Bifidobacterium breve HC2953, bacterial agent and application thereof

Abstract

The present invention belongs to the technical field of biological microbial agents, and specifically relates to a Bifidobacterium breve HC2953, a microbial agent and an application thereof. Bifidobacterium breve )HC2953, can reduce the concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in the body, increase the concentration of high-density lipoprotein cholesterol (HDL-C), increase the excretion of bile acid (TBA) in obese people, improve the state of lipid metabolism disorders in the body, and at the same time has extremely strong colonization and adhesion capabilities, thereby achieving the technical effect of lowering lipids, and can be used to prepare products for weight loss and treatment of obesity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological microbial agents, and in particular relates to Bifidobacterium breve HC2953, a microbial agent and applications thereof. Background Art

[0002] Bifidobacterium breve is a species of Bifidobacterium. Bifidobacterium is one of the dominant bacteria in the mammalian intestine and a member of the native microbiome. Its abundance changes dynamically with age and is closely associated with many physiological and pathological changes in the body. Bifidobacterium colonizes the host's intestinal membrane, forming a biological barrier. It has important physiological functions, including antagonizing pathogens, improving microecological balance, providing nutrition, boosting immunity, fighting tumors, and lowering cholesterol levels.

[0003] Lipids in the human body are divided into two categories: fat and phospholipids. Generally, body fat content fluctuates depending on factors such as nutritional status and energy consumption. Body fat percentage, also known as body fat percentage, refers to the ratio of body fat weight to total body weight. It reflects the amount of fat in the body. The normal range for adult body fat percentage is 20% to 25% for women and 15% to 18% for men. Excessive body fat percentage, exceeding the normal weight by more than 20%, is considered obesity, which increases the risk of various diseases.

[0004] Probiotics can affect appetite and energy expenditure by producing short-chain fatty acids such as acetate, propionate, and butyrate, inhibiting the absorption of dietary fat and increasing the amount of fat excreted in feces. Intestinal microorganisms secrete bioactive compounds through fermentation, inducing multiple reactions in the intestinal mucosa while affecting cellular metabolism in the liver and adipose tissue, thereby regulating the balance of lipids and glucose in the body. The intestinal flora can also regulate the intestinal barrier and endocrine function, affecting the transport and absorption of nutrients. Therefore, regulating the intestinal flora is an effective strategy for improving and managing obesity. However, there are few existing probiotic species with lipid-lowering effects, and the probiotic resource library is not rich enough. Summary of the Invention

[0005] The purpose of the present invention is to provide a Bifidobacterium breve HC2953, which has lipid-lowering effect and can enrich the probiotic species resource library.

[0006] The invention provides a Bifidobacterium breve (Bifidobacterium breve) HC2953. The preservation number of the Bifidobacterium breve HC2953 is CCTCCNO: M20222007.

[0007] The present invention also provides a bacterial agent containing the Bifidobacterium breve HC2953 described in the above technical solution.

[0008] Preferably, the viable bacterial count of Bifidobacterium breve HC2953 in the bacterial agent is 10 to 200 billion CFU / g.

[0009] Preferably, the bacterial agent includes the Bifidobacterium breve HC2953 bacterial cells and / or Bifidobacterium breve HC2953 fermentation broth.

[0010] The present invention also provides the use of the Bifidobacterium breve HC2953 described in the above technical solution or the bacterial agent described in the above technical solution in the preparation of a product with weight loss function.

[0011] Preferably, the product includes a product with lipid-lowering function.

[0012] Preferably, the product having lipid-lowering function includes a product that meets one or more of the following requirements:

[0013] Ⅰ: Reduce total cholesterol concentration in the body;

[0014] II: Reduce triglyceride concentration in the body;

[0015] III: Reduce the concentration of low-density lipoprotein cholesterol in the body;

[0016] IV: Reduce the level of alanine aminotransferase in the body;

[0017] Ⅴ: Increase the concentration of high-density lipoprotein cholesterol in the body;

[0018] VI: Increase bile acid excretion in the body.

