Application of bifidobacterium animalis in anti-inflammation, anti-helicobacter pylori and improvement of obesity, hyperlipidemia and bone loss

Abstract

The invention discloses a strain of bifidobacterium animalis dipro-60 and a preparation method thereof. The invention further discloses a preparation, and the preparation contains the bifidobacterium animalis dipro-60. The bifidobacterium animalis dipro-60 used in the invention has the ability of producing bile salt hydrolase BSH, the ability of reducing cholesterol, the ability of inhibiting helicobacter pylori and the ability of reducing inflammatory factor TNF-alpha; further animal experiments show that the dipro-60 can improve lipid metabolism related diseases, can significantly reduce weight gain of obese mice induced by high fat, significantly reduce the liver weight of the obese mice, reduce the content of triglyceride and blood sugar in blood, improve intestinal barriers and improve the lipid metabolism related diseases, and the dipro-60 can improve the lipid metabolism related diseases. Liver lipid accumulation caused by obesity or hyperlipidemia is improved, and bone loss caused by obesity or hyperlipidemia is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to application of an animal bifidobacterium in anti-inflammation, anti-Helicobacter pylori and improvement of obesity, hyperlipidemia and bone loss. Background Art

[0002] The association between obesity and bone metabolism has long been of interest. Traditionally, greater adipose tissue size has been thought to be protective against bone density and fractures due to the enhanced physical loading and cushioning effects of soft tissue. However, recent studies have shown that obesity, especially severe obesity, can affect both bone quality and quantity, which is known as osteopenic obesity.

[0003] The intestine is considered the largest digestive, endocrine, and immune organ in the human body. The intestinal microbiota can interact locally in the intestine and further communicate with distant organs. Microbiome dysbiosis is associated with impaired intestinal epithelial barrier function, allowing more microorganisms and bacterial metabolites to enter the host, which may be caused by the increased number of certain harmful bacteria and the reduced production of SCFAs. Translocated bacteria and osmotic metabolites (such as endotoxins) can activate intestinal immune cells, including helper T cells 1 (Th1), helper T cells 17 (Th17), and macrophages, to produce inflammatory cytokines. These microorganisms, bacterial products, immune cells, and cytokines enter the circulation, resulting in persistent, low-grade systemic inflammation. These alterations may contribute to obesity-associated osteopenia.

[0004] Bone metabolism in obese individuals is highly influenced by inflammatory responses. Studies have found that several key inflammatory markers, including TNF-α and IL-1β, are associated with obesity and obesity-related adverse outcomes, indicating that systemic low-grade inflammation is a risk factor for health, including bone health, in obese patients.

[0005] It has been recognized that intestinal flora is a potential target for obesity and osteoporosis. At present, there are few studies on the use of probiotics to treat osteopenic obesity. For example, Chinese patent CN115778987B "A strain of animal bifidobacterium that can relieve osteoporosis and its application" provides a strain of animal bifidobacterium XA-768 that can relieve osteoporosis, but the effect of this strain is verified on ovariectomized mice, simulating the situation of osteoporosis in postmenopausal women. The bacteria are limited to improving the situation of osteoporosis caused by estrogen reduction, and it is not mentioned whether it has an improvement effect on obesity and hyperlipidemia. For example, Chinese patent application CN117535200A "A strain of animal bifidobacterium lactis subspecies and its use" provides a strain of animal bifidobacterium that can inhibit the differentiation of 3T3-L1 preadipocytes, thereby achieving the effect of effectively inhibiting fat deposition and having a weight loss function, but it is not mentioned whether it can improve obesity-related osteopenia. The strains in the above-mentioned Chinese patents or Chinese patent applications do not have the effects of simultaneous anti-inflammatory, improvement of obesity, hyperlipidemia and bone loss.

[0006] Therefore, there is an urgent need to discover more probiotics that are more effective in treating and / or preventing osteopenic obesity, so as to provide new ideas or effective strategies for preventing and / or treating osteopenic obesity. Summary of the invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to provide a strain of animal Bifidobacterium that can simultaneously resist inflammation and Helicobacter pylori and improve obesity, hyperlipidemia and bone loss.

[0008] The technical problem that the present invention also solves is the application of the animal bifidobacterium in anti-inflammation, anti-Helicobacter pylori, and improvement of obesity, hyperlipidemia and bone loss.

[0009] Technical solution: To solve the above technical problems, the present invention provides a strain of Bifidobacterium animalis subsp. lactis dipro-60, which was deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms on December 25, 2023, with a deposit number of CGMCC No. 29382. The classification name of the Bifidobacterium animalis dipro-60 described in the present invention is Bifidobacterium animalis subsp. lactis.

