Composite probiotic composition for preventing, treating or improving osteoporosis and its application

Abstract

The present invention relates to the field of microbial application technology, and in particular to a composite probiotic composition for preventing, treating or improving osteoporosis and its application. The composite probiotic composition comprises composite probiotic powder, which comprises Lactobacillus johnsonii ( Lactobacillus johnsonii )HC1175 and Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) HC1983; the preservation number of Lactobacillus johnsonii HC1175 is CGMCC No.27354, and the preservation number of Lactobacillus delbrueckii subspecies bulgaricus HC1983 is CGMCC No.26584. The composite probiotic composition can significantly improve bone metabolism-related indicators, enhance bone density, improve multiple bone biomechanical indicators, and effectively alleviate or inhibit osteoporosis.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to a composite probiotic composition for preventing, treating or improving osteoporosis and an application thereof. Background Art

[0002] Osteoporosis is a common metabolic bone disease caused by a variety of reasons. It is divided into three categories: primary, secondary and idiopathic. Primary osteoporosis is further divided into postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis (including adolescent type); secondary osteoporosis is induced by metabolic diseases or long-term use of certain drugs and other factors; idiopathic osteoporosis mainly occurs in patients with a specific family history. With the increasing degree of aging, the number of osteoporosis patients is increasing, and the prevention, treatment or improvement of osteoporosis is becoming more and more important.

[0003] The onset of osteoporosis is accompanied by a variety of bone-related complications, which seriously affect the quality of life of patients. At present, the treatment of osteoporosis mainly relies on traditional drugs, which are mainly divided into two categories: inhibiting bone absorption and promoting bone formation. Such drugs will not only increase the financial burden of patients in terms of price, but also cause dependence if taken for a long time. In severe cases, they will cause organ damage, endocrine disorders and other side effects. Therefore, it is necessary to seek new safe and effective prevention and treatment methods to solve this dilemma. In recent years, many studies on the intervention of probiotics or prebiotics in osteoporosis have shown that they can safely and effectively prevent bone loss in various situations, showing the potential benefits of probiotics or prebiotics for bone health. Summary of the invention

[0004] In view of the technical problems that traditional drugs for treating osteoporosis are prone to dependence and have large side effects, the present invention provides a composite probiotic composition for preventing, treating or improving osteoporosis and its application.

[0005] In a first aspect, the present invention provides a composite probiotic composition for preventing, treating or improving osteoporosis, comprising a composite probiotic powder, wherein the composite probiotic powder comprises Lactobacillus johnsonii ( Lactobacillus johnsonii )HC1175 and Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) HC1983; the preservation number of Lactobacillus johnsonii HC1175 is CGMCC No.27354, and the preservation number of Lactobacillus delbrueckii subspecies bulgaricus HC1983 is CGMCC No.26584.

[0006] Furthermore, the ratio of the viable counts of Lactobacillus johnsonii HC1175 and Lactobacillus delbrueckii subsp. bulgaricus HC1983 was 1~3:1~3.

[0007] Further, the composite probiotic powder is prepared by mixing Lactobacillus johnsonii HC1175 powder and Lactobacillus delbrueckii subspecies bulgaricus HC1983 powder, and the bacterial content of Lactobacillus johnsonii HC1175 powder and Lactobacillus delbrueckii subspecies bulgaricus HC1983 powder is 5.0×10 9 ~3.0×10 10 cfu / g.

[0008] Further, the preparation method of Lactobacillus johnsonii HC1175 bacterial powder is as follows:

[0009] (1) The refrigerated Lactobacillus johnsonii HC1175 strain was warmed up at room temperature for 1 hour, inoculated into 2.5L liquid culture medium, and cultured aerobically at 37°C for 16-20 hours to prepare primary seeds, which were then transferred to a 100L seed tank and cultured aerobically at pH 6.58 and 37°C for 6-8 hours to obtain secondary seeds. Subsequently, the strain was transferred to a 1000L fermentation tank at a 10% inoculation rate, and cultured aerobically at pH 6.39 and 37°C for 6-8 hours until the logarithmic growth phase. The fermentation liquid was aseptically centrifuged to obtain Lactobacillus johnsonii HC1175 bacterial sludge;

[0010] (2) The first coating agent was sterilized by high-pressure air sterilization at 105°C for 30 min. The Lactobacillus johnsonii HC1175 sludge and the first coating agent were thoroughly stirred and mixed at a weight ratio of 1:1-2, and the pH was adjusted to 6.5-7.0 to obtain a sludge coating solution. After vacuum freeze-drying, the freeze-dried block was taken out and crushed to obtain Lactobacillus johnsonii HC1175 powder.

[0011] Further, the preparation method of Lactobacillus delbrueckii subspecies bulgaricus HC1983 bacterial powder is as follows:

[0012] (1) The refrigerated Lactobacillus delbrueckii subsp. bulgaricus HC1983 strain was warmed up at room temperature for 1 hour, inoculated into 2.5 L liquid culture medium, and cultured anaerobically at 37°C for 24-30 hours to prepare primary seeds, which were then transferred to a 100 L seed tank and cultured anaerobically at pH 6.45 and 37°C for 12-14 hours to obtain secondary seeds. Subsequently, the strain was transferred to a 1000 L fermentation tank at a 10% inoculation rate, and cultured anaerobically at pH 6.45 and 37°C for 6-8 hours until the logarithmic growth phase. The fermentation liquid was aseptically centrifuged to obtain Lactobacillus delbrueckii subsp. bulgaricus HC1983 bacterial sludge;

[0013] (2) The second coating agent was sterilized by high-pressure air sterilization at 105°C for 30 min. The Lactobacillus johnsonii HC1175 sludge and the second coating agent were thoroughly stirred and mixed at a weight ratio of 1:1-2, and the pH was adjusted to 6.5-7.0 to obtain a sludge coating solution. After vacuum freeze-drying, the freeze-dried blocks were taken out and crushed to obtain Lactobacillus delbrueckii subspecies bulgaricus HC1983 powder.

[0014] Furthermore, the first coating agent includes the following components in mass fractions: 10% skim milk powder, 8% trehalose, 2% oligofructose, 3% glycerol, 5% maltodextrin and 72% distilled water; the second coating agent includes the following components in mass fractions: 15% skim milk powder, 8% trehalose, 3% glycerol, 1% sodium ascorbate and 73% distilled water.

[0015] Furthermore, calculated by mass fraction, the composite probiotic composition includes: 4% composite probiotic powder, 67% fructooligosaccharides, 8% stachyose, 7% xylo-oligosaccharides, 8% galacto-oligosaccharides, 5% inulin, 0.2% fig fruit powder, 0.3% apple powder, 0.2% red date powder, and 0.3% wolfberry powder.

[0016] In a second aspect, the present invention provides an application of the above-mentioned composite probiotic composition in preparing a product for preventing, treating or improving osteoporosis.

