Lactobacillus salivarius combination HC1135 for regulating bone metabolism and improving bone loss as well as preparation and application of lactobacillus salivarius combination HC1135

Abstract

The invention relates to the technical field of screening and application of probiotics, in particular to combined lactobacillus salivarius HC1135 for regulating bone metabolism and improving bone loss as well as a preparation and application of the combined lactobacillus salivarius HC1135. The lactobacillus salivarius HC1135 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on February 20, 2023, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.26587. The lactobacillus salivarius HC1135 has the advantages that the lactobacillus salivarius HC1135 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on February 20, 2023, the preservation number is CGMCC No.26587; the strain can improve the bone metabolism state and reconstruct damaged bone microstructures by improving the level of indole-3-propionic acid (IPA) in a body, improving the balance of osteogenesis and osteoclast processes, relieving oxidative stress and inflammatory response, promoting bone formation and inhibiting bone resorption, and is suitable for preventing or assisting in improving related health problems such as bone loss.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotic screening and application, and particularly relates to a saliva combined lactobacillus HC1135 for regulating bone metabolism and improving bone loss as well as a preparation and application thereof. BACKGROUND

[0002] Bone loss and abnormal bone metabolism are the core pathological characteristics of many bone diseases such as osteoporosis, delayed fracture healing and inflammatory bone injury. The homeostasis of bone metabolism is maintained by the dynamic balance between bone formation and bone resorption. Osteoblasts are responsible for bone matrix synthesis, and osteoclasts are responsible for bone tissue resorption. When bone formation decreases or bone resorption increases, it can lead to decreased bone density, damaged bone microstructure and increased bone fragility, thereby causing osteoporosis and related complications. Especially in the case of long-term use of glucocorticoids, endocrine metabolic abnormalities, chronic inflammation and the like, bone metabolism disorder is aggravated, and the rate of bone loss is significantly increased, which seriously affects the motor function and quality of life of the body.

[0003] At present, the main drugs for improving bone loss in clinical use include bisphosphonates, calcitonin, parathyroid hormone analogs, selective estrogen receptor modulators, etc. These drugs mainly inhibit osteoclasts or promote osteoblasts, but still have the limitations of adverse reactions, poor compliance, high price, and the inability to simultaneously improve the bone microenvironment, inflammation level and oxidative stress. In addition, these drugs usually cannot effectively intervene in the imbalance of intestinal flora, lack of metabolites and other upstream factors of bone metabolism, and it is difficult to restore the homeostasis of bone metabolism from a systemic perspective.

[0004] In recent years, there have been some researches and reports on probiotics improving bone loss. For example, the patent application No. CN202011248842.0 discloses a lactobacillus rhamnosus for preventing and / or treating diseases caused by bone loss. The experimental results show that the strain has an inhibitory effect on bone loss in a mouse osteoporosis model induced by removing the ovary organ. In addition, the patent application No. CN202410628326.2 discloses a probiotic agent for improving osteoporosis and its application. The lactobacillus reuteri LR38 strain and the lactobacillus rhamnosus LRa05 strain are compounded, and it is found that the two strains have potential interaction and can cooperate with each other to synergistically improve osteoporosis caused by estrogen reduction.

[0005] Although some probiotics have been shown to have the potential to regulate bone metabolism and improve bone loss, the functional characteristics of probiotics are generally strain-specific. Different strains have significant differences in metabolic product production, immune regulation ability, mode of action on the intestinal barrier, and efficiency in regulating the "gut-bone axis". The reported effects of the strains are still limited, and the intensity, stability, safety, and industrialization of their ability to improve bone metabolism are still difficult to fully meet the market's demand for high-efficiency probiotics to intervene in bone metabolism.

[0006] With the increasing prevalence of unhealthy lifestyles, inadequate nutrition intake, and long-term stress, more and more individuals of different ages are at risk of bone metabolism disorders or bone loss. There is an urgent need for new probiotics with clear efficacy mechanisms, high safety, and scalable production. In particular, strains that can promote mineral absorption, improve bone microstructure, enhance osteogenic activity, inhibit osteoclast processes, and have anti-inflammatory and antioxidant abilities are considered important research directions for the next generation of bone health intervention products. Therefore, there is an urgent need to screen and develop innovative probiotic strains with biological safety, good tolerance, and significant bone metabolism regulation ability to improve bone loss, maintain bone metabolic homeostasis, and meet the application needs of future functional foods, nutritional supplements, special medical foods, and related industries. SUMMARY

[0007] To address the limitations of existing strains, unclear mechanisms, and difficulty in effectively improving bone metabolic homeostasis, the present application provides a saliva combined with lactobacillus HC1135, its preparation and application for regulating bone metabolism and improving bone loss. The strain can improve the balance between osteogenesis and osteoclast processes by increasing the level of indole-3-propionic acid (IPA) in the body, reduce oxidative stress and inflammation, promote bone formation and inhibit bone resorption, thereby improving bone metabolism and rebuilding damaged bone microstructure, and is suitable for preventing or assisting in improving bone loss and related health problems.

[0008] In a first aspect, the present application provides a saliva combined with lactobacillus HC1135 for regulating bone metabolism and improving bone loss. The saliva combined with lactobacillus (Lactobacillus salivarius) HC1135 has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on February 20, 2023, at the address of No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number of CGMCC No. 26587. Ligilactobacillus salivarius

[0009] Further, the 16s rDNA sequence of the saliva combined with lactobacillus HC1135 is SEQ ID NO: 3.

[0010] ​In a second aspect, the present application provides a probiotic preparation prepared from the above-mentioned Lactobacillus salivarius HC1135.

[0011] Further, the preparation method of the above-mentioned probiotic preparation comprises the following steps: (1) inoculate Lactobacillus salivarius HC1135 into MRS liquid medium at an inoculation amount of 1% by volume percentage, cultivate at 37℃ for 24h, then centrifuge the fresh bacterial liquid at a speed of 8000rpm for 10min, discard the supernatant, and obtain the bacterial body; after washing the bacterial body, resuspend it into a suspension, and obtain the bacterial suspension; (2) mix the bacterial suspension with edible carriers or excipients to prepare the probiotic preparation.

[0012] In a third aspect, the present application further provides a use of the above-mentioned Lactobacillus salivarius HC1135 in the preparation of a functional food or a drug for preventing or assisting in improving metabolic bone diseases, osteoporosis and related bone metabolic disorders.

[0013] Further, the functional food or the drug is a functional food or a drug for regulating bone metabolism and improving bone loss.

[0014] Further, the functional food or the drug is a functional food or a drug for reducing the serum pro-inflammatory factor IL-6 level and increasing the serum IPA level.

[0015] Further, the functional food or the drug is a functional food or a drug for promoting osteoblast activity and bone matrix deposition, inhibiting the number and function of osteoclasts, and reducing bone absorption.

[0016] The present application has the following advantages: 1. The Lactobacillus salivarius HC1135 provided by the present application has the effect of increasing the serum IPA level, which can promote osteoblast activity and bone matrix deposition to increase the bone formation level, and effectively inhibit the number and function of osteoclasts to reduce bone absorption and improve bone metabolism level. The Lactobacillus salivarius HC1135 can enhance bone density, improve bone microstructure, promote bone health development, and effectively prevent or assist in improving osteoporosis and other bone metabolism related diseases. The strain can be used alone without being compounded with prebiotics and / or other probiotics to boost the bone health development, which has important application value. Moreover, the Lactobacillus salivarius HC1135 has strong biological safety, can be used as a food raw material source, and has no side effects and excessive risk after long-term use.

