS99, 126-2, and 13-1, indicating that these four strains have a certain promoting effect on the differentiation of osteoblasts MC3T3-E1.

[0046] The butyrate production ability of the four strains was further analyzed for significant differences. Figure 5 As shown, the results showed that the butyrate production concentration of strain A508-1 was significantly higher than that of BS99, 126-2, and 13-1, indicating that it has better bone-building potential, while the difference in butyrate production ability among strains BS99, 126-2, and 13-1 was not significant (P>0.05).

[0047] In summary, combined with the analysis of the butyric acid content in the fermentation supernatant of different lactic acid bacteria strains and the ability of the fermentation supernatant to promote differentiation and calcium nodule production in mouse osteoblasts MC3T3-E1, four lactic acid bacteria with bone-building potential were selected from 80 lactic acid bacteria strains, namely A508-1, BS99, 126-2 and 13-1. Therefore, these four strains were selected for further experimental research.

[0048] Example 3 Strain Simulated Gastrointestinal Tolerance Experiment The activated strains A508-1, BS99, 126-2 and 13-1 were centrifuged at 8000 r / min for 10 min at 4 °C. After centrifugation, the bacteria were washed three times with sterilized PBS and resuspended in PBS with pH values ​​of 2.5, 3 and 4 and containing 3 g / L pepsin. The OD was adjusted to 1.0 and the cells were placed in a 37 °C constant temperature incubator. 2 mL of the bacterial solution was taken at 0 and 3 h, respectively, and diluted to 10 with sterilized PBS. -4 , 10 -5 , 10 -6 The cells were spread on MRS solid plates and cultured at 37°C for 36-48 hours before plate counting.

[0049] A suspension of pancreatic enzyme (1 g / L) was suspended in sterile PBS, and the pH was adjusted to 8.0 with NaOH. 100 μL of the bacteria cultured in simulated gastric fluid for 3 h was transferred to 900 μL of simulated pancreatic fluid and cultured at 37°C for 4 h. At 0 and 4 h, 100 μL of the bacterial solution was diluted to an appropriate dilution in sterile saline and spread on MRS solid plates. After culture at 37°C for 36-48 h, the plate count was performed.

[0050] The calculation formula of strain survival rate is shown in (1): Strain survival rate (%) = (logN1 / logN0) x 100% (1) N1——number of viable bacteria after strain treatment, CFU / mL; N0——initial viable bacterial count of the strain, CFU / mL; Table 3 Simulated gastrointestinal tolerance of strains

[0051] As shown in Table 3, strains A508-1, BS99, 126-2, and 13-1 showed significant differences in their survival in different simulated gastrointestinal environments. Overall, A508-1 could only survive in gastric juice at pH 4, while 13-1 had difficulty surviving in pancreatic juice. BS99 and 126-2 both had high survival rates in different simulated gastrointestinal environments. However, BS99 had a higher survival rate than 126-2 in both gastric juice at pH 2.5 and pancreatic juice at pH 8, indicating its strong tolerance. Therefore, BS99 was selected for in-depth experimental research.

[0052] Example 4 Experimental study on strain BS99 in rats Eight-week-old male SD rats (weighing 180-200g) were housed in a specific pathogen-free environment with a temperature of 23±2°C, humidity controlled at 50-70%, and a light cycle of 12 hours light / 12 hours dark. Before the start of the experiment, the rats were adaptively raised for one week and then randomly divided into three groups (8 in each group): a free-living group (Control group), a tail-suspension group, and a tail-suspension plus gavage BS99 group. The tail suspension method simulates osteoporosis conditions by suspending the rat's tail in a tail suspension rack at a 30° angle to the ground, allowing it to move freely. The experiment lasted 28 days. In the BS99 group, rats received 1×10 8 All rats were gavaged with a bacterial solution containing 100 CFU / mL. All rats had free access to food and water throughout the experiment. After the experiment, the rats were weighed and anesthetized with an intraperitoneal injection of 20% urethane (1 mL / 200 g body weight). Subsequently, femurs and tibias were collected for analysis. Microcomputed tomography (micro-CT) was used to assess bone microstructure. Femurs were scanned and analyzed using a micro-CT scanner with a resolution of 15 μm.