[0017] Furthermore, the product for preventing, treating or improving osteoporosis is a product that promotes osteoblast proliferation, increases osteoblast activity and inhibits osteoclast activity.

[0018] Furthermore, the product for preventing, treating or improving osteoporosis is a product that enhances bone density, maximum bone load and bone bending strength.

[0019] Furthermore, the product for preventing, treating or improving osteoporosis is a product for improving the levels of serum calcium, serum phosphorus, osteocalcin, calcitonin, serum alkaline phosphatase, and serum tartrate-resistant acid phosphatase in serum.

[0020] The beneficial effects of the present invention are:

[0021] The present invention provides a composite probiotic composition, which is compounded by Lactobacillus johnsonii HC1175, Lactobacillus delbrueckii subspecies bulgaricus HC1983 and a variety of prebiotics (fructooligosaccharides, stachyose, xylo-oligosaccharides, galacto-oligosaccharides, inulin, fig fruit powder, apple powder, red date powder and wolfberry powder). In in vitro tests, Lactobacillus johnsonii HC1175 and Lactobacillus delbrueckii subspecies bulgaricus HC1983 both have very excellent gastrointestinal survival and colonization capabilities, and can promote the proliferation of osteoblasts and inhibit the proliferation of osteoclasts, thereby playing an anti-osteoporosis role. Animal experiments have verified that the composite probiotic composition can significantly improve the content of blood calcium, blood phosphorus, osteocalcin, calcitonin, alkaline phosphatase and tartrate-resistant acid phosphatase in serum, effectively enhance bone density, improve multiple bone biomechanical indicators, and achieve the purpose of alleviating or inhibiting the occurrence of osteoporosis. And during the entire test process, the composite probiotic composition has no toxic effects and no liver-intestinal translocation phenomenon. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a graph showing the simulated gastric fluid survival rate of each group of probiotics in Example 3 of the present application.

[0024] Figure 2 This is a graph showing the simulated intestinal fluid survival rate of each group of probiotics in Example 3 of the present application.

[0025] Figure 3 This is a graph showing the simulated gastrointestinal fluid survival rate of each group of probiotics in Example 3 of the present application.

[0026] Figure 4 This is a graph showing the mucin adhesion rate and Caco-2 cell adhesion rate of each group of probiotics in Example 4 of the present application.

[0027] Figure 5 This is a graph of absorbance measurement results after osteoblast proliferation in Example 5 of the present application.

[0028] Figure 6 This is a graph showing the results of osteoblast ALP enzyme activity detection in Example 5 of the present application.

[0029] Figure 7 This is a graph showing the results of osteoclast TRAP enzyme activity detection in Example 5 of the present application.

[0030] Figure 8 This is a graph showing the bone density measurement results of the lumbar vertebrae and femurs of rats at week 13 of the test cycle in Example 6 of the present application.

[0031] Fig. 9 This is a graph showing the changes in body weight of rats in each group during the experiment in Example 6 of the present application.

[0032] Fig.10 This is a graph showing the bone density measurements of the lumbar vertebrae and femurs of rats in each group at week 25 of the test cycle in Example 6 of the present application.

[0033] Fig.11 This is a graph showing the maximum femoral load measurement results of rats in each group at week 25 of the test cycle in Example 6 of the present application.

[0034] Fig.12 This is a graph showing the femoral bending strength measurement results of rats in each group at the 25th week of the test cycle in Example 6 of the present application.

[0035] Fig.13 This is a graph showing the blood calcium content measurement results of rats in each group at the 25th week of the test cycle in Example 6 of the present application.

[0036] Fig.14 This is a graph showing the results of measuring blood phosphorus content in rats of each group at the 25th week of the test cycle in Example 6 of the present application.

[0037] Fig.15 This is a graph showing the results of measuring serum osteocalcin levels in rats of each group at week 25 of the test cycle in Example 6 of the present application.

[0038] Fig.16 This is a graph showing the results of measuring serum calcitonin levels in rats of each group at week 25 of the test cycle in Example 6 of the present application.

[0039] Fig.17 This is a graph showing the results of measuring serum alkaline phosphatase levels in rats of each group at the 25th week of the test cycle in Example 6 of the present application.

[0040] Fig.18 This is a graph showing the results of measuring the serum tartrate-resistant acid phosphatase content of rats in each group at the 25th week of the test cycle in Example 6 of the present application.

[0041] Fig.19 This is a graph showing the measurement results of the organ indexes of rats in each group at the 25th week of the test cycle in Example 7 of the present application. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in 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. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0043] In the following examples, Lactobacillus johnsonii ( Lactobacillus johnsonii ) HC1175 was isolated from the intestinal flora of a 6-month-old healthy infant in Lhasa, Tibet Autonomous Region, and was deposited on May 16, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Deposit Number: CGMCC No. 27354;

[0044] Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) HC1983 was isolated from a traditional fermented sour mare's milk sample from Hulunbuir and was deposited on February 20, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, Collection Number: CGMCC No.26584.

[0045] Example 1 Preparation of bacterial powder

[0046] 1. Preparation of Lactobacillus johnsonii HC1175 powder

[0047] (1) The refrigerated Lactobacillus johnsonii HC1175 strain was warmed up at room temperature for 1 hour, inoculated into 2.5L liquid culture medium, and cultured aerobically at 37°C for 16-20 hours to prepare primary seeds, which were then transferred to a 100L seed tank and cultured aerobically at pH 6.58 and 37°C for 6-8 hours to obtain secondary seeds. Subsequently, the strain was transferred to a 1000L fermentation tank at a 10% inoculation rate, and cultured aerobically at pH 6.39 and 37°C for 6-8 hours until the logarithmic growth phase. The fermentation liquid was aseptically centrifuged to obtain Lactobacillus johnsonii HC1175 bacterial sludge;

[0048] (2) The first coating agent was sterilized by high-pressure air sterilization at 105°C for 30 min. The Lactobacillus johnsonii HC1175 sludge and the first coating agent were thoroughly stirred and mixed at a weight ratio of 1:1-2, and the pH was adjusted to 6.5-7.0 to obtain a sludge coating solution. After vacuum freeze-drying, the freeze-dried block was taken out and crushed to obtain Lactobacillus johnsonii HC1175 powder.

[0049] The first coating agent includes the following components in mass fractions: 10% skimmed milk powder, 8% trehalose, 2% oligofructose, 3% glycerol, 5% maltodextrin and 72% distilled water.

[0050] By adjusting the concentration of the bacterial mud coating liquid, Lactobacillus johnsonii HC1175 bacterial powders with bacterial contents of 5 billion / g, 10 billion / g, 20 billion / g, 25 billion / g and 30 billion / g were obtained respectively.