[0017] 2、The saliva combined lactobacillus HC1135 provided by the application has strong resistance to artificial gastrointestinal fluid and can germinate in the artificial gastrointestinal fluid; the strain is sensitive to common antibiotics such as erythromycin, gentamicin and ampicillin, does not produce hemolysin, cannot dissolve blood cells, and has good biological safety. The strain can metabolize common carbon sources such as raffinose, raffinose, mannitol, sucrose, galactose and maltose, can ferment glucose to produce acid, and does not produce gas. The maximum tolerance salt concentration of the strain is 5%, the antioxidant capacity is relatively strong, the lipid peroxidation inhibition rate of the bacterial suspension is 80.67%, the lipid peroxidation inhibition rate of the fermentation supernatant is 82.67%; the DPPH clearance rate of the bacterial suspension reaches 87.67%, the DPPH clearance rate of the fermentation supernatant reaches 76.33%; the hydroxyl radical clearance rate of the bacterial suspension reaches 69.00%, and the hydroxyl radical clearance rate of the fermentation supernatant reaches 61.00%. The strain can also degrade cholesterol, and the degradation rate reaches 73.76%; in addition, the cell surface hydrophobicity of the strain is 78.43%, and the adhesion capacity is 75.23. The strain has an aflatoxin B1 clearance capacity of 85.00.

[0018] 3、In the study of improving bone health, the saliva combined lactobacillus HC1135 has good intervention effect on bone metabolism and bone loss. Through continuous administration, it is found that HC1135 can reduce the excretion level of calcium and phosphorus in the urine of rats, increase the content of calcium and phosphorus in the serum, help to improve the mineral utilization efficiency and alleviate bone loss. At the same time, HC1135 can reduce the bone absorption metabolic intensity and promote the bone metabolism balance to restore to the bone formation direction.

[0019] 4、In terms of inflammation and metabolic factors, the level of pro-inflammatory factor IL-6 in the serum of rats given HC1135 decreases, and the level of IPA increases, indicating that HC1135 has the effect of improving inflammatory bone damage and regulating intestinal-bone axis metabolism. Further evaluation found that the metabolic state of osteoblasts and osteoclasts of rats given HC1135 is improved: osteoblast-related differentiation and osteogenic ability are enhanced, while osteoclast activity is weakened, thereby promoting bone formation and inhibiting bone resorption. In addition, the mineral deposition rate of bone tissue is improved, which is conducive to bone mass accumulation and bone structure stability.

[0020] 5. In terms of bone mineral density and bone microstructure, the bone mineral density (BMD), bone volume fraction (BV / TV), trabecular number (Tb.N) and trabecular thickness (Tb.Th) of the rats intervened by HC1135 were increased compared with the model control group; the trabecular separation (Tb.Sp) and structure model index (SMI) were decreased, indicating that the number, thickness and connectivity of the bone trabecula were improved. Compared with IPA alone, HC1135 showed better performance in improving the above indicators. In summary, HC1135 can enhance bone anabolic activity, increase bone mass and bone density, promote the recovery of bone trabecular structure, make the formation activity of bone trabecula higher than the decomposition activity, and improve bone metabolism and bone microstructure at multiple levels. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows. Obviously, those drawings can also provide other drawings for those skilled in the art without creative effort.

[0022] Figure 1 It is a colony morphology diagram of HC1135 strain.

[0023] Figure 2 It is a crystal violet staining microscope photograph of HC1135 strain.

[0024] Figure 3 It is a RAPD fingerprint map of HC1135 strain.

[0025] Figure 4 It is a rep-PCR fingerprint map of HC1135 strain.

[0026] Figure 5 It is the body weight change result of rats in the experimental period.

[0027] Figure 6 It is the determination result diagram of urine calcium, urine phosphorus, blood calcium and blood phosphorus content of rats in each group; wherein, (a) is the determination result of urine calcium content, (b) is the determination result of urine phosphorus content, (c) is the determination result of blood calcium content, and (d) is the determination result of blood phosphorus content.

[0028] Figure 7 It is the determination result diagram of serum IL-6 and IPA content of rats in each group; wherein, (a) is the determination result of IL-6 content, and (b) is the determination result of IPA content.

[0029] Figure 8 It is the bone metabolism level change diagram of rats in each group, wherein, (a) is the determination result of osteoclast number per bone perimeter, (b) is the determination result of osteoblast number per bone perimeter, and (c) is the determination result of mineral deposition rate.

[0030] Figure 9 Figures of Micro-CT scanning analysis results of femoral trabecular bone of rats in each group; (a) is the bone density measurement result, (b) is the bone volume fraction measurement result, (c) is the trabecular bone separation measurement result, (d) is the trabecular bone thickness measurement result, (e) is the trabecular bone structure model index measurement result, and (f) is the trabecular bone quantity measurement result. DETAILED DESCRIPTION

[0031] In order for those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor should fall within the scope of protection of the present application.

[0032] Example 1 Isolation and screening of saliva combined with Lactobacillus HC1135 1. Preliminary screening Prepare MRS agar medium, adjust pH to 6.2-6.5, and sterilize at 121°C for 15 min.

[0033] Take 1 g of fresh fermented cheese sample obtained from the Inner Mongolia herdsman's family, dilute with sterile normal saline, and put into a sterile sample bag. Mix well with a homogenizer. Take 100 μL of the mixed solution and gradient dilute, spread on MRS agar medium, and incubate at 37°C for 48 h in anaerobic culture. Wait for the plate to grow single colony and observe under a microscope. According to the observation results, 40 potential Lactobacillus strains were screened out, named HC1100, HC1101, …, HC1139, HC1140, respectively.

[0034] 2. Rescreening Prepare 1 L of MRS liquid medium, sterilize at 121°C for 15 min, and then add 3.2 g of porcine mucosa pepsin, shake to dissolve, and place in a 37°C water bath shaker for 1 h to prepare an acid-resistant medium. Inoculate the 40 Lactobacillus strains obtained by screening into the acid-resistant medium at an inoculation amount of 6%, and incubate at 37°C for 48 h in anaerobic culture. Take the fermentation broth for bacterial count.

[0035] The results show that among the 40 Lactobacillus strains obtained by preliminary screening, the HC1135 strain has the highest viable cell count after rescreening with acid-resistant medium, with a log value of 10.59 Log CFU / mL, indicating that the HC1135 strain has the highest acid resistance.

[0036] The HC1135 strain was inoculated on MRS agar medium, and after 24 h of anaerobic culture at 37°C, the HC1135 single colony was milky white, with a colony diameter of about 1.5-2 mm, a smooth and moist surface, a round shape, no transparency, a neat edge, a surface elevation, and a colony photograph as shown in Figure 1 After crystal violet staining, the HC1135 strain was short rod-shaped under a microscope, with smooth ends, arranged in single, pairs, or chains, gram-positive, and no spores formed. The optical microscope photograph is as shown in Figure 2 .