[0010] The animal bifidobacterium of the present invention is separated from the feces of healthy children. The whole genome sequencing analysis and BLAST nucleic acid sequence comparison of the strain through the NCBI gene database show that the strain is animal bifidobacterium (Bifidobacterium animalis subsp. lactis).

[0011] The Bifidobacterium animalis subsp. lactis dipro-60 can produce bile salt hydrolase, and the diameter of the bile salt hydrolase ring is 15 to 35 mm.

[0012] The strain can directly reduce the cholesterol level in the culture medium, and the cholesterol reduction ratio is 10% to 100%.

[0013] The strain can inhibit Helicobacter pylori, and the diameter of its inhibition zone is 12 to 30 mm.

[0014] The present invention also includes products containing the Bifidobacterium animalis subsp. lactis dipro-60, and the products include fermented products.

[0015] The fermented product comprises bacterial supernatant, bacterial precipitation or bacterial suspension of Bifidobacterium animalis subsp. lactis dipro-60.

[0016] The dosage forms of the product include powder, capsule, tablet, pill, film-coated agent, aerosol, granule, liquid, liposome, transdermal agent, suppository or lyophilized powder injection.

[0017] The present invention also includes the use of the Bifidobacterium animalis subsp. lactis dipro-60 product in the preparation of products for anti-inflammatory, anti-Helicobacter pylori, and improving obesity, hyperlipidemia and / or bone loss.

[0018] The present invention also includes the application of the Bifidobacterium animalis subsp. lactis dipro-60 in the product having at least one of the following functions:

[0019] 1) Products that reduce cellular inflammation;

[0020] 2) Products with the ability to reduce body weight;

[0021] 3) Products that can improve dyslipidemia;

[0022] 4) Products that improve blood sugar levels;

[0023] 5) Products that improve the intestinal barrier;

[0024] 6) Products that can improve intestinal flora structure disorders;

[0025] 7) Products that can improve liver tissue damage caused by obesity or hyperlipidemia;

[0026] 8) Products that can improve the content of tartrate phosphatase or type I collagen N-terminal propeptide, which are bone metabolism indicators in the blood.

[0027] The product having the ability to reduce cellular inflammation is specifically a product having the ability to reduce TNF-α at the cellular level and in the intestine.

[0028] The product having the effect of reducing body weight is specifically a product having the effect of reducing weight gain caused by a high-fat diet.

[0029] The product having the function of improving dyslipidemia is specifically a product having the function of reducing the content of triglycerides, total cholesterol and low-density lipoprotein in the blood.

[0030] The product having the effect of improving blood sugar is specifically a product having the effect of reducing the increase of blood sugar caused by a high-fat diet.

[0031] Among them, the product with improved intestinal barrier is specifically a product with increased expression of intestinal barrier-related genes mTff3, Muc2, and ZO-1 in the intestine.

[0032] Among them, the product capable of improving the disorder of intestinal flora structure is specifically a product capable of regulating the abundance of beneficial bacteria in the intestine and reducing the abundance of harmful bacteria.

[0033] Among them, the product that can improve liver tissue damage caused by obesity or hyperlipidemia is specifically a product that can improve aspartate aminotransferase and liver cell fat accumulation.

[0034] Among them, the product that can improve the content of tartrate phosphatase or type I collagen N-terminal propeptide, a bone metabolism indicator in the blood, is specifically a product that can improve the content of tartrate phosphatase or type I collagen N-terminal propeptide, a bone metabolism indicator caused by obesity or hyperlipidemia.

[0035] The present invention reports for the first time that the intake of animal Bifidobacterium dipro-60 improves the intestinal flora structure of mice fed a high-fat diet (HFD), reduces their blood lipids and blood sugar, improves the intestinal barrier and reduces the level of inflammatory factors, and on this basis can further improve bone loss caused by lipid metabolism disorders.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the present invention provides a strain of animal bifidobacterium dipro-60 that has the ability to produce bile salt hydrolase BSH, lower cholesterol, inhibit Helicobacter pylori, and reduce the inflammatory factor TNF-α; further animal experiments show that dipro-60 can improve lipid metabolism-related diseases, wherein dipro-60 can significantly reduce the weight gain of obese mice modeled with high fat, significantly reduce the liver weight of obese mice, and simultaneously reduce the triglyceride and blood sugar content in the blood, improve the intestinal barrier, improve the liver lipid accumulation caused by obesity or hyperlipidemia, and improve the bone loss caused by obesity or hyperlipidemia. Therefore, the use of animal bifidobacterium dipro-60 strains for the preparation of products for improving obesity, hyperlipidemia and bone loss has a good prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : Figure A shows the bile salt hydrolase (BSH) production capacity of animal Bifidobacterium dipro-60 and the reference strain animal Bifidobacterium lactis subsp. BPL1; Figure B shows the statistical results of the diameter of the BSH hydrolysis ring produced by dipro-60 and its reference strains B420 and BPL1.