[0051] 2. Preparation of Lactobacillus delbrueckii subspecies bulgaricus HC1983 bacterial powder

[0052] (1) The refrigerated Lactobacillus delbrueckii subsp. bulgaricus HC1983 strain was warmed up at room temperature for 1 hour, inoculated into 2.5 L liquid culture medium, and cultured anaerobically at 37°C for 24-30 hours to prepare primary seeds, which were then transferred to a 100 L seed tank and cultured anaerobically at pH 6.45 and 37°C for 12-14 hours to obtain secondary seeds. Subsequently, the strain was transferred to a 1000 L fermentation tank at a 10% inoculation rate, and cultured anaerobically at pH 6.45 and 37°C for 6-8 hours until the logarithmic growth phase. The fermentation liquid was aseptically centrifuged to obtain Lactobacillus delbrueckii subsp. bulgaricus HC1983 bacterial sludge;

[0053] (2) The second coating agent was sterilized by high-pressure air sterilization at 105°C for 30 min. The Lactobacillus johnsonii HC1175 sludge and the second coating agent were thoroughly stirred and mixed at a weight ratio of 1:1-2, and the pH was adjusted to 6.5-7.0 to obtain a sludge coating solution. After vacuum freeze-drying, the freeze-dried blocks were taken out and crushed to obtain Lactobacillus delbrueckii subspecies bulgaricus HC1983 powder.

[0054] The second coating agent includes the following components in mass fractions: 15% skimmed milk powder, 8% trehalose, 3% glycerol, 1% sodium ascorbate and 73% distilled water.

[0055] By adjusting the concentration of the bacterial mud coating liquid, bacterial powders of Lactobacillus delbrueckii subspecies bulgaricus HC1983 with bacterial contents of 5 billion / g, 10 billion / g, 20 billion / g, 25 billion / g and 30 billion / g were obtained respectively.

[0056] Example 2 Preparation of composite probiotic composition

[0057] (1) Preparation of composite probiotic powder: Lactobacillus johnsonii HC1175 powder and Lactobacillus delbrueckii subsp. bulgaricus HC1983 powder were mixed evenly to prepare composite probiotic powder.

[0058] (2) Preparation of a composite probiotic composition: Calculated by mass, the composite probiotic composition includes 4% composite probiotic powder, 67% fructooligosaccharide, 8% stachyose, 7% xylo-oligosaccharide, 8% galacto-oligosaccharide, 5% inulin, 0.2% fig fruit powder, 0.3% apple powder, 0.2% red date powder, and 0.3% wolfberry powder. The raw materials are mixed evenly according to the proportion to prepare a composite probiotic composition.

[0059] Different weight portions of Lactobacillus johnsonii HC1175 powder and Lactobacillus delbrueckii subsp. bulgaricus HC1983 powder were mixed to obtain probiotic compositions having different ratios of viable counts of HC1175 and HC1983. The specific ratios are shown in Table 1.

[0060] Table 1 Bacterial formula in probiotic composition

[0061]

[0062] Example 3 Determination of probiotic colonization ability under simulated digestion conditions of gastrointestinal fluid

[0063] Probiotics need to be digested in the stomach and small intestine, and colonize to a certain number in the gastrointestinal tract during this process before they can truly exert their probiotic properties. This requires probiotics to have a certain tolerance to gastric acid, bile salts, and proteases in the stomach and intestines. Therefore, tolerance to gastric acid and bile salts is an important indicator for measuring probiotics.

[0064] 1. Gastric juice simulates digestion

[0065] (1) Prepare simulated gastric fluid: accurately weigh 0.5144g KCl, 0.1225g KH2PO4, 2.75085g NaCl, 2.1002g NaHCO3, 0.0203g MgCl2(H2O)6, 0.0480g (NH4)2CO3, 0.0083g CaCl2, and dilute to 1000mL to prepare gastric fluid buffer (pH 3.0). Dissolve 3g pepsin in 1000mL gastric fluid buffer, adjust the pH to 3.0 with 1mol / L HCl, and filter through a 0.22μm filter to sterilize to prepare simulated gastric fluid, which is prepared before use.

[0066] (2) Weigh 1.00g of Lactobacillus delbrueckii subsp. bulgaricus HC1983 powder (5 billion / g), add to 9mL simulated gastric fluid, recorded as DJ-BJLY group; weigh 1.00g of Lactobacillus johnsonii HC1175 powder (5 billion / g), add to 9mL simulated gastric fluid, recorded as DJ-YS group; weigh 0.5g of Lactobacillus delbrueckii subsp. bulgaricus HC1983 powder (5 billion / g) and 0.5g of Lactobacillus johnsonii HC1175 powder (5 billion / g), mix, add to 9mL simulated gastric fluid, recorded as FP-mixed group. Set up 3 parallel groups for each group, keep in a constant temperature water bath at 37°C, oscillate at a speed of 100r / min, and count the live bacteria by the dilution spread plate method after treatment for 0h, 1h, 2h, 3h, and 4h, respectively. Set up 4 replicates for counting. The simulated gastric fluid survival rate of probiotics was calculated according to the following formula, and the results are as follows Figure 1 shown.

[0067]

[0068] 2. Intestinal fluid simulated digestion

[0069] (1) Simulated intestinal fluid (0.1% bile salt): Accurately weigh 0.5069g KCl, 0.1089g KH2PO4, 2.2442g NaCl, 7.1408g NaHCO3, 0.0067g MgCl2(H2O)6, and 0.0333g CaCl2, and dilute to 1000mL to prepare intestinal fluid buffer. Take 1000mL of intestinal fluid buffer, add 1g trypsin and 1g bile salt to dissolve, adjust the pH of the simulated intestinal fluid to 8.0 with 1mol / L NaOH, filter and sterilize with a 0.22μm filter membrane to prepare simulated intestinal fluid, which is prepared before use.

[0070] (2) Weigh 1.00g of Lactobacillus delbrueckii subsp. bulgaricus HC1983 powder (5 billion / g), add to 9mL of simulated intestinal fluid, recorded as DJ-BJLY group; weigh 1.00g of Lactobacillus johnsonii HC1175 powder (5 billion / g), add to 9mL of simulated intestinal fluid, recorded as DJ-YS group; weigh 0.5g of Lactobacillus delbrueckii subsp. bulgaricus HC1983 powder (5 billion / g) and 0.5g of Lactobacillus johnsonii HC1175 powder (5 billion / g), mix, add to 9mL of simulated intestinal fluid, recorded as FP-mixed group. Set up 3 parallel groups for each group, keep in a constant temperature water bath at 37°C, oscillate at a speed of 100r / min, and count the live bacteria by the dilution spread plate method after treatment for 0h, 12h, 24h, 36h, and 48h, respectively, with 4 replicates set for counting. The simulated intestinal fluid survival rate of probiotics was calculated according to the following formula, and the results are as follows Figure 2 shown.