[0037] Example 2 Identification of Saliva Combined with Lactobacillus HC1135 In this embodiment, the preparation of the inoculum is as follows: under sterile conditions, an appropriate amount of fresh HC1135 strain bacterial liquid was centrifuged at 5000 rpm for 5 min, washed twice with PBS buffer, and then diluted 50 times with the same volume of PBS buffer after re-suspending the bacterial bodies, to serve as the inoculum.

[0038] 1. Temperature growth range experiment Under sterile conditions, the inoculum was inoculated into 10 mL of MRS liquid medium at a 10% inoculation amount, and 10 mL of MRS liquid medium without inoculation was used as a control, which was placed in a 15°C constant temperature shaking incubator for 7 days, and in 37°C, 45°C, and 60°C constant temperature shaking incubators for 2 days, respectively, to observe whether the culture liquid became turbid.

[0039] The results show that after 7 days of constant temperature culture at 15°C, the culture medium remained clear; after 2 days of constant temperature culture at 37°C, the culture medium became turbid; and after 2 days of constant temperature culture at 45°C and 60°C, the culture medium remained clear. Thus, it is shown that the HC1135 strain cannot grow at 15°C and below and at 45°C and above, and can grow normally at 37°C.

[0040] 2. Catalase determination experiment Fresh bacterial liquid was taken and dropped on a glass slide, and then 3%-15% hydrogen peroxide was added. The results show that the HC1135 strain does not produce bubbles, and the catalase reaction is negative.

[0041] 3. Carbon source metabolism experiment The formula of the phenol red-containing basic medium used in this embodiment is as follows: Proteose peptone 1.5 g; yeast extract 0.6 g; Tween 80 0.1 g; salt solution 0.5 mL; phenol red 18 mg; distilled water 100 mL; pH 7.4±0.2. The composition of the salt solution: MgSO4·7H2O 11.5 g, MnSO4·4H2O 8 g, distilled water 100 mL.

[0042] Prepare 10 g / mL sugar, alcohol and glycoside carbohydrate solution and filter with 0.22 μm sterile filter. Under sterile conditions, add 20 μL of the sterilized carbohydrate solution to a 96-well plate, 4 parallels for each carbohydrate, then add 170 μL of sterilized base medium containing phenol red, add 10 μL of inoculum, and the un-inoculated wells serve as controls. Add 50 μL of liquid paraffin to each well to prevent water evaporation during incubation. Incubate at 37°C, and observe the color change of the medium using phenol red as an indicator. The results are shown in Table 1.

[0043] Table 1 Metabolism of carbon sources by HC1135 strain

[0044] 4. Glucose acid and gas production test The medium used in this test is as follows: Peptone 0.5 g; yeast extract 0.3 g; Tween 80 0.1 mL; salt solution A 0.5 mL; salt solution B 0.5 mL; sodium acetate 0.5 g; glucose 2.5 g; 2% bromocresol green (w / v) 0.05 mL; distilled water 100 mL; pH 6.8-7.0.

[0045] Dispense the prepared medium into large test tubes containing inverted small test tubes, 3 mL per tube, and autoclave at 121°C for 15 min.

[0046] Salt solution A: KH2PO4 10 g, K2HPO4 1.0 g, dissolved in distilled water and made up to 100 mL.

[0047] Salt solution B: MgSO4·7H2O 11.5 g, MnSO4·2H2O 2.4 g, FeSO4·7H2O 0.68 g, dissolved in distilled water and made up to 100 mL.

[0048] Under sterile conditions, inoculate the medium with the inoculum at a 10% inoculum, and the un-inoculated medium serves as a control, then seal the top with 2 mL of sterile liquid paraffin, and incubate at 37°C. Observe the color change of the medium every day for 6 days. The results show that after 6 days of incubation at 37°C, the medium changes from green to yellow, and there is no gas in the small inverted tube, indicating that the HC1135 strain produces acid but not gas during glucose fermentation.

[0049] 5. Molecular biology identification Pick a single colony of the HC1135 strain from the plate and incubate it in MRS broth at 37°C for 24 hours, then take 500 μL of the fermentation broth and follow the procedures of the Genomic DNA Extraction Kit (Catalog No. DP302) to obtain the genome of the strain, which is used for subsequent molecular biology identification.

[0050] 5.1 16s rDNA gene sequence identification 16s rDNA gene amplification 1) Primer sequence: 27F: AGAGTTTGATCCTGGCTCA (SEQ ID NO: 1); 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 2); 2) Reaction system (50 μL) Table 2 16s rDNA PCR amplification system

[0051] 3) Electrophoresis verification The nucleic acid electrophoresis result of the PCR product is about 1500 bp, which meets the requirements.

[0052] 4) PCR product sequencing The sequencing result shows that the 16s rDNA sequence of HC1135 strain is as follows:

[0053] The sequence was subjected to BLAST alignment in the NCBI database, and the highest similarity was found with Lactobacillus salivarius. Therefore, the HC1135 strain was preliminarily determined as Lactobacillus salivarius.

[0054] 5.2 RAPD fingerprint identification 1) Primer sequence: GAGGGTGGCGGTTCT (SEQ ID NO: 4).

[0055] 2) RAPD reaction system Table 3 RAPD reaction system (20 μL)

[0056] 3) Electrophoresis A 1.5% agarose gel plate was prepared, DL2000 DNA Marker was used as a result control, and constant voltage electrophoresis was performed at 100 V for 80 min. Finally, the electrophoretogram was detected by using a gel imaging system, and the RAPD fingerprint of the HC1135 strain is shown in Figure 3 .

[0057] 5.3 rep-PCR fingerprint identification 1) rep-PCR primer sequence: GTGGTGGTGGTGGTG (SEQ ID NO: 5) 2) rep-PCR reaction system Table 4 rep-PCR reaction system (20 μL)

[0058] 3) Electrophoresis DL2000 DNA Marker was used as a result control, and the amplification result was detected by electrophoresis at a voltage of 100 V for 80 min, and the rep-PCR fingerprint of the HC1135 strain is shown in Figure 4 .

[0059] The colony morphology and physiological and biochemical characteristics of the HC1135 strain were uploaded to the website http: / / www.tgw1916.net / bacteria_logare_desktop.htmL, and the results published in the literature De Clerck E, et al. Systematic and Applied Microbiology, 2004, 27 (1) 50 were compared. Based on the molecular biology identification results, the HC1135 strain was determined to be a new Lactobacillus salivarius (Lactobacillus gasseri) Ligilactobacillus salivarius). The strain was named Lactobacillus salivarius HC1135.

[0060] The HC1135 strain was sent to the China General Microbiological Culture Collection Center (CGMCC) for preservation on February 20, 2023, and the preservation address was No. 3, Beichen West Road, Chaoyang District, Beijing, China, and the preservation number was CGMCC No. 26587, and the classification name was Lactobacillus salivarius Ligilactobacillus salivarius .

[0061] Example 2 Salinity tolerance test of Lactobacillus salivarius HC1135 An appropriate amount of fresh HC1135 strain bacterial liquid was centrifuged at 5000 rpm for 5 min, washed twice with PBS buffer, and then diluted 50 times with the same volume of PBS buffer as the inoculum. Under sterile conditions, 190 μL of BSM liquid medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% was added to the 96-well plate, with 3 replicates for each salt concentration, and then 10 μL of inoculum was added. The un-inoculated wells served as controls. Each well was added with 50 μL of autoclaved paraffin oil to prevent water evaporation during the culture process. The plate was incubated at 37°C, and the turbidity of the culture medium was observed.