[0038] Figure 2 : Cholesterol-lowering ability of Bifidobacterium animalis dipro-60 and its reference strains B420 and BPL1.

[0039] Figure 3 :The inhibition zone size of animal Bifidobacterium dipro-60 and its reference bacterium Lactobacillus reuteri 17648 against Helicobacter pylori.

[0040] Figure 4 :Anti-inflammatory ability of animal Bifidobacterium dipro-60 and its reference strains B420 and BPL1 in in vitro cell experiments.

[0041] Figure 5 : Figure A shows the changes in the percentage of weight gain in the obese mouse model after continuous gavage of PBS, orlistat and dipro-60 for 11 weeks; Figure B shows the percentage of weight gain in the obese mouse model at the gavage endpoint; Figure C shows the liver weight results of the obese mouse model at the gavage endpoint.

[0042] Figure 6 :Figure A shows the results of measuring triglyceride content in the blood of obese mouse model; Figure B shows the results of measuring fasting blood glucose content in the blood of obese mouse model; Figure C shows the statistical results of the area under the curve of oral glucose tolerance test (OGTT) in obese mouse model.

[0043] Figure 7: Figure A shows the results of HE staining of liver tissue pathology in obese mouse models under different treatment groups; Figure B shows the NAS scoring results of HE staining of liver tissue pathology in obese mouse models under different treatment groups.

[0044] Figure 8 : Expression of intestinal barrier-related genes and inflammatory factor TNF-α in high-fat diet modeled mice under different treatments; Figure A shows the expression results of mTff3 in the intestine; Figure B shows the expression results of ZO-1 in the intestine; Figure C shows the expression results of Muc2 in the intestine; Figure D shows the expression results of TNF-α in the intestine.

[0045] Fig. 9 :Changes in bone metabolism indicators in the blood of obese mouse models under different treatments; Figure A shows the results of determination of the content of bone formation marker type I procollagen amino-terminal propeptide (PINP) in different treatment groups; Figure B shows the results of determination of the content of tartrate-resistant acid phosphatase (TRAP), a characteristic marker of osteoclasts in different treatments.

[0046] Fig.10 : Results of lefse analysis of intestinal flora in obese mouse model under different treatments.

[0047] Fig.11 :Changes in lipid metabolism in the blood of hyperlipidemia mouse model under different treatments; Figure A shows the measurement results of total cholesterol (TC) content in the blood of hyperlipidemia mouse model under different treatments; Figure B shows the measurement results of low-density lipoprotein (LDL) content in the blood of hyperlipidemia mouse model under different treatments; Figure C shows the measurement results of the ratio of total cholesterol (TC) to high-density lipoprotein (HDL) in the blood of hyperlipidemia mouse under different treatments.

[0048] Fig.12 :Determination results of lipopolysaccharide (LPS) content in blood of hyperlipidemic mouse models with different treatments.

[0049] Fig.13 :The results of determination of aspartate aminotransferase (AST) content in the blood of hyperlipidemia mouse models treated with different methods.

[0050] Fig.14 : Pathological results of HE staining of liver tissue in hyperlipidemia mouse models with different treatments.

[0051] Fig.15 :The results of determination of the content of tartrate-resistant acid phosphatase (TRAP), a characteristic marker of osteoclasts in the blood of hyperlipidemic mice treated with different methods. DETAILED DESCRIPTION

[0052] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0053] Example 1 Microbiological Identification of Strain dipro-60

[0054] dipro-60 was isolated from the feces of healthy children and was identified as Bifidobacterium animalis subsp. lactis by MALDI-TOF, 16SrRNA sequencing and whole genome sequencing.

[0055] The animal Bifidobacterium dipro-60 of the present invention was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 25, 2023, with the deposit number: CGMCC No.29382.