[0053] Depend on Figure 6 (A is the Control group; B is the tail-hanging group; C is the tail-hanging and gavage BS99 group) It can be seen that the rats in the Control group had a larger cancellous bone density and a larger number of trabeculae. In contrast, the rats in the tail-hanging group had a smaller bone density and were more porous. After the tail-hanging rats were gavaged with the BS99 strain, it was clearly observed that the bone density of the rats in the tail-hanging and gavage BS99 group increased significantly and the number of trabeculae increased, indicating that the BS99 strain has a significant effect on increasing bone density and improving osteoporosis.

[0054] Depend on Figure 7In animal experiments, rats in the control group had higher bone density, indicating normal bone health. In contrast, rats in the tail-suspension group had a significant decrease in bone density, showing typical characteristics of osteoporosis. After 28 days of tail-suspension plus oral gavage with the BS99 strain, the bone density of rats in the tail-suspension and oral gavage BS99 group increased, indicating that the BS99 strain can have a positive impact on bone density by regulating bone metabolism. In summary, the BS99 strain can promote the recovery of bone density and improve bone health in osteoporosis caused by tail suspension.

[0055] Depend on Figure 8 In animal studies, the control group had the highest bone volume fraction, while the tail-suspension group had the lowest. However, after 28 days of tail-suspension plus oral gavage with BS99, the BS99 group showed a significant increase in bone volume fraction, approaching normal levels. This suggests that the BS99 strain has a certain bone-protective effect, effectively alleviating tail-suspension-induced osteoporosis and improving bone structure and function.

[0056] Figure 9 In the figure, (a) is the trabecular thickness map, (b) is the trabecular number map, and (c) is the trabecular separation map. Figure 9 In animal studies, rats in the control group exhibited healthy trabecular bone structure, with greater trabecular thickness, a greater number of trabeculae, and a lower degree of trabecular separation, indicating a compact and well-mineralized skeletal structure and high bone density. Rats in the tail-suspension group showed significant changes in trabeculae, with decreased trabecular thickness, a significant decrease in trabecular number, and an increase in trabecular separation, indicating that the lack of mechanical load leads to trabecular degeneration and osteoporosis. After oral administration of the BS99 strain, the trabecular thickness of rats in the tail-suspension and oral administration groups recovered, the trabecular number rebounded, and the trabecular separation was significantly reduced, indicating that the BS99 strain can improve the symptoms of osteoporosis caused by tail suspension, promote trabecular remodeling and mineralization, and alleviate trabecular rarefaction.

[0057] Example 5 Identification of strain BS99 (1) Colony morphology identification Take one tube of glycerol bacteria with the collection number BS99 from the bacterial library, inoculate it into MRS liquid culture medium at a 3% (v / v) inoculation rate for activation, culture it in a shaking incubator at 37°C and 180 r / min for 24 h, then streak it on an MRS solid culture medium plate, and culture it upside down in a constant temperature incubator at 37°C for 24 h to obtain a single colony.

[0058] The colony is round in appearance, milky white, with a smooth and raised surface, neat edges and opaque.

[0059] (2) Physiological and biochemical tests Single colonies of the strain BS99 isolated and purified by the plate streak method with good growth were picked for Gram staining and catalase test, and its physiological and biochemical indices were measured. The test results were compared with the "Bergey's Manual of Systematic Bacteriology, 8th Edition" for preliminary identification of the strain. It was found that the strain BS99 was Gram-positive with a purple color, a spherical cell shape, negative for catalase and oxidase, and no spore formation.