[0062] The results of the salinity tolerance test showed that the maximum salt concentration tolerated by the HC1135 strain was 5%.

[0063] Example 3 Gastrointestinal fluid tolerance test of Lactobacillus salivarius HC1135 An appropriate amount of freshly cultured Lactobacillus salivarius HC1135 bacterial liquid was centrifuged to collect the bacterial cells, which were then resuspended in sterile physiological saline as the test bacterial suspension and subjected to viable cell counting. Equal volumes of artificial gastric juice and artificial intestinal juice were taken in sterile centrifuge tubes. The artificial gastric juice was adjusted to pH 2.0 and contained 0.5% pepsin, while the artificial intestinal juice was adjusted to pH 7.5 and contained 0.3% pancreatin and 0.5% bovine bile salt. The test bacterial suspension was added to the artificial gastric juice at a volume ratio of 1:1 and incubated at 37°C for 2 h with shaking. Then, an equal amount of the mixture was taken and immediately subjected to viable cell counting. Another portion of the bacterial suspension was added to the artificial intestinal juice at the same volume ratio and incubated at 37°C for 3 h with shaking. After the incubation, viable cell counting was also performed. The viable cell counting method followed the national standard "GB4789.35-2016 Food Microbiological Examination Lactic Acid Bacteria Examination".

[0064] Table 5 Viable cell count after artificial gastric and intestinal juice digestion (Log CFU / mL)

[0065] As shown in Table 5, the saliva combined Lactobacillus HC1135 used in the application can resist artificial gastric juice and artificial intestinal juice, and has good gastric acid resistance and bile salt resistance.

[0066] Example 4 Hemolytic property and antibiotic resistance experiment of saliva combined Lactobacillus HC1135 1. Hemolytic property experiment The components of TBS basic medium were weighed, dissolved, and autoclaved at 121°C for 15 min. When the medium was cooled to 50°C, 5% sterile defibrillated sheep blood was added and mixed, and then poured into a plate. The test strain was streaked on the prepared blood cell plate, and cultured in a 37°C incubator. After 24-48 h, whether the test strain had hemolytic property was observed.

[0067] The results show that the blood cell plate has no change, and the saliva combined Lactobacillus HC1135 cannot grow on the blood cell plate, indicating that the saliva combined Lactobacillus HC1135 does not produce hemolysin and cannot dissolve blood cells, and has good biological safety.

[0068] 2. Antibiotic resistance experiment The minimum inhibitory concentration (MIC) of antibiotics on the saliva combined Lactobacillus HC1135 was determined by micro-broth dilution method, and the specific results are shown in Table 6.

[0069] Table 6 Antibiotic MIC value (μg / mL) of saliva combined Lactobacillus HC1135

[0070] As shown in the results of Table 6, the saliva combined Lactobacillus HC1135 provided by the application is sensitive to common antibiotics such as erythromycin, gentamicin, ampicillin and clindamycin, and has good biological safety.

[0071] Example 5 Determination of antioxidant function of saliva combined Lactobacillus HC1135 1. Determination of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging ability of the strain 1 mL of PBS bacterial suspension of HC1135 strain and 1 mL of fermentation supernatant were taken, 1 mL of freshly prepared 0.4 mM DPPH radical solution was added, and then the mixture was uniformly mixed and then placed in a room temperature light-shielded reaction for 30 min. Then the absorbance A sample of the sample at a wavelength of 517 nm was determined, and 3 parallel measurements were performed. The control sample was mixed with PBS solution and DPPH-ethanol in equal volumes, and PBS bacterial suspension and ethanol were mixed in equal volumes to zero. The clearance rate was calculated according to the following formula: clearance rate % = [1- (A sample-A blank) / A control] x 100%. The results are shown in Table 7.

[0072] Table 7 DPPH radical scavenging rate

[0073] 2. Determination of the ability of strains to scavenge hydroxyl radicals After mixing 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water and 200 μL of lactic acid bacterial PBS bacterial suspension, 100 μL of hydrogen peroxide solution (3 mM) was added. After 15 min of 37°C water bath, the absorbance of the sample was measured at 510 nm. The calculation formula of the hydroxyl radical scavenging rate was: scavenging rate (%) = (A sample - A control) / (A blank - A control) x 100%, wherein A control is deionized water instead of the sample, and A blank is deionized water instead of the sample and H2O2. The results are shown in Table 8.

[0074] Table 8 Hydroxyl radical scavenging rate

[0075] 3. Identification of strains by anti-lipid peroxidation experiment Preparation of linoleic acid emulsion: 0.1 mL of linoleic acid, 0.2 mL of Tween 20, 19.7 mL of deionized water.

[0076] 0.5 mL of PBS solution (pH 7.4) was added with 1 mL of linoleic acid emulsion, 1 mL of FeSO4 (1%), and then 0.5 mL of sample was added. After 1.5 h of 37°C water bath, 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) were added to the mixture. After 30 min of 100°C water bath, the mixture was quickly cooled, centrifuged at 4000 rpm for 15 min, and the supernatant was collected and the absorbance was measured at 532 nm, which was A. The control group was 0.5 mL of distilled water instead of the sample, which was A0. The calculation formula was: inhibition rate (%) = (A0-A) / A0x100%, wherein A is the absorbance of the sample group, and A0is the absorbance of the control group. The results are shown in Table 9.

[0077] Table 9 Anti-lipid peroxidation inhibition rate

[0078] Example 6 In vitro cholesterol degradation test of saliva combined with Lactobacillus HC1135 1. Preparation of cholesterol micellar solution: 1 g of cholesterol was accurately weighed, dissolved in anhydrous ethanol, and made up to 100 mL. Under sterile conditions, it was filtered to remove bacteria with a 0.22 μm microporous filter membrane.

[0079] 2. Take 10.0 g of proteose peptone, 10.0 g of beef extract, 5.0 g of yeast extract, 2.0 g of diammonium hydrogen citrate, 20.0 g of glucose, 1.0 mL of Tween 80, 5.0 g of sodium acetate, 0.1 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of potassium phosphate dibasic, 1000 mL of distilled water, adjust the pH value to 7.3, sterilize at 115°C for 30 min, then add a cholesterol solution to make the final concentration of cholesterol 0.1%. Inoculate fresh bacterial liquid according to the inoculation amount of 0.1%, and cultivate the Lactobacillus salivarius HC1135 at 37°C for 48 hours. Then take 0.2 mL of bacterial liquid, add 1.8 mL of anhydrous ethanol, mix well, stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and take the supernatant for determination of cholesterol content. The determination method of cholesterol is according to GB / T 5009.128-2003 "Determination of Cholesterol in Food".

[0080] The results show that the cholesterol degradation rate of the Lactobacillus salivarius HC1135 provided by the application in vitro is 73.76±2.57 (%).

[0081] Example 7: Test of hydrophobic cell surface of Lactobacillus salivarius HC1135 1. Preparation of bacterial liquid to be tested: pick the purified Lactobacillus salivarius HC1135 colonies, inoculate in freshly prepared MRS liquid medium, and cultivate at 40°C for 24-48 h. Then inoculate in MRS liquid medium according to the inoculation amount of 1% (V / V), continue to cultivate at 40°C for 24-48 h, centrifuge at 6000 x g for 10 min, collect the bacterial cells, wash twice with sterile normal saline, resuspend the bacterial cells with 1 mL of sterilized 0.1M KNO3 solution, and use as the bacterial liquid to be tested.