[0056] When conducting various experiments, the glycerol-preserved bacteria of animal Bifidobacterium dipro-60 were taken out from the -80°C refrigerator, melted in a 37°C water bath, and 20 μL was spread on MRS+ (MRS (Guangdong Huankai Microbiological Technology Co., Ltd., catalog number: 027315) + 0.5 g / L cysteine) plates, and cultured anaerobically at 37°C for 48 h; then, a single clone was picked up with an inoculation loop for streaking and subcultured, and after 48 h, a single clone colony was picked up and transferred to MRS broth liquid culture medium (MRS+) supplemented with 0.5 g / L cysteine, and incubated at 37°C constant temperature for 48 h for expansion culture, and then subsequent experiments could be carried out.

[0057] Example 2 Determination of the bile salt hydrolase (BSH) production and cholesterol-lowering ability of animal Bifidobacterium dipro-60

[0058] Bile salt hydrolase (BSH) production experiment: Use BSH screening plates, the preparation method is as follows: MRS solid medium + 0.37g / L CaCl2 (calcium chloride) + 5g / L sodium taurodeoxycholate (TDCA); place a 6mm drug-sensitive paper in the center of the BSH screening plate, and then wash animal Bifidobacterium dipro-60 and the reference strain grown to the logarithmic phase with PBS twice, and then adjust the bacterial suspension to OD600=0.5 with PBS, then take 10μL of the bacterial suspension and drop it on the drug-sensitive paper, and measure the hydrolysis ring diameter after culturing at 37℃ anaerobic conditions for 48h. The results are as follows Figure 1 A; at the same time, further statistical analysis of the hydrolysis ring diameter of animal Bifidobacterium dipro-60 and its reference strain animal Bifidobacterium lactis subspecies B420 (IFF International Flavors and Fragrances Company) and animal Bifidobacterium lactis subspecies BPL1 (ADM, USA) were performed. Figure 1As shown in B, the diameter of the BSH hydrolysis ring of animal Bifidobacterium dipro-60 was higher than that of the reference strains BPL1 and B420, indicating that animal Bifidobacterium dipro-60 has a good ability to produce BSH.

[0059] Cholesterol-lowering ability experiment: Animal Bifidobacterium dipro-60 and reference strains B420 and BPL1 were inoculated into freshly prepared MRS+ liquid culture medium containing 0.3% (w / v) bile salts and 0.1g / L cholesterol (soluble in anhydrous ethanol, Sigma-Aldrich), and incubated at 37°C for 24 hours. Then centrifuged at 4°C, 4000g for 15 minutes, and the cholesterol level in the supernatant was detected using a cholesterol level detection kit (E-BC-K109-M). The blank culture medium without bacteria but cultured in parallel was used as the control group (control group); the test results are shown in the figure. Figure 2 As shown, the remaining percentage of cholesterol in the culture supernatant of the reference strains B420 and BPL1 was 95%, and the remaining percentage of cholesterol in the culture supernatant of Bifidobacterium animalis dipro-60 was 88%, indicating that Bifidobacterium animalis dipro-60 has a stronger cholesterol-lowering ability.

[0060] Example 3 Determination of the ability of animal Bifidobacterium dipro-60 to inhibit Helicobacter pylori

[0061] The Oxford cup method was used to test the in vitro anti-Helicobacter pylori ability of probiotics: Helicobacter pylori (H.pylori SS1, Beina Biotech) was spread on Columbia blood agar plates (BNCC, catalog number: BNCC352241) and cultured under microaerobic conditions at 37°C.

[0062] The dipro-60 bacterial solution in the exponential growth phase was inoculated at a 2% inoculation rate into MRS+ liquid culture medium and anaerobically cultured for 48 h for later use. Similarly, the control strain Lactobacillus reuteri DSM 17648 (Novozymes, Denmark) in the exponential growth phase was inoculated at a 2% inoculation rate into MRS liquid culture medium and anaerobically cultured for 24 h for later use.

[0063] With the help of an Oxford cup, holes were made in Columbia blood agar plates at a distance of 2.5 cm. A suspension of H. pylori (1 × 10 9 CFU / mL) was inoculated onto blood agar using a sterile swab. Then the cultured dipro-60 bacterial solution and the reference strain Lactobacillus reuteri DSM 17648 bacterial solution (3×10 8CFU / mL) was taken and added to an Oxford cup, and phosphate buffer PBS (Procell, 10 mM, pH 7.2-7.4, catalog number: PB180327) was used as a blank control. The diameter of the growth inhibition zone was measured after incubation at 37°C for 48 hours in a microaerobic environment.

[0064] like Figure 3 As shown, the average diameter of the inhibition zone of dipro-60 against Helicobacter pylori is 14.49 mm, while the average diameter of the inhibition zone of the reference strain Lactobacillus reuteri 17648 against Helicobacter pylori is 13.53 mm, indicating that dipro-60 exhibits a good antibacterial effect against Helicobacter pylori.