[0071]

[0072] 3. Gastric juice-intestinal juice two-step method to simulate digestion

[0073] Weigh 1.00g of the Lactobacillus delbrueckii subspecies bulgaricus HC1983 powder (5 billion / g) prepared in Example 1, recorded as DJ-BJLY group; weigh 1.00g of the Lactobacillus johnsonii HC1175 powder (5 billion / g) prepared in Example 1, recorded as DJ-YS group; weigh 0.5g of the Lactobacillus delbrueckii subspecies bulgaricus HC1983 powder (5 billion / g) and 0.5g of the Lactobacillus johnsonii HC1175 powder (5 billion / g) and mix them, recorded as FP-mixed group, and set 3 parallels for each group. Each group was added with 9mL of simulated gastric fluid, 37°C constant temperature water bath, 100r / min oscillation for 3h, and then quickly centrifuged, and then 9mL of simulated intestinal fluid was added, 37°C constant temperature water bath, 100r / min oscillation for 24h, and the live bacteria were counted by dilution coating plate method, and 4 repetitions were set for counting. The survival rate of probiotics in simulated gastrointestinal fluid was calculated according to the following formula. Figure 3 shown.

[0074]

[0075] from Figure 1-3It can be clearly seen that as the treatment time of simulated gastric juice and simulated intestinal juice increases, the survival rate of each group of probiotics shows a downward trend. The survival rate of Lactobacillus delbrueckii subspecies HC1983 and Lactobacillus johnsonii HC1175 single strains in simulated gastric juice digestion for 4 hours is more than 80%, and the survival rate in simulated intestinal juice digestion for 48 hours is more than 60%, and the survival rate after digestion by simulated gastric juice-intestinal juice two-step method can also reach more than 60%. The composite probiotic composition prepared by mixing and compounding the above two strains, whether it is digested in simulated gastric juice for 4 hours, digested in simulated intestinal juice for 48 hours, or digested by simulated gastric juice-intestinal juice two-step method, its survival rate is higher than the survival rate of a single strain. Based on the comprehensive test results, single strains of Lactobacillus johnsonii HC1175 and Lactobacillus delbrueckii subspecies bulgaricus HC1983 have good tolerance to gastric acid, bile salts and pepsin, and have a higher survival rate in the stomach and intestines; the compound probiotics made by mixing the two strains have higher tolerance and survival rate.

[0076] Example 4 Determination of strain adhesion ability

[0077] The site where probiotic lactic acid bacteria exert their probiotic effects is the intestine. They can only work after colonizing a certain part of the intestine to form a stable flora, and adhesion is the key to colonization. Probiotics can prolong their residence time in the body through adhesion, and then affect the health of the host by improving the local microbial flora or its metabolites.

[0078] 1. Determination of the ability of strains to adhere to mucin

[0079] (1) Establishment of mucin model: Weigh 10 mg of mucin and prepare a 1 mg / mL mucin solution with sterile PBS buffer and store at -20°C. Take 500 µL of mucin solution and fix it on a 24-well cell culture plate for 1 hour, culture it at 4°C overnight, then add an equal volume of mucin solution and continue to culture it at 37°C for 2 hours to fill the blank site, then wash it twice with sterile PBS buffer.

[0080] (2) Determination of adhesion ability: Lactobacillus johnsonii HC1175 and Lactobacillus delbrueckii subspecies bulgaricus HC1983 were cultured overnight, the cells were collected, and resuspended in sterile PBS. The cell count was adjusted to 10 8CFU / mL, spread and count and record. The mucin model was divided into three groups, namely DJ-BJLY group, DJ-YS group and FP-mixed group. 500µL of Lactobacillus delbrueckii subsp. bulgaricus HC1983 suspension, 500µL of Lactobacillus johnsonii HC1175 suspension and 500µL of composite bacterial suspension (Lactobacillus delbrueckii subsp. bulgaricus HC1983 suspension: Lactobacillus johnsonii HC1175 suspension = 1:1) were added to the mucin models of the DJ-BJLY group, DJ-YS group and FP-mixed group, respectively. After incubation at 37°C for 1 hour, the mixture was washed 5 times with sterile citric acid buffer to remove unbound bacteria, and 1mL of 0.5% Tween 80 was added to collect the adherent bacteria. The counts were performed by the dilution plate method, and each sample was spread 3 times. The mucin adhesion rate of probiotics in each group was calculated according to the following formula. The results are shown in the figure. Figure 4 shown.

[0081]

[0082] 2. Determination of the adhesion ability of strains to Caco-2 cells

[0083] 0.5 mL of Caco-2 cell suspension was inoculated into a 24-well cell culture plate, where the concentration of Caco-2 cell suspension was 2×10 5 cell / mL, cultured to a monolayer, washed twice with PBS solution, and then divided into three groups, namely DJ-BJLY group, DJ-YS group, and FP-mixed group. 500 μL of Lactobacillus delbrueckii subsp. bulgaricus HC1983 bacterial suspension (10 8 CFU / mL), 500µL of Lactobacillus johnsonii HC1175 suspension (10 8 CFU / mL) and 500 µL of composite bacterial suspension (Lactobacillus delbrueckii subsp. bulgaricus HC1983 suspension: Lactobacillus johnsonii HC1175 suspension = 1:1, 10 8 CFU / mL), cultured at 37℃ for 2h, washed 3 times with PBS to remove non-adherent bacteria, added 150µL trypsin cell digestion solution, and added 350µL MEM complete culture solution to terminate digestion after the cells were completely detached. Counted by dilution plate method, each sample was plated 3 times. The Caco-2 cell adhesion rate of each group of probiotics was calculated according to the following formula. The results are shown in the figure. Figure 4 shown.

[0084]

[0085] from Figure 4It can be seen that Lactobacillus johnsonii HC1175, Lactobacillus delbrueckii subsp. bulgaricus HC1983 and the mixed bacteria of the two have good adhesion, and the adhesion rate to mucin and Caco-2 cells is high; and from the data, it can be seen that the adhesion rate of mixed bacteria to mucin and Caco-2 cells is higher than that of a single strain.

[0086] Example 5 Effects of different probiotic compositions on osteoblast and osteoclast differentiation

[0087] According to Example 2, the viable bacteria count was 5×10 9 The probiotic composition with different strain ratios (G1, G2, G3, G4, G5, G6, G7) was adjusted to 1×10 CFU / g with sterile saline. 8 CFU / mL, and obtain the probiotic composition supplement liquid with different strain ratios.

[0088] A prebiotic composition was prepared by mixing 67 g of fructooligosaccharide, 8 g of stachyose, 7 g of xylo-oligosaccharide, 8 g of galacto-oligosaccharide, 5 g of inulin, 0.2 g of fig fruit powder, 0.3 g of apple powder, 0.2 g of red date powder, 0.3 g of wolfberry powder and 4 g of soluble starch, and then diluted with sterile physiological saline according to the dilution multiple of the probiotic composition to obtain a prebiotic composition additive solution.