[0082] 2. Determination of surface hydrophobicity: take 50 μL of the above bacterial suspension and add 2450 μL of 0.1M KNO3, and record OD 600 as A0, take 1.5 mL of the bacterial suspension and mix with 500 μL of xylene, then stand at room temperature for 10 min (at this time, a two-phase system is formed). Vortex the two-phase system for 2 min, then stand for 20 min to re-form the water phase and the organic phase. Carefully take the water phase (without sucking the organic phase) and measure the absorbance at 600 nm as A1. The cell hydrophobicity is calculated according to the formula Hydrophobicity% = (A0-A1) / A1 x 100%, and the average value of three tests is taken.

[0083] The results show that the cell surface hydrophobicity of the Lactobacillus salivarius HC1135 provided by the application is 78.43±6.22 (%).

[0084] Example 8: Test of cell adhesion of Lactobacillus salivarius HC1135 Caco-2 cells were seeded in six-well plates at a seeding density of 2 x 10 6 cells / well after resuscitation, culture and counting, and incubated in a carbon dioxide incubator for 24 h for cell adhesion experiments. The Lactobacillus salivarius HC1135 strain in the logarithmic growth phase was resuspended in MRS medium to 5 x 10 7 CFU / mL. 1 mL of the above strain was added to the six-well plate with the cells adhered, and incubated in a carbon dioxide incubator for 2 h. PBS was repeatedly washed 3 times to remove the unadhered bacteria. 500 μL of trypsin was added for 3 min, 1.5 mL of cell culture solution was then added to terminate the digestion and repeatedly blown, and the resulting solution was collected into a sterile EP tube, and the collected solution was gradient diluted by 10 times, 100 times, 1000 times and 10000 times, plated and counted, and the cells of the blank group were counted. The adhesion ability of the test strain was calculated according to the following formula: adhesion ability (CFU / cell) = total number of adhered bacteria in each culture well / total number of cells in each culture well.

[0085] The results show that the adhesion ability of Lactobacillus salivarius HC1135 is 75.23 ± 0.02 (CFU / cell).

[0086] Example 9 Determination of aflatoxin B1 clearance ability of Lactobacillus salivarius HC1135 1. Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus and other fungi. There are 18 kinds of aflatoxins isolated so far, among which aflatoxin B1 (AFB1) is confirmed as a class I carcinogen by the International Cancer Research Agency due to its strong carcinogenic, mutagenic and teratogenic properties. A trace amount of aflatoxin will have harmful effects on humans and animals.

[0087] 2. Prepare AFB1 working solution according to the kit instructions, and take AFB1 standard into a certain volume of sterilized PBS solution to make the final concentration of AFB1 1 μg / mL. Take 1 mL of fresh bacterial solution and centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the bacterial body with PBS buffer solution twice, centrifuge at 8000 rpm for 5 min, and collect the bacterial body. Then resuspend the bacterial body in 1 mL of the above AFB1 working solution, and place it in a 37°C constant temperature incubator. After 1 h, take it out, centrifuge at 8000 rpm for 10 min, and take the supernatant for testing. Set up a blank control, AFB1 PBS solution without bacteria (AFB1 concentration 5 μg / mL), and set up 3 parallels.

[0088] 3. According to the instructions of the aflatoxin B1 enzyme-linked immunoassay kit (purchased from Wuhan Huamei Biological Engineering Co., Ltd., product number: CSB-E14087), the AFB1 concentration in the reaction solution was determined. The residual AFB1 concentration in the reaction solution was calculated using the special analysis software provided by the kit manufacturer, and then the AFB1 clearance rate of each strain was calculated.

[0089] The results showed that the aflatoxin B1 clearance ability of saliva combined with Lactobacillus HC1135 was 85.00±7.42 (%).

[0090] Example 10 Effect of Lactobacillus salivarius HC1135 on regulating bone metabolism in rats 1. Experimental animals SPF grade SD rats, half male and half female, 14 weeks old, 24 rats, weighing 190-210 g, provided by Qinglongshan Animal Breeding Center, license number SCXK (Su) 2017-0001. Conventional pellet feed (Jiangsu Cooperation Biological Co., Ltd.) was fed, and the rats were raised in SPF animal room. 12h / 12h light / dark cycle, free feeding and drinking water, temperature maintained at 20-26°C, relative humidity 40%-70%.

[0091] 2. Experimental method After 7 days of adaptive feeding, the rats were randomly divided into normal group (NOR), modeling group (PLA), low-dose probiotic group (L-PRO) and high-dose probiotic group (H-PRO), 6 rats in each group. After adaptive feeding, from the day of modeling, the L-PRO group and the H-PRO group were injected with glucocorticoids for 8 weeks, twice a day, with an injection dose of 4.5 mg / kg, and at the same time, the probiotic bacteria liquid was given by gavage at a dose of 0.2 mL / 10 g per day, the probiotic bacteria concentration of the L-PRO group was 1×10 9 CFU / mL, and the probiotic bacteria concentration of the H-PRO group was 1×10 11 CFU / mL; the PLA group of rats was injected with glucocorticoids for 8 weeks, twice a day, with an injection dose of 4.5 mg / kg, and at the same time, the same amount of MRS liquid medium was given by gavage; the NOR group was not modeled, and normal saline was injected for 8 weeks, twice a day, with an injection dose of 200 μL. At the same time, normal saline was given by gavage at a dose of 0.2 mL / 10 g per day. After the injection of the rats was completed, the rats were normally fed, and the state of the rats was observed daily, and the body weight of the rats was recorded every week.

[0092] 3. Detection index At the end of the experiment, 10 mL of blood was taken from the abdominal aorta of the rats, and serum samples were obtained by centrifugation. The levels of interleukin-6 (IL-6) and IP A were measured using ELISA kits, and the calcium and phosphorus contents in the blood of the rats were measured. At the end of the experiment, the overnight urine of the rats was collected to measure the calcium and phosphorus contents.

[0093] The femur tissues of the rats were fixed in formaldehyde and then decalcified in a decalcification solution for 1 month, with the decalcification solution being replaced every 3 days. Subsequently, the tissues were dehydrated, transparentized, and embedded in paraffin. Sections with a thickness of 5 pm were prepared and stained with hematoxylin-eosin. The number of osteoblasts was quantitatively analyzed using OsteoMeasure software. Tartrate-resistant acid phosphatase staining was used to label osteoclasts. Positive cells were defined as having 3 or more multinucleated cells with a red-purple color. The number of osteoclasts was quantitatively analyzed using OsteoMeasure software. At 10 days before the end of the experiment, 15 mg / kg of calcein was injected intraperitoneally. Immediately after the rats were sacrificed, the femurs were removed and fixed in 40 g / L paraformaldehyde. Hard tissue sections were prepared without decalcification. The distance between the two fluorescent calcium bands was measured to quantify the mineral deposition rate and assess the bone formation rate.