[0065] Example 4 Determination of the anti-inflammatory ability of animal Bifidobacterium dipro-60

[0066] The dipro-60 bacterial suspension and the reference strains B420 and BPL1 bacterial suspensions in Example 1 were inoculated into MRS+ liquid culture medium at 2%. After the culture was completed, the bacterial suspension was centrifuged at 6000 rpm / min for 5 min, the supernatant was discarded, and the suspension was resuspended and washed twice by centrifugation with PBS for later use.

[0067] RAW246.7 cells that were in good growth condition and in the logarithmic growth phase were cultured at 1×10 5 The cells were inoculated into 96-well plates at a density of 1 × 10 / well and cultured until adhered to the wall. The culture medium was discarded, 100 μL of DMEM solution was added to the negative wells (control group), 100 μL of DMEM solution containing 0.5 μg / mL LPS was added to the positive wells (LPS group), and 100 μL of 1×10 7 CFU Bifidobacterium animalis dipro-60, 1×10 7 CFU B420, 1×10 7 The DMEM solution containing CFU BPL1 and 0.5 μg / mL LPS was placed in a 37°C, 5% CO2 incubator and incubated for 18 h. The supernatant after the above culture was collected and the TNF-α concentration was detected using a kit. The test results are as follows Figure 4 As shown, compared with the positive LPS group, dipro-60 can significantly reduce the concentration of inflammatory factor TNF-α in cells, indicating that dipro-60 has a good anti-inflammatory effect.

[0068] Example 5 Animal Bifidobacterium dipro-60 effectively reduces the body weight of an obese animal model induced by a high-fat diet

[0069] Twenty-four 8-week-old male C57BL / 6J mice were randomly divided into four groups: normal diet group (NC group); model control group (PBS group); positive drug group (oselita group); probiotic group (dipro-60 group). The normal diet group (NC group) maintained normal feed feeding and PBS (Procell, 10mM, pH7.2-7.4, catalog number: PB180327) gavage treatment for 11 weeks, and the other three groups used Dyets 60% kcal fat calorie high-fat mouse diet (Dyets, catalog number: HF60) for 11 weeks of obesity modeling and corresponding gavage treatment.

[0070] Oral administration: Animal Bifidobacterium dipro-60 was fermented anaerobically at 37°C, the fermentation broth was centrifuged and resuspended in phosphate buffered saline (PBS) (Procell, 10 mM, pH 7.2-7.4, catalog number: PB180327), and the OD600 was adjusted to 2.5, and the bacterial concentration was 5*10 9 CFU / ml, 200 μL was administered orally to each mouse for 11 weeks.

[0071] The results of weight gain percentage of each treatment group for 11 consecutive weeks are as follows Figure 5 As shown in A, it can be seen that dipro-60 began to reduce the weight gain of mice caused by high-fat diet after 7 weeks of oral administration. Further analysis of the weight gain percentage of different treatment groups at the end point of oral administration is shown in Figure 5 As shown in B, after 11 weeks of oral administration, dipro-60 can significantly reduce the weight gain of mice caused by a high-fat diet. At the same time, the liver weights of mice in different treatment groups were measured, and it was found that dipro-60 can also reduce the weight gain of mice liver caused by a high-fat diet.

[0072] Example 6 Animal Bifidobacterium dipro-60 effectively reduces blood lipids and blood sugar in an obese animal model induced by a high-fat diet

[0073] The experimental model was the same as that described in Example 5. The blood lipid and blood glucose indexes of mice in different treatment groups were tested. The blood of mice in different treatment groups was collected by ocular venous blood sampling, and then the triglyceride (TG) content in the blood of mice in each treatment group was measured by Catic dry biochemical analyzer. The test results are shown in Figure 6 As shown in A, compared with the PBS control group, the oral dipro-60 treatment group was able to significantly reduce the triglyceride content in the blood of obese mice and maintain the level consistent with that of the normal group NC, indicating that dipro-60 has a good effect on improving blood lipids in obese mice.

[0074] Fasting blood glucose determination: One day before the end of intragastric administration, the mice were fasted for 12 hours, and the blood glucose levels of the mice in different treatment groups were measured using a Roche Zhihang blood glucose meter. Figure 6 As shown in B, compared with the PBS control group, the oral dipro-60 treatment group was able to significantly reduce the blood glucose content in obese mice and maintain the level consistent with that of the normal group NC, indicating that dipro-60 has a good effect on improving blood glucose in obese mice.