[0089] 1. Effects of different probiotic compositions on osteoblast proliferation and ALP enzyme activity

[0090] (1) Cultivation and proliferation of osteoblasts

[0091] Five SPF SD rats within 72 hours of birth were selected, regardless of gender. After being killed by cervical dislocation, they were rinsed with deionized sterile water and immersed in 75% alcohol for 20 minutes. The rats were then dissected under sterile conditions and the skull bones were separated. The periosteum and other tissues were removed and washed with D-Hank's solution until there was no other foreign matter on the bone surface. The bone slices were cut as much as possible for later use. The bone slices were digested with 5 mL 0.25% trypsin solution for 20 minutes, rinsed repeatedly with D-Hank's solution, and then transferred to a culture dish with 5 mL 0.1% type II collagenase solution for digestion for 60 minutes. After digestion, the digestion solution was aspirated and filtered, and the filtrate was centrifuged and the supernatant was discarded. The white precipitate obtained was fresh osteoblasts. Low-glucose DMEM medium (1% double antibody, 10% FBS) was added to the fresh osteoblasts to make a cell suspension, which was transferred to a culture bottle and cultured in a 5% CO2 incubator at 37°C. After the cells covered the bottom of the bottle, they were passaged for later use.

[0092] (2) Preparation of low-glucose DMEM culture medium containing a probiotic composition or prebiotics: Add probiotic composition supplements or prebiotic composition supplements of different strain ratios (G1, G2, G3, G4, G5, G6, G7) to low-glucose DMEM culture medium (1% double antibody, 10% FBS) at an addition amount of 5% (v / v) to prepare a low-glucose DMEM culture medium containing a probiotic composition or prebiotics.

[0093] (3) After the second generation of osteoblasts spread all over the flat bottom, subculture and adjust the cell density to 2×10 4 / mL, transplanted into 96-well plates, divided into blank control group (NK), prebiotic group (CK) and 7 experimental groups (G1 group, G2 group, G3 group, G4 group, G5 group, G6 group, G7 group), a total of 9 groups. When the cells grew to 50% confluence, 100 μL of low-glucose DMEM medium (1% double antibody, 10% FBS) was added to the NK group, 100 μL / well of low-glucose DMEM medium containing prebiotics was added to the CK group, and 100 μL / well of low-glucose DMEM medium containing composite probiotic compositions (G1, G2, G3, G4, G5, G6, G7) was added to the G1-G7 groups respectively, and 3 parallels were set for each group. After seven days of culture, the MTT cell proliferation and cytotoxicity detection kit (purchased from Shanghai Shangbao Biotechnology Co., Ltd.) was used to detect the cell proliferation of each group using the MTT method. The number of living cells is determined by the absorbance value (OD value) measured by the microplate reader. The larger the OD value, the stronger the cell activity. Figure 5 shown.

[0094] from Figure 5 It can be seen that compared with the NK group, the number of osteoblasts in the CK group and the G1-G7 groups increased, indicating that the probiotic composition (G1, G2, G3, G4, G5, G6, G7) and the prebiotic composition can promote the proliferation of osteoblasts. The influence of each group of compositions is ranked as follows: G4>G5>G3>G6>G2>G1>G7>CK, among which the composite probiotic composition of the G4 group (live bacteria ratio HC1983:HC1175=1:2) had the most significant effect on promoting the proliferation of osteoblasts, and the composite probiotic composition of the G5 group (live bacteria ratio HC1983:HC1175=1:3) had the second most significant effect on promoting the proliferation of osteoblasts.

[0095] (3) After the third generation of osteoblasts spread all over the flat bottom, subculture and adjust the cell density to 2×10 4The cells were transplanted into a 96-well plate at 100 μL / mL and divided into a blank control group (NK), a prebiotic group (CK) and 7 experimental groups (G1, G2, G3, G4, G5, G6, and G7), for a total of 9 groups. When the cells grew to 50% confluence, 100 μL of low-glucose DMEM medium (1% double antibody, 10% FBS) was added to the NK group, 100 μL / well of low-glucose DMEM medium containing prebiotics was added to the CK group, and 100 μL / well of low-glucose DMEM medium containing a probiotic composition (G1, G2, G3, G4, G5, G6, and G7) was added to the G1-G7 groups, respectively, with 3 parallels set up for each group. After seven days of culture, the ALP enzyme activity of osteoblasts in each group was detected by the disodium phenyl phosphate colorimetric method using an alkaline phosphatase (ALP) detection kit (purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.). The results are shown in the figure. Figure 6 shown.

[0096] from Figure 6 It can be seen that compared with the NK group, the ALP enzyme activity of osteoblasts in the CK group and G1-G7 groups increased, indicating that the probiotic composition (G1, G2, G3, G4, G5, G6, G7) and the prebiotic composition can improve the ALP enzyme activity of osteoblasts. The influence of each group of compositions is ranked as follows: G4>G5>G3>G6>G2>G7>G1>CK, among which the composite probiotic composition of the G4 group (live bacteria ratio HC1983:HC1175=1:2) had the most significant effect on promoting the ALP enzyme activity of osteoblasts, and the composite probiotic composition of the G5 group (live bacteria ratio HC1983:HC1175=1:3) had the second best effect on promoting the ALP enzyme activity of osteoblasts.

[0097] 2. Effects of different probiotic compositions or prebiotics on osteoclast TRAP activity

[0098] (1) Five SPF SD rats within 72 hours of birth were selected, regardless of gender. They were killed by cervical dislocation, rinsed with deionized sterile water, and immersed in 75% alcohol for 20 min. The rats were then dissected under sterile conditions and the tibiae of the four limbs were isolated. Osteoclast induction medium (α-MEM medium containing 10 nmol / L 1,25-dihydroxyvitamin D3, 100 nmol / L dexamethasone, and 10% fetal bovine serum) was repeatedly drawn out with a syringe to flush out the cells on the bone surface and in the bone marrow cavity. The bone marrow suspension was filtered using a 200-mesh cell sieve and inoculated into a culture flask. The flask was placed in a 5% CO2 incubator and cultured at 37°C for 24 h. The non-adherent cells were removed and centrifuged. The cell density was adjusted to 10 using osteoclast induction medium. 6The cell suspension was re-inoculated into the corresponding culture plate and cultured for another 2 days. The non-adherent cells and culture medium were removed by aspiration, and D-Hank's solution was added for repeated washing to obtain osteoclasts for later use.

[0099] (2) Preparation of osteoclast induction medium containing probiotic composition or prebiotics: Add probiotic composition supplement or prebiotic composition supplement of different strain ratios (G1, G2, G3, G4, G5, G6, G7) to osteoclast induction medium at an addition amount of 5% (v / v) to prepare osteoclast induction medium containing probiotic composition or prebiotics.