[0060] (3) 16S rRNA identification 1) Extract strain gene DNA. The specific process is as follows: Step 1: Take 2 mL of bacterial culture medium, centrifuge at 12000 rpm for 1 min, remove the supernatant, and retain the bacteria; Step 2: Add 200 μL of lysozyme (final concentration: 20 mg / mL) to the cells and incubate at 37°C for at least 30 min. (Lysozyme buffer: 20 mM Tris, pH 8.0; 2 mM Na2-EDTA; 1.2% Triton X-100). Step 3: Add 200 μL of solution A to the cells, oscillate or pipette to fully suspend the cells, add 20 μL of RNase A (10 mg / mL) to the suspension, mix thoroughly by inversion, and let it stand at room temperature for 15-30 minutes; Step 4: Add 20 μL of proteinase K (10 mg / mL) to the tube, mix thoroughly, and digest at 55°C for 30-60 min. During the digestion period, invert the tube several times to mix until the sample is completely digested and the bacterial solution becomes clear and viscous. Step 5: Add 200 μL of solution B to the tube and mix thoroughly by inversion. If a white precipitate appears, place it at 75°C for 15-30 minutes. The precipitate will disappear and will not affect subsequent experiments. Step 6: Add 200 μL of anhydrous ethanol to the tube and mix thoroughly. Flocculent precipitates may appear at this time, which does not affect DNA extraction. Add both the solution and flocculent precipitates to the adsorption column and let it stand for 2 minutes. Step 7: Centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the adsorption column into the collection tube; Step 8: Add 600 μL of rinse solution to the adsorption column (check and add 60 mL of anhydrous ethanol before use). Centrifuge at 12,000 rpm for 1 minute, discard the waste solution, and place the adsorption column in a collection tube. Step 9: Add 600 μL of rinse solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the adsorption column in a collection tube; Step 10: Centrifuge at 12,000 rpm for 2 minutes. Leave the adsorption column open at room temperature for 15 minutes to remove any remaining rinse solution. Otherwise, the ethanol in the rinse solution may affect subsequent experiments such as enzyme digestion and PCR. Step 11: Place the adsorption column in a clean centrifuge tube, add 100 μL of eluent preheated in a 65°C water bath to the center of the adsorption membrane, let it stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 1 minute; Step 12: Add the eluate obtained by centrifugation to the adsorption column, place it at room temperature for 2 minutes, and centrifuge it at 12000 rpm for 2 minutes to obtain high-quality bacterial genomic DNA.

[0061] 2) PCR amplification of the 16S rRNA gene using the gene DNA as a template.

[0062] The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') were used as amplification primers.

[0063] The PCR reaction system was: DNA 2 μL, 27F 2 μL, 1492R 2 μL, Premix Ex Taq 25 μL, and ddH2O 19 μL.

[0064] PCR reaction conditions: pre-denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min; and final extension at 72°C for 5 min.

[0065] After DNA genomic DNA was extracted from the aforementioned strain BS99, 16S rRNA was amplified. The PCR amplification product was then sequenced (by Xiamen Sangon Biotech Co., Ltd.).

[0066] The sequencing results were searched for similar sequences in the NCBI database using Blast software, and the strain BS99 sequence was compared with the 16S rRNA gene sequences of related species obtained from the gene bank.

[0067] The gene sequence is as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1).

[0068] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).

[0069] The results of 16S rRNA gene sequencing are as follows:

[0070] In summary, based on the comprehensive analysis of the colony morphology, cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence and other data of strain BS99, and with reference to the Bergey's Manual of Systematic Bacteriology, 8th edition, the strain BS99 was identified as Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. lactis ).

[0071] Example 6 Preparation of a bacterial agent using the Lactococcus lactis subsp. lactis of the present invention Lactococcus lactis subsp. lactis BS99 was inoculated at 3% of the culture medium mass into MRS liquid medium sterilized at 121°C for 15 min. 12% enzymatically hydrolyzed skim milk, 0.8% glucose, 1.5% soy peptone, 0.6% yeast extract, and the balance water were added to the total mass of the culture medium and adjusted to pH 6.8. Then, after culturing at 37°C for 48 h, the culture medium was centrifuged at 6000 r / min for 20 min at 4°C, the supernatant was discarded, and the culture medium was washed 2-4 times with pH 7.2 phosphate buffer to obtain bacterial sludge, which was then resuspended in a protective agent to a concentration of 10 10 CFU / mL. The protective agent contained 150 g / L skim milk, 30 g / L fructooligosaccharides, 60 g / L sorbitol, and 10 g / L inulin. Finally, the suspension was pre-incubated at 37°C for 60 minutes and then freeze-dried. The survival rate of BS99 cells was calculated after freeze-drying.

[0072] The calculation formula for bacterial survival rate is: Bacterial survival rate (%) = (N / N0) × 100% (2) In formula (2), N is the number of viable bacteria after reconstitution of the freeze-dried bacterial agent, CFU / mL; N0 is the number of viable bacteria in the bacterial suspension before freeze-drying, CFU / mL.

[0073] The data measured in the above experiment were obtained by formula (2), and the survival rate of the lyophilized bacterial strain BS99 of the present invention was 92.6%, which well maintained the high activity of the strain.

[0074] It should be noted that the freeze-drying protective agent can adopt other protective agent compositions or formulas, including but not limited to the above-mentioned embodiment schemes.