[0094] 4. Data analysis Statistical software was used for data analysis. The results in this evaluation process are expressed as the mean ± standard error (x ± SEM). Single-factor analysis of variance was used to compare differences between multiple groups. When the variances were equal, the least significant difference method was used for pairwise comparison. When the variances were not equal, the rank sum test was used. P < 0.05 indicates that the difference is significant, and P < 0.01 represents that the difference is extremely significant. Software was used for plotting.

[0095] 5. Test results 5.1 Changes in the body weight of rats The changes in the body weight of rats during the entire test period are shown in Table 1. Figure 5The body weight of the rats in the NOR group increased from 202.67 g in the first week to 257.17 g in the eighth week, an increase of 54.5 g or 26.89% compared with the first week. The body weight of the rats in the PLA group increased from 196.83 g in the first week to 258.50 g in the eighth week, an increase of 61.7 g or 31.33% compared with the first week. The body weight of the rats in the L-PRO group increased from 195.33 g in the first week to 259.50 g in the eighth week, an increase of 64.2 g or 32.85% compared with the first week. The body weight of the rats in the H-PRO group increased from 197.17 g in the first week to 260.33 g in the eighth week, an increase of 63.2 g or 32.04% compared with the first week. In the eighth week, there was no significant difference in the body weight of the rats in each group. During the entire experimental period, the body weight of all the rats showed a stable growth trend, and the body weight at each week was within the normal range of the body weight of rats, indicating that the rats in each group grew normally and the modeling process did not affect the growth of the rats.

[0096] 5.2 Determination of the calcium and phosphorus contents in the urine of rats At the end of the experiment, the calcium content in the urine of the rats in the PLA group, the L-PRO group and the H-PRO group was significantly higher than that in the NOR group, indicating that there was a loss of calcium in the body of the rats in the PLA group, the L-PRO group and the H-PRO group. Among them, the urine calcium content of the PLA group was significantly increased compared with the NOR group, and the difference was extremely significant (P<0.01), indicating that the modeling was successful. The calcium content in the urine of the L-PRO group was decreased compared with the PLA group, and the difference was extremely significant (P<0.01). The calcium content in the urine of the H-PRO group was decreased compared with the PLA group, and the difference was extremely significant (P<0.01). The calcium content in the urine of the H-PRO group was decreased compared with the L-PRO group, and the difference was not significant (P>0.05). The determination results are shown in (a) of FIG. 5. Figure 6

[0097] At the end of the experiment, the phosphorus content in the urine of the rats in the PLA group, the L-PRO group and the H-PRO group was significantly higher than that in the NOR group, indicating that there was a loss of phosphorus in the body of the rats in the PLA group, the L-PRO group and the H-PRO group. Among them, the urine phosphorus content of the PLA group was significantly increased compared with the NOR group, and the difference was extremely significant (P<0.01), indicating that the modeling was successful. The phosphorus content in the urine of the L-PRO group was decreased compared with the PLA group, and the difference was extremely significant (P<0.01). The phosphorus content in the urine of the H-PRO group was decreased compared with the PLA group, and the difference was extremely significant (P<0.01). The phosphorus content in the urine of the H-PRO group was decreased compared with the L-PRO group, and the difference was also extremely significant (P<0.01). The determination results are shown in (b) of FIG. 5. Figure 6 ​(b) shown in FIG. 1.

[0098] From the changes of urine calcium and urine phosphorus of rats in each group, it can be seen that the rats in PLA group, L-PRO group and H-PRO group have calcium element and phosphorus element loss phenomenon. After the intervention of low dose and high dose of saliva combined with lactobacillus HC1135, the calcium element and phosphorus element loss phenomenon are significantly improved. And for the calcium element loss phenomenon, the intervention effect of low dose and high dose of probiotics is the same, and for the phosphorus element loss phenomenon, the intervention effect of high dose of probiotics is better than that of low dose of probiotics.

[0099] 5.3 Determination of blood calcium and phosphorus content of rats At the end of the experiment, the calcium content in the blood of rats in PLA group, L-PRO group and H-PRO group decreased compared with NOR group, indicating that there was calcium loss in the body of rats in PLA group, L-PRO group and H-PRO group. Among them, the blood calcium content of PLA group decreased significantly compared with NOR group, with extremely significant difference (P<0.01), indicating that the modeling was successful. Compared with PLA group, the calcium content in the blood of L-PRO group increased, with significant difference (P<0.05). Compared with PLA group, the calcium content in the blood of H-PRO group increased, with extremely significant difference (P<0.01). Compared with L-PRO group, the calcium content in the blood of H-PRO group increased, with no significant difference (P>0.05), and the determination results are shown in FIG. 1. Figure 6 (c) shown in FIG. 1.

[0100] At the end of the experiment, compared with NOR group, the phosphorus content in the blood of rats in PLA group, L-PRO group and H-PRO group decreased significantly, indicating that there was phosphorus element loss in the body of rats in PLA group, L-PRO group and H-PRO group. Among them, compared with NOR group, the blood phosphorus content of PLA group decreased significantly, with extremely significant difference (P<0.01), indicating that the modeling was successful. Compared with PLA group, the phosphorus content in the blood of L-PRO group increased, with significant difference (P<0.05). Compared with PLA group, the phosphorus content in the blood of H-PRO group increased, with extremely significant difference (P<0.01). Compared with L-PRO group, the phosphorus content in the blood of H-PRO group increased, with no significant difference (P>0.05), and the determination results are shown in FIG. 1. Figure 6 (d) shown in FIG. 1.

[0101] From the changes of blood calcium and blood phosphorus of rats in each group, it can be seen that the rats in PLA group, L-PRO group and H-PRO group have calcium element and phosphorus element loss phenomenon. After the intervention of low dose and high dose of saliva combined with lactobacillus HC1135, the calcium element and phosphorus element loss phenomenon are significantly improved.

[0102] Serum calcium, phosphorus, urinary calcium and phosphorus are important biochemical markers of bone metabolism, and changes in their levels can directly reflect the state of bone metabolism. For example, phosphate, as the main component of bone, plays a crucial role in maintaining bone strength. The excretion of urinary calcium and phosphorus can reflect the intensity of bone resorption, and excessive excretion of urinary calcium and phosphorus may indicate enhanced bone resorption activity. After modeling, the excretion of urinary calcium and phosphorus in rats increased, and the content of calcium and phosphorus in blood decreased. The use of low-dose and high-dose saliva combined with lactobacillus HC1135 can reverse the loss of calcium and phosphorus elements in rats, reduce the intensity of bone resorption metabolism in rats, and help improve the level of bone metabolism in rats.