[0100] (3) The obtained osteoclasts were taken out and divided into blank control group (NK), prebiotic group (CK) and 7 experimental groups (G1 group, G2 group, G3 group, G4 group, G5 group, G6 group, G7 group), a total of 9 groups. When the cells grew to 50% confluence, 100 μL of osteoclast induction medium was added to the NK group, 100 μL / well of osteoclast induction medium containing prebiotics was added to the CK group, and 100 μL / well of osteoclast induction medium containing a probiotic composition (G1, G2, G3, G4, G5, G6, G7) was added to the G1-G7 groups respectively, with 3 parallels set up for each group. After 48 hours of culture, the TRAP enzyme activity of osteoclasts in each group was detected by PNP colorimetry using a tartrate-resistant acid phosphatase (TRAP) detection kit (purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.). The results are as follows: Figure 7 shown.

[0101] Depend on Figure 7 It can be seen that compared with the NK group, the osteoclast TRAP enzyme activity of the CK group and the G1-G7 groups was reduced, indicating that the probiotic composition (G1, G2, G3, G4, G5, G6, G7) and the prebiotic composition can inhibit the osteoclast TRAP enzyme activity. The influence of each group of compositions is ranked as follows: G4>G3>G5>G7>G2>G6>G1>CK, among which the composite probiotic composition of the G4 group (live bacteria ratio HC1983:HC1175=1:2) had the most significant inhibitory effect on the osteoclast TRAP enzyme activity, and the composite probiotic composition of the G3 group (live bacteria ratio HC1983:HC1175=1:1) had the second largest inhibitory effect on the osteoclast TRAP enzyme activity.

[0102] Example 6 Effects of the composite probiotic composition on body weight and various bone markers

[0103] 1. Animal experiment modeling and grouping

[0104] (1) Sixty 10-week-old SPF female SD rats with an initial body weight of 200±10g were fed a rat-specific diet and were allowed to drink water and eat freely. The bedding was changed regularly, and abnormal rats were removed after one week of adaptive feeding. The 60 rats were randomly divided into five groups, and one of the groups was randomly selected as the sham operation group (SHAM group). The rats in the SHAM group underwent surgery after injection of 1% sodium pentobarbital (50 mg / kg), exposing the ovaries but only removing a small amount of fat near the ovaries; the remaining rats underwent surgery to remove both ovaries after injection of 1% sodium pentobarbital. For three consecutive days after surgery, the surgical rats were injected with 80,000 units of sodium penicillin intramuscularly to prevent postoperative inflammation and infection. The postoperative status of the rats was monitored at any time to prevent them from tearing the wound, and surgical repair was performed in time if the wound broke. If the rats were stable after three days, the rats in the SHAM group would be fed with basic feeding until the end of the experimental period. In the other part, the rats that underwent ovariectomy were randomly divided into 4 groups, namely the surgery group (OVX group), the low-dose probiotic group (CPP-L group), the medium-dose probiotic group (CPP-M group) and the continuous probiotic group (CPP-S group). The continuous probiotic group (CPP-S group) was gavaged with a medium-dose composite probiotic composition (HC1983:HC1175=1:2, diluted to 5×10 7 CFU / mL, 1mL / 100g weight), while the rats in the other groups were uniformly fed with basal feeding after surgery.

[0105] (2) In the 13th week of the experimental cycle, three rats from each group were randomly selected and anesthetized with 1% sodium pentobarbital injection. The bone density of the lumbar spine and femur of the rats was measured using a bone densitometer to determine whether the rat osteoporosis model was successfully established. Since the OVX group, CPP-L group, and CPP-M group were uniformly managed and fed until the model was successfully established during the postoperative modeling process, the data were uniformly processed. The results are as follows: Figure 8 As shown. Figure 8 It can be seen that the femoral bone density and lumbar spine bone density of the model group rats after surgery were significantly lower than those of the SHAM group, and the reduction effect was significant. This phenomenon can indicate that the ovariectomized osteoporosis model of this experiment was successfully established.

[0106] (3) After the model was successfully established, the rats were randomly divided into three groups: the surgical group (OVX group), the low-dose probiotic group (CPP-L group), and the medium-dose probiotic group (CPP-M group). The rats in the OVX group were fed with a basic diet, and the rats in the CPP-L group were gavaged twice a day with a low-dose composite probiotic composition (HC1983:HC1175=1:2, diluted to 1×10 7CFU / mL, 1mL / 100g weight), the CPP-M group rats were gavaged twice a day to assist in feeding the medium-dose composite probiotic composition (HC1983:HC1175=1:2, diluted to 5×10 with sterile saline). 7 CFU / mL, 1mL / 100g weight). The intragastric dose was calculated at any time according to the changes in the rats' body weight during the entire experimental period, and the intragastric dose was fixed at 9 am and 8 pm.

[0107] 2. Weight changes of rats

[0108] Weigh the rats at a fixed time every week to monitor their weight changes. Fig. 9 As shown. Fig. 9 It can be seen that during the entire test period, the weight of rats in all experimental groups showed a trend of steady growth, but the differences between the groups were significant. The SHAM group had a relatively stable weight growth trend among all groups, and the weight of each week of age was in line with the weight range of normal rats. The weight gain of the OVX group, CPP-L group, and CPP-M group after surgery during the modeling process of 2-13 weeks after surgery was significantly higher than that of the SHAM group, and after the modeling was completed, it was clearly seen that the weight growth trend of the CPP-L group and the CPP-M group was slower than that of the OVX group since the 14th week, and the weight reduction of the CPP-M group was significantly higher than that of the CPP-L group, which shows that taking the composite probiotic composition provided by the present invention can inhibit the weight gain of ovariectomized osteoporosis model rats, and maintain their weight as close to the normal weight range as possible, and the higher the dosage, the better the effect of inhibiting weight gain. The CPP-S group began to gavage and assist in feeding a medium-dose composite probiotic composition twice a day after surgery, which had an extremely significant effect on slowing down the weight gain of the ovariectomized osteoporosis model rats. Its weight gain rate was slightly higher than that of the SHAM group, but significantly lower than that of the CPP-L group and the CPP-M group, almost close to the normal weight range of rats. This shows that the composite probiotic composition intervention before modeling can regulate the unconventional weight gain of the ovariectomized osteoporosis model rats throughout the growth process and maintain the weight balance during its growth process. Based on the above test results, it can be shown that the composite probiotic composition intervention provided by the present invention has a certain alleviating effect on the symptoms of unconventional weight gain caused by the decrease in the secretion level of estrogen after osteoporosis, and the earlier the intervention time, the better the effect.

[0109] 3. Changes in bone density in rats

[0110] Bone density is an important indicator of bone strength. It can not only effectively detect the body's bone loss, but also be used to predict whether osteoporosis occurs in the body. Monitoring changes in bone density is an important method for studying osteoporosis and evaluating drug effects.