[0075] Example 7 Preparation of probiotic products using Lactococcus lactis subsp. lactis of the present invention The probiotic product is a composition of Lactococcus lactis subspecies lactis and other ingredients; in the product, the number of viable Lactococcus lactis subspecies lactis is not less than 1×10 10 CFU / mL or 1×10 10 CFU / g.

[0076] The ingredients may include one or more of prebiotics, fillers, acidulants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, and filter aids.

[0077] The dosage form of the probiotic product can be compressed candy, solid beverage, beverage, granule, capsule, tablet, pill or oral liquid, etc.

[0078] More specifically, the present invention also provides an operational example of a method for manufacturing Lactococcus lactis subsp. lactis tablet candy: The Lactococcus lactis subsp. lactis strain is cultured in a liquid culture medium, the cells are collected and washed, auxiliary materials are added, and the cells are dried to prepare an active bacterial agent; 6 parts of the bacterial agent prepared in Example 6, 30 parts of sorbitol, 12 parts of whole milk powder, 8 parts of fermented kiwi fruit powder, 2 parts of oligomaltodextrose, 2 parts of magnesium stearate, 0.6 parts of vitamin C, 1 part of oligogalactose, and 1 part of oligofructose are mixed uniformly to obtain a Lactococcus lactis subsp. lactis tablet candy. The number of viable Lactococcus lactis subsp. lactis in the tablet candy is not less than 1×10 10 CFU / g.

[0079] Example 8 Preparation of bone-strengthening products using Lactococcus lactis subsp. lactis BS99 and peptides The bone-strengthening functional product is a composition of Lactococcus lactis subspecies lactis, small molecule active functional peptides and other ingredients; More specifically, the present invention also provides an operational example of a method for manufacturing a bone-strengthening functional product: Take 4g of bacterial agent, 3g of collagen peptide, 1g of sturgeon tendon peptide, 0.5g of sturgeon cartilage peptide, 1g of oligofructose, 0.3g of chitosan oligosaccharide, and 0.2g of vitamin D in 10g of the product and mix them evenly. The number of viable Lactococcus lactis subspecies in the bone strengthening product is not less than 1×10 10 CFU / g.

[0080] Example 9 A Chinese medicinal composition having the effect of strengthening bones and treating osteoporosis The traditional Chinese medicine composition consists of the following ingredients in parts by weight: 40-100 parts of Lactococcus lactis subspecies BS99 bacterial agent, 30-100 parts of astragalus, 20-100 parts of schisandra chinensis, 50-100 parts of wolfberry, 30-100 parts of trichosanthes, 25-100 parts of anemarrhena, 20-100 parts of kudzu root, 60-100 parts of angelica sinensis and 30-100 parts of salvia miltiorrhiza.

[0081] Example 10 A postbiotic product for improving bone health The Lactococcus lactis subsp. lactis BS99 bacterial agent in Example 6 was replaced with an inactivated bacterial agent, and maltodextrin and a sweetener were added to prepare a postbiotic product.

[0082] The inactivated fungicide is prepared by inoculating Lactococcus lactis subspecies BS99 into a fermentation broth containing skim milk, followed by fermentation, centrifugation, inactivation, and freeze-drying. The inactivation condition is to maintain the temperature at 115°C for 15 minutes. The number of inactivated bacteria is not less than 1×10 10 CFU / g.

[0083] It should be noted that the above concept is to prepare a probiotic product by combining Lactococcus lactis subsp. lactis with other ingredients, and the selection of ingredients and the ratio of the ingredient components can be adaptively adjusted, including but not limited to the embodiments.

[0084] In summary: The Lactococcus lactis subspecies BS99 of the present invention is a natural strain with high food safety. Its fermentation supernatant has a high butyrate production capacity and the ability to promote differentiation and calcium nodule production in mouse osteoblasts. It is also resistant to gastric acid and bile salts and has good intestinal colonization ability. In addition, it can improve the symptoms of osteoporosis in rats caused by tail suspension, promote the remodeling and mineralization of trabecular bone, alleviate the sparseness of trabecular bone, and improve the structure and function of bones. It has important application value and high commercial value in the fields of food, health products and medicines such as assisting bone health, promoting bone development and growth, and improving osteoporosis. For example: (1) Lactococcus lactis subspecies BS99 is used as a raw material component to prepare bacterial agents, probiotic products and traditional Chinese medicine compositions having the above functions; (2) Lactococcus lactis subspecies BS99 is fermented to obtain a fermentation product having the above functions; (3) Lactococcus lactis subspecies BS99 and small molecule active functional peptides are used as raw material components to prepare functional products with the above functions; (4) Lactococcus lactis subspecies BS99 is used as a raw material component to prepare postbiotics with the above functions; (3) Lactococcus lactis subspecies BS99 is used as a raw material component of a microecological preparation to prepare a microecological preparation with the above-mentioned efficacy.