[0103] 5.4 Rat serum cytokine factor determination At the end of the experiment, the serum IL-6 content of the PLA group was significantly higher than that of the NOR group (P < 0.01). Compared with the PLA group, the serum IL-6 content of the L-PRO group was reduced, but the difference was not significant (P > 0.05). Compared with the PLA group, the serum IL-6 content of the H-PRO group was reduced, and the difference was significant (P < 0.05). Compared with the L-PRO group, the serum IL-6 content of the H-PRO group was reduced, but the difference was not significant (P > 0.05). The determination results are shown in (a) of FIG. 5. Figure 7

[0104] At the end of the experiment, the serum IPA content of the PLA group was significantly lower than that of the NOR group (P < 0.01). Compared with the PLA group, the serum IPA content of the L-PRO group was increased, and the difference was significant (P < 0.01). Compared with the PLA group, the serum IPA content of the H-PRO group was increased, and the difference was significant (P < 0.01); compared with the L-PRO group, the serum IPA content of the H-PRO group was increased, but the difference was not significant (P > 0.05). The determination results are shown in (b) of FIG. 5. Figure 7

[0105] 5.5 Rat bone metabolism level At the end of the experiment, the number of osteoclasts per bone perimeter of the PLA group was significantly increased compared with the NOR group (P < 0.01), and the bone resorption activity of the PLA group was abnormal. Compared with the PLA group, the number of osteoclasts per bone perimeter of the L-PRO group was decreased, and the difference was not significant (P > 0.05), and the bone resorption activity was improved. Compared with the PLA group, the number of osteoclasts per bone perimeter of the H-PRO group was decreased, and the difference was significant (P < 0.05), and the bone resorption activity was significantly improved; compared with the L-PRO group, the number of osteoclasts per bone perimeter of the H-PRO group was decreased, and the difference was not significant (P > 0.05). The determination results are shown in (a) of FIG. 6. Figure 8 ​​(a) shown in FIG. 1 (b).

[0106] At the end stage, compared with the NOR group, the number of osteoblasts per bone perimeter of the rats in the PLA group was significantly decreased, and the difference was extremely significant (P < 0.01), and the bone formation of the rats in the PLA group was inhibited. Compared with the PLA group, the number of osteoblasts per bone perimeter of the rats in the L-PRO group was increased, and the difference was not significant (P > 0.05). Compared with the PLA group, the number of osteoblasts per bone perimeter of the rats in the H-PRO group was increased, and the difference was significant (P < 0.05); compared with the L-PRO group, the number of osteoblasts per bone perimeter of the rats in the H-PRO group was increased, and the difference was not significant (P > 0.05). The determination results are shown in FIG. 1 (c). Figure 8

[0107] At the end stage, compared with the NOR group, the mineral deposition rate of the rats in the PLA group was significantly decreased, and the difference was extremely significant (P < 0.01), and the bone formation ability of the rats was impaired. Compared with the PLA group, the mineral deposition rate of the rats in the L-PRO group was increased, and the difference was not significant (P > 0.05), and the bone formation ability of the rats was improved. Compared with the PLA group, the mineral deposition rate of the rats in the H-PRO group was increased, and the difference was not significant (P > 0.05), and the bone formation ability of the rats was improved; compared with the L-PRO group, the mineral deposition rate of the rats in the H-PRO group was increased, and the difference was not significant (P > 0.05). The determination results are shown in FIG. 1 (c). Figure 8

[0108] The animal experiment results show that the saliva combined with Lactobacillus HC1135 can enhance the synthesis and metabolism levels of rat osteoblasts and osteoclasts, reduce the calcium and phosphorus levels in the urine of rats, and reduce the bone loss phenomenon. After the intervention of the saliva combined with Lactobacillus HC1135, the IL-6 pro-inflammatory cytokine level in the serum of rats is decreased, and the IPA level is increased. On the one hand, IPA can promote the activity of osteoblasts and the deposition of bone matrix to improve the bone formation level, and on the other hand, IPA can effectively inhibit the number and function of osteoclasts to reduce bone absorption, so as to improve the bone metabolism level and realize the growth of the skeleton.

[0109] Example 11 Effect of saliva combined with Lactobacillus HC1135 on improving the bone structure of rats 1. Test animals The same as in Example 10.

[0110] 2. Experimental method ​​The rats were randomly divided into normal group (NOR), modeling group (PLA), indole-3-propionic acid group (IPA) and probiotic group (PRO) after adaptive feeding for 7 days, 6 rats in each group. After adaptive feeding, from the day of modeling, the IPA group and the PRO group were injected with glucocorticoids for 8 weeks, twice a day, with an injection dose of 4.5 mg / kg, the IPA group was given IPA (20 mg / kg) by gavage according to 0.2 mL / 10g per day, and the PRO group was given probiotic bacteria liquid (1×10 11 CFU / mL) by gavage according to 0.2 mL / 10g per day; the PLA group was injected with glucocorticoids for 8 weeks, twice a day, with an injection dose of 4.5 mg / kg, and was given the same amount of MRS liquid medium by gavage at the same period; the NOR group was not modeled, and was injected with normal saline for 8 weeks, twice a day, with an injection dose of 200 μL, and was given normal saline by gavage according to a dose of 0.2 mL / 10g per day at the same period. After the injection of the rats was completed, the rats were normally fed, and the state of the rats was observed daily. After the end of the test, the femurs of the rats in each group were taken for Micro-CT scanning and three-dimensional reconstruction of the bone microstructure by software, and the results of the calculation of the Micro-CT indicators of the rats in each group are shown in Figure 9 .

[0111] 3. Result analysis As shown in (a) of Figure 9 , at the end of the test, compared with the NOR group, the bone mineral density (BMD) of the rats in the PLA group decreased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the BMD of the rats in the IPA group increased, and the difference was not significant (P>0.05). Compared with the PLA group, the BMD of the rats in the PRO group increased, and the difference was significant (P<0.05). Compared with the IPA group, the BMD of the rats in the PRO group increased, and the difference was not significant (P>0.05).

[0112] As shown in (b) of Figure 9 , at the end of the test, compared with the NOR group, the bone volume fraction (BV / TV) of the rats in the PLA group decreased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the BV / TV of the rats in the IPA group increased, and the difference was not significant (P>0.05). Compared with the PLA group, the BV / TV of the rats in the PRO group increased, and the difference was extremely significant (P<0.01). Compared with the IPA group, the BV / TV of the rats in the PRO group increased, and the difference was not significant (P>0.05).

[0113] As shown in (c) of Figure 9As shown in (c) of FIG. 6, at the end stage, compared with the NOR group, the Tb.Sp of the rats in the PLA group increased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.Sp of the rats in the IPA group decreased, and the difference was not significant (P>0.05). Compared with the PLA group, the Tb.Sp of the rats in the PRO group decreased, and the difference was significant (P<0.05). Compared with the IPA group, the Tb.Sp of the rats in the PRO group decreased, and the difference was not significant (P>0.05).

[0114] As shown in (d) of FIG. 6, at the end stage, compared with the NOR group, the Tb.Th of the rats in the PLA group decreased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.Th of the rats in the IPA group increased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.Th of the rats in the PRO group increased, and the difference was extremely significant (P<0.01). Compared with the IPA group, the Tb.Th of the rats in the PRO group increased, and the difference was extremely significant (P<0.01). Figure 9 As shown in (e) of FIG. 6, at the end stage, compared with the NOR group, the SMI of the rats in the PLA group increased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the SMI of the rats in the IPA group decreased, and the difference was not significant (P>0.05). Compared with the PLA group, the SMI of the rats in the PRO group decreased, and the difference was significant (P<0.05). Compared with the IPA group, the SMI of the rats in the PRO group decreased, and the difference was not significant (P>0.05).

[0115] Figure 9 As shown in (f) of FIG. 6, at the end stage, compared with the NOR group, the Tb.N of the rats in the PLA group decreased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.N of the rats in the IPA group increased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.N of the rats in the PRO group increased, and the difference was extremely significant (P<0.01). Compared with the IPA group, the Tb.N of the rats in the PRO group increased, and the difference was not significant (P>0.05).