[0111] At the 25th week of the experimental period, all rats were anesthetized by 1% sodium pentobarbital injection, and the bone density of the lumbar spine and femur of the rats was measured by bone densitometer. Fig.10 As shown. Compared with the SHAM group, the femoral bone density and lumbar bone density of the OVX group were significantly reduced, indicating that the bone density value would decrease under osteoporosis; and the femoral bone density and lumbar bone density of the OVX group were the lowest among all the experimental groups, that is, the bone density value of the osteoporosis model rats after surgery was significantly lower than that of the other four groups, which can indirectly illustrate the successful modeling of this experiment. The CPP-L group, CPP-M group and CPP-S group taking the composite probiotic composition all alleviated the decrease in femoral bone density and lumbar bone density of the model rats to varying degrees. Among them, taking a medium-dose composite probiotic composition is better than a low-dose composite probiotic composition in alleviating the decrease in femoral bone density and lumbar bone density, and continuous use of a medium-dose composite probiotic composition has the best effect.

[0112] 4. Rat bone biomechanics

[0113] The maximum load of bone reflects the structural continuity of bone, and the bending strength of bone reflects the ability of bone to resist compression and deformation. Both belong to the category of bone biomechanics and are mechanical properties of bones under the action of external forces. Changes in the internal structure of bone and reduction in bone mass will change the above two characteristics. Comprehensive evaluation of bone density, maximum load of bone and bending strength of bone can better judge bone strength and fracture possibility.

[0114] At the 25th week of the experimental period, all rats were anesthetized by 1% sodium pentobarbital injection and then killed by cervical dislocation. The right femur was removed to remove the muscle and connective tissue and the maximum load and bending strength of the femur were measured. The results are as follows: Figure 11-12 As shown. Figure 11-12 It can be seen that compared with the SHAM group, the maximum femoral load and femoral bending strength of the OVX group were greatly reduced, indicating that the bone strength and bone compression and deformation resistance of the osteoporosis model rats after surgery were greatly reduced. The maximum femoral load and femoral bending strength of the rats in the CPP-L group were significantly improved compared with the OVX group, and the maximum femoral load and femoral bending strength of the rats in the CPP-M group were much higher than those in the OVX group. The increase in the maximum femoral load and femoral bending strength of the rats in the CPP-M group was much greater than that in the CPP-L group, which shows that the effect of taking a medium-dose composite probiotic composition on improving the maximum femoral load and femoral bending strength is better than that of a low-dose composite probiotic composition. The CPP-S group was better than the OVX group, the CPP-L group, and the CPP-M group, which shows that the continuous use of a medium-dose composite probiotic has a better effect on improving the maximum femoral load and femoral bending strength.

[0115] Based on the above results, the intervention with the composite probiotic composition provided by the present invention can slow down the degree of femoral structure destruction, increase bone mass and bone strength, enhance the femoral resistance to bending and deformation, reduce the fracture risk of osteoporosis model rats, and improve their bone biomechanical properties. The earlier the intervention time, the better the effect.

[0116] Example 7 Effects of the composite probiotic composition on blood parameters

[0117] The role of bones is not only to support body activities and protect organs, but also to participate in certain hematopoietic functions. It is well known that mineral salts such as calcium and phosphorus are the main components of bone inorganic salts, which can show the hardness of bones. The detection of blood calcium and blood phosphorus in serum can indirectly reflect the changes in bone minerals. Osteocalcin is a regulatory hormone synthesized and secreted by osteoblasts. It is usually relatively stable and not affected by bone absorption factors. It can be used to understand the overall activity state of osteoblasts. Calcitonin is a polypeptide hormone that plays an important role in the regulation of bone metabolism. It can inhibit the formation of osteoclasts, enhance the osteogenesis process, reduce bone loss, increase bone mineralization, and increase bone density. When the blood calcium concentration increases, the secretion of calcitonin also increases. Alkaline phosphatase is produced by osteoblasts and can promote matrix mineralization by decomposing inorganic phosphorus of phosphate esters. It is an important protein involved in bone metabolism. Tartrate-resistant acid phosphatase is a protein secreted by osteoclasts that can participate in bone metabolism. It can participate in the degradation of solid calcium and phosphate minerals in the bone matrix during bone absorption and is a marker of osteoclast function. During the construction of osteoporosis model rats, bone metabolism will be unbalanced, and this phenomenon will be reflected to a certain extent in the relevant indicators of serum bone metabolism. Therefore, it is necessary to reflect the body's bone status by measuring various bone metabolism-related indicators in serum.

[0118] At the 25th week of the experimental cycle, all rats were anesthetized by sodium pentobarbital injection, and blood was collected from the heart. After standing at 4°C for 3 hours, the blood was centrifuged at 4°C and 3000 rpm for 16 minutes, and the serum was separated and packaged, and stored at -80°C for use to avoid repeated freezing and thawing. The serum calcium (Ca) test kit and serum phosphorus (P) test kit (both purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) were used to determine the serum calcium and serum phosphorus levels in the serum of rats in each group. The rat osteocalcin (OC / BGP) enzyme-linked immunosorbent assay kit and rat calcitonin (CT) enzyme-linked immunosorbent assay kit (both purchased from Wuhan Yilai Ruite Biotechnology Co., Ltd.) were used to determine the levels of osteocalcin and calcitonin in rat serum. The rat tartrate-resistant acid phosphatase (TRAP) ELISA test kit and rat alkaline phosphatase (ALP) ELISA test kit (both purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.) were used to determine the levels of TRAP enzyme and ALP enzyme in rat serum. The results are shown in Table 1. Figure 13-18 shown.

[0119] Depend on Figure 13-18 It can be seen that the serum osteocalcin and tartrate-resistant acid phosphatase levels in the serum of rats in the OVX group were significantly higher than those in the SHAM group, while the serum calcium, blood phosphorus, alkaline phosphatase and calcitonin levels in the OVX group were significantly lower than those in the SHAM group, indicating that the serum indicators of osteoporosis model rats after surgery did change significantly compared with normal rats. It is speculated that the above changes may be due to the imbalance of bone metabolism pathways caused by changes in hormone levels in ovariectomized rats after surgery, which changes the activity of osteoblasts and osteoclasts. The serum calcium, blood phosphorus, serum osteocalcin, calcitonin, alkaline phosphatase and tartrate-resistant acid phosphatase levels in the CPP-L group rats treated with low-dose composite probiotic composition were improved to varying degrees, gradually approaching normal levels. The improvement in serum calcium, blood phosphorus, serum osteocalcin, calcitonin, alkaline phosphatase and tartrate-resistant acid phosphatase levels in the CPP-M group rats treated with a medium-dose composite probiotic composition was better than that in the CPP-L group, while the CPP-S group, which was gavaged with a medium-dose composite probiotic composition since surgery, had a more significant improvement effect on the above serum indicators than the CPP-M group.

[0120] Based on the above results, taking the composite probiotic composition provided by the present invention can improve various bone metabolism-related serum indicators (blood calcium, blood phosphorus, serum osteocalcin, calcitonin, alkaline phosphatase and tartrate-resistant acid phosphatase) of osteoporosis, and the effect of medium dose is better than that of low dose, and the effect of continuous taking of medium dose of composite probiotic composition is even better.