[0085] It should be noted that: (1) The culture medium involved in the embodiments of the present invention is specifically: MRS liquid medium: 20.0 g glucose, 10.0 g tryptone, 10.0 g beef extract, 5.0 g yeast extract powder, 1.0 mL Tween 80, 2.0 g dipotassium hydrogen phosphate, 2.0 g ammonium citrate, 5.0 g anhydrous sodium acetate, 0.5 g magnesium sulfate, 0.25 g manganese sulfate monohydrate, 1 L deionized water, pH 6.5 (add 1.5% agar powder to make MRS solid medium); Unless otherwise specified, the experimental operation methods involved in the embodiments of the present invention are conventional experimental operation methods in the field, and the reagents or instruments involved can be purchased from regular channels.

[0086] (2) Definition of terms: As used herein, the term "food" in the context of "functional food" is used in a broad sense, encompassing food and drink for humans. In certain embodiments, the food product is suitable for and designed for human consumption. The present invention can be used to prepare solid preparations such as powders and tablets, and can also be dispersed in liquids to prepare liquid preparations suitable for oral administration to humans, including but not limited to powders.

[0087] In addition, the Lactococcus lactis subspecies lactis of the present application can be used as a raw material component of probiotic products, fermented products, functional foods and other products, but its efficacy includes but is not limited to increasing bone density, improving osteoporosis and other obvious effects, and other effects that can be beneficial to the human body.

[0088] Although terms such as fermentation broth, probiotic product, fermentation product, functional food, and postbiotic are frequently used herein, the use of other terms is not excluded. These terms are used merely to more conveniently describe and explain the essence of the present invention; interpreting them as any additional limitations is contrary to the spirit of the present invention.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )BS99, characterized by: Its deposit number is CGMCC No.33932.

2. A Lactococcus lactis subspecies BS99 bacterial agent, characterized in that: Its components include the Lactococcus lactis subspecies BS99 as described in claim 1.

3. A probiotic product of Lactococcus lactis subspecies BS99, characterized in that: Its components include the Lactococcus lactis subspecies BS99 as described in claim 1.

4. A functional product of Lactococcus lactis subspecies BS99, characterized in that: The functional product is a functional food or a health product, and its components include small molecule active functional peptides and the Lactococcus lactis subspecies BS99 as claimed in claim 1.

5. The functional product of Lactococcus lactis subspecies BS99 according to claim 4, characterized in that: The functional food includes one or more combinations of functional foods that assist in bone health, promote bone development and growth, improve osteoporosis symptoms, promote trabecular remodeling and mineralization, alleviate trabecular sparsity, and enhance bone structure, and foods that are resistant to acid and bile salts.

6. A fermentation liquid, characterized in that: Inoculate the Lactococcus lactis subspecies BS99 of claim 1 into a culture solution, culture at 30-40° C. for 24-48 hours, separate the culture solution into solid and liquid, collect the liquid, and obtain a fermentation solution; Wherein, the fermentation broth is a fermentation broth containing butyric acid.

7. A Chinese medicine composition, characterized in that: Its components include the Lactococcus lactis subspecies BS99 as described in claim 1.

8. A postbiotic product, characterized in that: The lactic acid Lactococcus lactis subspecies BS99 is inoculated into a culture solution containing skim milk, and the solution is fermented, centrifuged, inactivated, and freeze-dried; The Lactococcus lactis subspecies BS99 is the Lactococcus lactis subspecies BS99 described in claim 1.

9. A probiotic preparation of Lactococcus lactis subspecies BS99, characterized in that: The components include one or more of Lactococcus lactis subspecies BS99, fermentation broth of Lactococcus lactis subspecies BS99, precipitate of fermentation broth of Lactococcus lactis subspecies BS99, bacterial agent of Lactococcus lactis subspecies BS99, and freeze-dried bacterial powder of Lactococcus lactis subspecies BS99; The Lactococcus lactis subsp. lactis BS99 is the Lactococcus lactis subsp. lactis BS99 according to claim 1.

10. Use of the probiotic preparation as claimed in claim 9 in a bone strengthening product for improving osteoporosis and / or strengthening bone structure.

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