[0075] Oral glucose tolerance test (OGTT): One day before the gavage endpoint, mice that had fasted for 12 hours were gavaged with 20% glucose solution at a dose of 1 g / kg. Blood glucose levels at 0, 30, 60, 90, and 120 minutes after gavage were measured to analyze the insulin resistance level of mice. The results of the area under the OGTT curve (AUC) of different treatment groups were statistically analyzed as follows: Figure 6 As shown in C, compared with the PBS control group, the oral dipro-60 treatment group was able to significantly reduce the OGTT area under the curve (AUC) level of obese mice, indicating that dipro-60 has a good effect in improving insulin resistance in obese mice.

[0076] Example 7 Animal Bifidobacterium dipro-60 effectively improves the liver lipid accumulation level of the obese animal model induced by high-fat diet

[0077] The experimental model was the same as that described in Example 5. At the end of intragastric administration, liver samples of each treatment group were collected, fixed in 4% paraformaldehyde, and then HE-stained. After taking pictures, NAS scores were calculated. Figure 7 As shown in A, the liver of mice in the PBS control group of the obesity model showed large areas of vacuoles, which was caused by lipid accumulation in the liver. At the same time, many inflammatory cells were observed to be infiltrated, indicating that they had a more serious inflammation. In contrast, the number of vacuoles in the liver of mice in the dipro-60 treatment group was reduced, and there was little inflammatory cell infiltration. Further NAS score statistics were performed as follows Figure 7 As shown in B, the NAS score of the dipro-60-treated group was significantly lower than that of the PBS group, indicating that dipro-60 has a good effect in improving liver lipid accumulation and inflammatory infiltration in obese mice.

[0078] Example 8 Animal Bifidobacterium dipro-60 effectively improves the intestinal barrier of high-fat diet-induced obese animal models and reduces the level of inflammatory factors

[0079] The experimental model was the same as that described in Example 5. Colon tissues of each treatment group were collected at the end of intragastric administration to analyze the expression of colon-related genes. The experimental steps were as follows: About 5 mm2 of mouse colon tissue was taken into a 1.5 mL EP tube, 1 mL of Trizol reagent (Invitrogen, Cat. No.: 15596026) and 4 2.5 mm stainless steel beads were added to each tube, and the program was set to 60 MHz for 45 seconds of homogenization, with a rest period of 15 seconds, and repeated 5 times. RNA was then extracted and reverse transcribed using QuantStudio TM 5Real-Time PCR System was used to quantitatively analyze gene expression levels, and the detected genes included ZO-1, mTff3, TNF-α, Muc2 and the internal reference gene beta-Actin.

[0080] The statistical analysis results are as follows Figure 8 As shown, Figure 8 A indicates that compared with the PBS control group, the oral administration of dipro-60 can significantly increase the expression level of trefoil peptide factor (mTff3) in the mouse colon, indicating that dipro-60 can promote intestinal mucosal repair. The expression of the intestinal barrier tight junction protein gene ZO-1 was analyzed as follows Figure 8 As shown in Figure B, compared with the PBS group, the expression of ZO-1 gene in the dipro-60 group was significantly increased, indicating that dipro-60 gavage treatment can promote the expression of tight junction proteins. Figure 8 As shown in C, compared with the PBS group, the expression of Muc2 gene in the dipro-60 group was significantly increased, indicating that dipro-60 gavage treatment can promote the expression of mucus protein Muc2; analysis of the level of proinflammatory cytokine TNF-α found that compared with the PBS group, the level of proinflammatory cytokine TNF-α in the colon tissue of mice was significantly reduced in the dipro-60 group, indicating that gavage of dipro-60 can reduce the level of inflammation in the colon of mice caused by a high-fat diet; In summary, dipro-60 can maintain the health of the intestinal barrier by increasing the expression of intestinal barrier-related genes and inhibiting the expression of inflammatory cytokines.

[0081] Example 9 Animal Bifidobacterium dipro-60 effectively improves bone loss in the blood of obese animal models induced by high-fat diet

[0082] The experimental model was the same as that described in Example 5. Blood samples were collected from each treatment group at the end of intragastric administration, and the bone metabolism indexes in the mouse serum were detected using an ELISA kit. Fig. 9 As shown, Fig. 9A indicates that compared with the control group PBS group, the oral administration of dipro-60 group can significantly increase the level of type I collagen N-terminal propeptide (PINP) in the blood of obese mice. Type I collagen N-terminal propeptide (PINP) is often used to reflect the therapeutic effect of osteoporosis, especially for the evaluation of bone formation-promoting treatment, and is an important clinical detection indicator reflecting bone formation.