[0116] As shown in (f) of FIG. 6, at the end stage, compared with the NOR group, the Tb.N of the rats in the PLA group decreased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.N of the rats in the IPA group increased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.N of the rats in the PRO group increased, and the difference was extremely significant (P<0.01). Compared with the IPA group, the Tb.N of the rats in the PRO group increased, and the difference was not significant (P>0.05). Figure 9 As shown in (f) of FIG. 6, at the end stage, compared with the NOR group, the Tb.N of the rats in the PLA group decreased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.N of the rats in the IPA group increased, and the difference was extremely significant (P<0.01). Compared with the PLA group, the Tb.N of the rats in the PRO group increased, and the difference was extremely significant (P<0.01). Compared with the IPA group, the Tb.N of the rats in the PRO group increased, and the difference was not significant (P>0.05).

[0117] ​BV / TV is an index for evaluating the bone mass of cortical bone and cancellous bone, which can reflect the amount of bone trabecula in different samples. The greater the value, the greater the bone synthesis metabolism than the decomposition metabolism, and the bone mass increases, and vice versa. Tb.N, Tb.Th and Tb.Sp are the main indexes for evaluating the spatial morphological structure of bone trabecula. When the bone decomposition metabolism is greater than the bone synthesis metabolism, the values of Tb.N and Tb.Th decrease, and the value of Tb.Sp increases. SMI is a parameter for describing the proportion of lamellar structure and rod structure in the bone trabecula structure, which becomes larger when the bone metabolism is abnormal. After the intervention of saliva combined with lactobacillus HC1135, the bone synthesis metabolism of rats is enhanced, the bone mass is increased, and the bone density is improved. The bone trabecula structure is significantly improved, and the bone synthesis metabolism level of the bone trabecula is significantly higher than the decomposition metabolism level.

[0118] After the intervention of saliva combined with lactobacillus HC1135, the bone density, BV / TV, Tb.N and Tb.Th indexes of rats increase, the Tb.Sp and SMI indexes decrease, the bone trabecula structure is significantly improved, the bone synthesis metabolism level of the bone trabecula is significantly higher than the decomposition metabolism level, and the bone development and growth are improved. Moreover, the effect of intragastric administration of saliva combined with lactobacillus HC1135 on improving the bone microstructure of rats is better than that of IPA alone.

[0119] In summary, the saliva combined with lactobacillus HC1135 has strong tolerance to the simulated artificial gastrointestinal fluid, which lays a foundation for the successful passage of the probiotic strain through the gastrointestinal tract and the colonization in the colon to play the probiotic function. The antibiotic resistance test proves that the saliva combined with lactobacillus HC1135 is sensitive to common antibiotics, does not produce hemolysin, and has good biological safety. At the same time, the saliva combined with lactobacillus HC1135 can scavenge DPPH free radicals and hydroxyl free radicals, inhibit lipid peroxidation, has certain antioxidant functional activity, can degrade cholesterol, and has the probiotic property of reducing serum cholesterol.

[0120] It is proved by animal experiments that the saliva combined with lactobacillus HC1135 can reverse the loss of calcium and phosphorus elements in rats, reduce the bone absorption metabolism intensity of rats, promote the maturation and differentiation of osteoblasts by increasing the serum IPA level of rats, inhibit the maturation and differentiation of osteoclasts, promote the mineral deposition in the bone, improve the bone synthesis metabolism level and improve the bone microstructure of rats, so as to promote the growth and development of the bone. This shows that the saliva combined with lactobacillus HC1135 has potential application value in regulating bone metabolism, improving bone loss and improving bone health level, and can be used for improving the bone mass reduction, bone density reduction and bone microstructure damage caused by metabolic abnormalities, inflammatory reactions or hormone use, and is suitable for preventing or assisting in improving metabolic bone diseases, osteoporosis and related bone metabolism disorders.

[0121] Example 12 A probiotic preparation prepared from saliva combined with lactobacillus HC1135 (1) Lactobacillus salivarius HC1135 was inoculated into MRS liquid medium at an inoculation amount of 1% by volume percentage, and cultured at 37°C for 24 h, then the fresh bacterial liquid was centrifuged at a speed of 8000 rpm for 10 min, and the supernatant was discarded to obtain bacterial bodies; the bacterial bodies were washed and resuspended to obtain a bacterial suspension of Lactobacillus salivarius HC1135.

[0122] (2) The bacterial suspension and malt dextrin were mixed at a mass ratio of 1:3, uniformly stirred, and then placed in a freeze dryer, pre-frozen at -40°C for 2 h, and then freeze-dried at 0.1 mbar for 20 h to obtain a freeze-dried powder. The freeze-dried powder was divided into aluminum foil bags to obtain a probiotic preparation prepared from Lactobacillus salivarius HC1135.

[0123] The obtained probiotic preparation was stored at 4°C for 6 months, and the viable cell count was still maintained at 1×10 9 CFU / g or more.

[0124] Although the present application has been described in detail by referring to the preferred embodiments thereof, it is to be understood that the present application is not limited to them. Variations and modifications are possible in the embodiments of the present application without departing from the spirit and scope thereof. Therefore, it should be understood that the application is not limited to the specific embodiments described above. Any modifications and replacements obvious to those skilled in the art within the technical scope of the present application should be encompassed within the scope of the present application.

Claims

1. Lactobacillus salivarius HC1135 for regulating bone metabolism and improving bone loss, characterized in that, Lactobacillus salivarius (ATCC 431 24) Ligilactobacillus salivarius Lactobacillus salivarius (ATCC 431 24) HC1135 was deposited at China General Microbiological Culture Collection Center on February 20, 2023, at the address of No. 1 Yard, West Beichen Road, Chaoyang District, Beijing, with the preservation number of CGMCC No. 26587.

2. The Lactobacillus salivarius HC1135 according to claim 1, characterized in that, The 16s rDNA sequence of Ligilactobacillus salivarius HC1135 is SEQ ID NO:

3.

3. A probiotic preparation prepared from the Ligilactobacillus salivarius HC1135 of claim 1.

4. The probiotic preparation as described in claim 3, characterized in that, The preparation method comprises the following steps: (1) inoculate Ligilactobacillus salivarius HC1135 into MRS liquid medium at an inoculation amount of 1% by volume percentage, cultivate at 37°C for 24h, then centrifuge the fresh bacterial liquid at a speed of 8000rpm for 10min, discard the supernatant, and obtain the bacterial body; wash the bacterial body and resuspend it into a suspension, thereby obtaining a bacterial suspension; (2) mix the bacterial suspension with edible carriers or excipients to prepare a probiotic preparation.

5. Use of the Ligilactobacillus salivarius HC1135 of claim 1 in the preparation of a functional food or a drug for preventing or assisting in improving metabolic bone disease, osteoporosis and related bone metabolic disorders.

6. The use according to claim 5, wherein the compound is ###0002### The functional food or drug is a functional food or drug for regulating bone metabolism and improving bone loss.

7. The use according to claim 5, wherein the compound is ###0002### The functional food or drug is a functional food or drug for reducing serum pro-inflammatory factor IL-6 level and increasing serum IPA level.

8. The use according to claim 5, wherein the compound is ###0002### The functional food or drug is a functional food or drug for promoting osteoblast activity and bone matrix deposition, inhibiting the number and function of osteoclasts, and reducing bone absorption.

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