[0121] Example 8 Effects of the composite probiotic composition on various organ indexes and liver probiotics

[0122] 1. Determination of organ indexes in rats

[0123] The stability of the rat organ index directly indicates the normality of its own immunity and the health of its own body condition, and is an important reference indicator. In the 25th week of the test cycle, the rats were fasted but not watered for 12 hours. After being killed by dislocating the neck, the rat organs - heart, liver, spleen, kidney, lung, thymus - were accurately and quickly removed during the dissection process, weighed and recorded, and the rat organ index (the percentage of each organ weight to body weight) was calculated. The results are as follows: Fig.19 shown.

[0124] Combination Fig.19, from the comparison of the organ indexes between the groups, the heart index, spleen index, and thymus index of each treatment group were basically the same or very small, which shows that the establishment of the osteoporosis model and the grouping process did not cause serious damage to the heart, spleen, thymus, and lungs of the rats, and their various organ indexes were in a normal state. The liver index and kidney index between the treatment groups were slightly different. The liver index and kidney index of the OVX group were slightly lower than those of the SHAM group. The CPP-L group, CPP-M group, and CPP-S group treated with the composite probiotic composition for gavage had improved the downward trend of the visceral index and kidney index, and the effect of the medium-dose composite probiotic composition was better than that of the low-dose composite probiotic composition, and the effect of continuous use of the medium-dose composite probiotic was better. This shows that the composite probiotic composition provided by the present invention can not only help rats maintain a relatively normal liver index and kidney index, but also has no toxic side effects on the heart, spleen, thymus, and lungs of rats, and can keep rats in a good immune state and a healthy state.

[0125] 2. Liver probiotics detection

[0126] Some studies have shown that after model rats are fed certain probiotics, bacteria will grow in the liver, leading to liver failure. Therefore, it is very important to evaluate whether probiotics will grow in the liver and affect liver function. In the 25th week of the test cycle, rats were fasted but not watered for 12 hours. After the rats were killed, the rat livers were dissected and quickly placed in sterile plates. After recording the data, 1g of the liver was cut and added to the sterile PBS buffer for suspension and homogenization. 1mL of the solution was applied to the MRS solid plate. Two plates were set up for each group, and they were cultured at 37°C for 48 hours and 37°C for 48 hours respectively. The plate was tested for bacterial growth. In this experiment, the liver tissues of each group of rats were aseptically coated on the MRS plate, and no colony formation was found in the aerobic culture for 48 hours and the anaerobic culture for 48 hours. This shows that the composite probiotic composition provided by the present invention does not cause hepatointestinal translocation in animals.

[0127] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A composite probiotic composition for preventing, treating or improving osteoporosis, characterized in that: Including compound probiotic powder, compound probiotic powder is composed of Lactobacillus johnsonii ( Lactobacillus johnsonii )HC1175 and Lactobacillus delbrueckii subsp. bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ) HC1983 bacterial powder; the preservation number of Lactobacillus johnsonii HC1175 is CGMCC No.27354, and the preservation number of Lactobacillus delbrueckii subspecies bulgaricus HC1983 is CGMCC No.26584; the bacterial content of Lactobacillus johnsonii HC1175 and Lactobacillus delbrueckii subspecies bulgaricus HC1983 is 5.0×10 9 ~3.0×10 10 cfu / g, the ratio of viable bacteria count is 1~3:1~3.

2. The composite probiotic composition according to claim 1, characterized in that The preparation method of Lactobacillus johnsonii HC1175 bacterial powder is as follows: (1) The refrigerated Lactobacillus johnsonii HC1175 strain was warmed up at room temperature for 1 hour, inoculated into 2.5L liquid culture medium, and cultured aerobically at 37°C for 16-20 hours to prepare primary seeds, which were then transferred to a 100L seed tank and cultured aerobically at pH 6.58 and 37°C for 6-8 hours to obtain secondary seeds. Subsequently, the strain was transferred to a 1000L fermentation tank at a 10% inoculation rate, and cultured aerobically at pH 6.39 and 37°C for 6-8 hours until the logarithmic growth phase. The fermentation liquid was aseptically centrifuged to obtain Lactobacillus johnsonii HC1175 bacterial sludge; (2) The first coating agent was sterilized by high-pressure air sterilization at 105°C for 30 min. The Lactobacillus johnsonii HC1175 sludge and the first coating agent were thoroughly stirred and mixed at a weight ratio of 1:1-2, and the pH was adjusted to 6.5-7.0 to obtain a sludge coating solution. After vacuum freeze-drying, the freeze-dried blocks were taken out and crushed to obtain Lactobacillus johnsonii HC1175 powder. The first coating agent included the following components in mass fractions: 10% skimmed milk powder, 8% trehalose, 2% oligofructose, 3% glycerol, 5% maltodextrin and 72% distilled water.

3. The composite probiotic composition according to claim 1, characterized in that The preparation method of Lactobacillus delbrueckii subspecies bulgaricus HC1983 bacterial powder is as follows: (1) The refrigerated Lactobacillus delbrueckii subsp. bulgaricus HC1983 strain was warmed up at room temperature for 1 hour, inoculated into 2.5 L liquid culture medium, and cultured anaerobically at 37°C for 24-30 hours to prepare primary seeds, which were then transferred to a 100 L seed tank and cultured anaerobically at pH 6.45 and 37°C for 12-14 hours to obtain secondary seeds. Subsequently, the strain was transferred to a 1000 L fermentation tank at a 10% inoculation rate, and cultured anaerobically at pH 6.45 and 37°C for 6-8 hours until the logarithmic growth phase. The fermentation liquid was aseptically centrifuged to obtain Lactobacillus delbrueckii subsp. bulgaricus HC1983 bacterial sludge; (2) The second coating agent was sterilized by high-pressure air sterilization at 105°C for 30 min. The Lactobacillus johnsonii HC1175 sludge and the second coating agent were thoroughly stirred and mixed at a weight ratio of 1:1-2, and the pH was adjusted to 6.5-7.0 to obtain a sludge coating solution. After vacuum freeze-drying, the freeze-dried blocks were taken out and crushed to obtain Lactobacillus delbrueckii subspecies bulgaricus HC1983 powder. The second coating agent included the following components in mass fractions: 15% skimmed milk powder, 8% trehalose, 3% glycerol, 1% sodium ascorbate and 73% distilled water.

4. The composite probiotic composition according to claim 1, characterized in that Calculated by mass fraction, the composite probiotic composition includes: 4% composite probiotic powder, 67% fructooligosaccharides, 8% stachyose, 7% xylo-oligosaccharides, 8% galacto-oligosaccharides, 5% inulin, 0.2% fig fruit powder, 0.3% apple powder, 0.2% red date powder, and 0.3% wolfberry powder.

5. Use of the composite probiotic composition according to any one of claims 1 to 4 in preparing a product for preventing, treating or improving osteoporosis, characterized in that: Products for preventing, treating or improving osteoporosis are products for preventing, treating or improving postmenopausal osteoporosis.

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