[0083] During bone resorption, osteoclasts secrete tartrate-resistant acidphosphatase (TRAP) into the blood as an active enzyme, so TRAP is often used as an important indicator to reflect the number of osteoclasts. Fig. 9 As shown in B, compared with the PBS group, the intragastric administration of dipro-60 significantly reduced the level of tartrate-resistant acid phosphatase (TRAP) in the blood of obese mice. Therefore, in summary, dipro-60 can improve bone loss caused by obesity.

[0084] Example 10 Animal Bifidobacterium dipro-60 effectively improves the intestinal flora of obese animal models induced by high-fat diet

[0085] The experimental model was the same as that described in Example 5. Fecal samples from each treatment group were collected at the end of intragastric administration and sent to Qingke Biotechnology Co., Ltd. for sequencing. The samples were subjected to lefse analysis using bioinformatics analysis software (such as QIIME2, etc.) to compare the differential flora between the groups. The analysis results are shown in Fig.10 As shown in the figure, compared with PBS, the abundance of 20 bacteria was significantly increased after oral administration of dipro-60, among which Bifidobacterium had obvious probiotic properties, and the abundance of 11 bacteria was significantly reduced, among which the abundance of harmful bacteria such as Tuzzerella and Eubacterium_nodatumgroup was reduced. This shows that dipro-60 can regulate the dysbiosis of obese mice to a certain extent while exerting the effect of reducing weight and lipid.

[0086] Example 11 Animal Bifidobacterium dipro-60 effectively improves blood lipid content in hyperlipidemia mouse model

[0087] Eighteen 8-week-old male C57BL / 6J mice were randomly divided into three groups: normal feeding group (NC), hyperlipidemia model group (PBS), and dipro-60 treatment group (dipro-60). The NC group was fed with normal feed, while the PBS group and dipro-60 group were fed with Dyets high-fat (40 kcal%), high-cholesterol (1.25%), and high-sodium cholate (0.5%) mouse diet (Dyets, catalog number: ASHF4) for 8 weeks of hyperlipidemia modeling. At the same time, gavage treatment was performed. The gavage conditions were as follows: animal Bifidobacterium dipro-60 was anaerobic fermented at 37°C, and the fermentation broth was centrifuged and resuspended in phosphate buffered saline (PBS, Procell, 10 mM, pH 7.2-7.4, catalog number: PB180327), and the OD600 was adjusted to 2.5, and the bacterial concentration was 5*10 9 CFU / ml, 200 μL was administered intragastrically to each mouse.

[0088] Blood samples were collected from mice at the end of intragastric administration, and the results were Fig.11 As shown, compared with the PBS group, intervention with Bifidobacterium longum dipro-60 in the ASHF-induced hyperlipidemia mouse model can significantly reduce the levels of total cholesterol (TC), low-density lipoprotein (LDL) and the ratio of total cholesterol (TC) to high-density lipoprotein (HDL) in the blood, indicating that dipro-60 has a positive effect on the blood lipid intervention of hyperlipidemia model mice.

[0089] Example 12 Animal Bifidobacterium dipro-60 effectively improves the LPS content in the blood of hyperlipidemia mouse model

[0090] The experimental model was the same as that described in Example 11. Blood samples were collected from mice at the end of intragastric administration to measure the content of lipopolysaccharide (LPS) in the blood. The results were as follows: Fig.12 As shown, intervention with Bifidobacterium longum dipro-60 in the ASHF-induced hyperlipidemic mouse model could significantly reduce the level of LPS in the blood compared with the PBS group.

[0091] Example 13 Animal Bifidobacterium dipro-60 effectively improves the AST content in the blood of a hyperlipidemia mouse model The experimental model was the same as that described in Example 11. Blood samples were collected from mice at the end of intragastric administration to measure the level of aspartate aminotransferase (AST) in the blood. The results are as follows: Fig.13 As shown in the results, compared with the PBS group, dipro-60 can effectively reduce the level of aspartate aminotransferase (AST) in the blood of ASHF-induced hyperlipidemia mouse model. Aspartate aminotransferase is an important type of transaminase. It is an indicator of liver function test in clinical medicine and is used to determine whether the liver is damaged. This shows that the intervention of dipro-60 alleviated the liver damage caused by ASHF diet in mice.

[0092] Example 14 Animal Bifidobacterium dipro-60 effectively improves lipid accumulation in the liver of hyperlipidemia mouse model

[0093] The experimental model was the same as that described in Example 11. The liver tissues of mice were collected at the end of oral administration and fixed with 4% paraformaldehyde before slice preparation. Fig.14 As shown in the figure, after hyperlipidemia modeling, a large number of cavities caused by lipid droplets and some inflammatory cell aggregation appeared in the liver (PBS group). After intervention with dipro-60, the vacuolar structure and inflammatory cell aggregation were significantly reduced, indicating that dipro-60 intervention can effectively reduce the accumulation of lipids and inflammation in the liver.

[0094] Example 15 Animal Bifidobacterium dipro-60 effectively improves the content of TRAP in the blood of hyperlipidemia mouse model

[0095] The experimental model was the same as that described in Example 11. Blood samples of mice were collected at the end point of intragastric administration, and bone metabolism indicators in mouse serum were detected using an ELISA kit (Enzyme-linked Biologicals, catalog number ml095180).

[0096] The results are as follows Fig.15 As shown, compared with the PBS group, the highly expressed TRAP in the blood of hyperlipidemic mice was significantly decreased after dipro-60 intervention (P<0.05).

Claims

1. A strain of Bifidobacterium animalis subsp. lactis dipro-60, characterized in that: The animal Bifidobacterium (Bifidobacterium animalis subsp. lactis) dipro-60 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 25, 2023, with the deposit number: CGMCC No.29382.

2. The Bifidobacterium animalis subsp. lactis dipro-60 according to claim 1, characterized in that The strain can produce bile salt hydrolase, and the diameter of its bile salt hydrolysis ring is 15 to 35 mm; preferably, the strain can directly reduce the level of cholesterol in the culture medium, and the proportion of cholesterol reduction is 10% to 100%; preferably, the strain can inhibit Helicobacter pylori, and the diameter of its inhibition zone is 12 to 30 mm.

3. A product containing Bifidobacterium animalis subsp. lactis dipro-60 according to claim 1, characterized in that: The products include fermented products.

4. The product according to claim 3, wherein the fermented product comprises a fermentation supernatant, a fermentation precipitate or a bacterial suspension of Bifidobacterium animalis subsp. lactis dipro-60.

5. The product according to claim 3, characterized in that The dosage forms of the product include powder, capsule, tablet, pill, film-coated agent, aerosol, granule, liquid, liposome, transdermal agent, suppository or lyophilized powder injection.

6. The product according to claim 3, characterized in that The product also includes a protective agent, a functional auxiliary agent / or an auxiliary additive, a drug carrier and / or a pharmaceutical excipient.

7. Use of Bifidobacterium animalis subsp. lactis dipro-60 according to claim 1 and the product according to any one of claims 2 to 5 in the preparation of products for anti-inflammatory, anti-Helicobacter pylori, improvement of obesity, hyperlipidemia and / or bone loss.

8. The use according to claim 7, characterized in that: The application of the product is a product that has at least one of the following functions: 1) Products that reduce inflammation; 2) Products with the ability to reduce body weight; 3) Products that can improve dyslipidemia; 4) Products that improve blood sugar levels; 5) Products that improve the intestinal barrier; 6) Products that can improve intestinal flora structure disorders; 7) Products that can improve liver tissue damage caused by obesity or hyperlipidemia; 8) Products that can improve the content of tartrate phosphatase or type I collagen N-terminal propeptide, which are bone metabolism indicators in the blood.

9. The use according to claim 8, characterized in that: The product having the function of reducing inflammation is specifically a product having the function of reducing TNF-α at the cellular level and in the intestine; preferably, the product having the function of reducing body weight is specifically a product having the function of reducing weight gain caused by a high-fat diet; preferably, the product having the function of improving dyslipidemia is specifically a product having the function of reducing triglyceride content, total cholesterol content and low-density lipoprotein content in the blood; preferably, the product having the function of improving blood sugar is specifically a product having the function of reducing elevated blood sugar caused by a high-fat diet; preferably, the product having the function of improving the intestinal barrier is specifically a product having the function of increasing the expression of intestinal barrier-related genes mTff3, Muc2 and ZO-1 in the intestine; preferably, the product having the function of improving intestinal flora structure disorder is specifically a product having the function of regulating the abundance of beneficial bacteria in the intestine and reducing the abundance of harmful bacteria; preferably, the product having the function of improving liver tissue damage caused by obesity or hyperlipidemia is specifically a product having the function of improving aspartate aminotransferase and fat accumulation in liver cells.

10. The use according to claim 8, characterized in that: The product having the ability to improve the content of tartrate phosphatase or type I collagen N-terminal propeptide, a bone metabolism indicator in the blood, is specifically a product having the ability to improve the content of tartrate phosphatase or type I collagen N-terminal propeptide, a bone metabolism indicator caused by obesity or hyperlipidemia.

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