[0019] The present invention also provides a product with weight loss function, comprising the Bifidobacterium breve HC2953 described in the above technical solution or the bacterial agent and auxiliary materials described in the above technical solution.

[0020] Preferably, the viable count of Bifidobacterium breve HC2953 in the product is 10 to 200 billion CFU / g.

[0021] Preferably, the product comprises a pharmaceutical product.

[0022] Beneficial effects:

[0023] The present invention provides Bifidobacterium breve HC2953, which has been biologically preserved. The Bifidobacterium breve HC2953 can reduce the concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in the body, increase the concentration of high-density lipoprotein cholesterol (HDL-C), increase the excretion of bile acid (TBA) in obese people, and improve the state of lipid metabolism disorder in the body. At the same time, it has extremely strong colonization and adhesion capabilities, thereby achieving the technical effect of lowering lipids. The Bifidobacterium breve HC2953 can be used to prepare medicines for weight loss and treating obesity.

[0024] Biological deposit information

[0025] Bifidobacterium breve HC2953 was deposited in the China Center for Type Culture Collection on December 23, 2022, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20222007. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 are briefly introduced below.

[0027] Figure 1 This is the standard curve for determining cholesterol content using the o-phthalaldehyde colorimetric method in Example 1;

[0028] Figure 2 is the phylogenetic tree of the HC2953 strain in Example 1;

[0029] Figure 3 This is the colony map of the HC2953 strain in Example 1;

[0030] Figure 4 This is a microscopic morphological image of the HC2953 strain in Example 1;

[0031] Figure 5-1 The results of body weight measurement of the high-fat model mice in Application Example 1;

[0032] Figure 5-2 The results of total cholesterol and triglyceride content determination in the mouse hyperlipidemia model in Application Example 1;

[0033] Figure 6 The weight of mice in the fourth week of the experimental period in Application Example 1 is measured;

[0034] Figure 7 The results of the feed utilization rate test for mice in the fourth week of the experimental period in Application Example 1 are as follows;

[0035] Figure 8 The results of body fat percentage measurement of mice in the 10th week of the experimental cycle in Application Example 1;

[0036] Figure 9 The Lee's index and BMI index of mice in the 10th week of the test cycle in Application Example 1 are measured;

[0037] Figures 10-11 The results of the mouse organ index measurement in the 10th week of the test cycle in Application Example 1;

[0038] Figure 12 The results of the blood-related indicators of mice in the 10th week of the test cycle in Application Example 1;

[0039] Figures 13-1 to 13-2 The results of the relevant index determination of mouse liver tissue in the 10th week of the test cycle in Application Example 1;

[0040] Figure 14 This is the result of bile acid detection in mouse feces in Application Example 1. DETAILED DESCRIPTION

[0041] The invention provides Bifidobacterium breve HC2953. The preservation number of the Bifidobacterium breve HC2953 is CCTCC NO: M 20222007.

[0042] The Bifidobacterium breve HC2953 described in the present invention is preferably isolated by screening from the feces of healthy adults. The 16S rDNA of the Bifidobacterium breve HC2953 described in the present invention is preferably as shown in SEQ ID NO.1, specifically: 5’-CATGGCTCAGGATGAACGCTGG CGGCGTGCTTAACACATGCAAGTCGAACGGGATCCATCGGGCTTTGCTTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACCGACCTGCCCCATGCACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTCCATCACACCGCATGGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCCTATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTGTGTTGAGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTCGCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGGCTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATATCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCC-3'. The homology of this strain to Bifidobacterium breve ATCC15700 in NCBI database is as high as 99%. Figure 1 As shown, this strain has a very high similarity with Bifidobacterium breve, so this strain was named Bifidobacterium breve HC2953.

[0043] The colony morphology of the Bifidobacterium breve HC2953 on MRS solid culture medium is: relatively regular circular colonies with neat edges, a convex and shiny surface, an opaque milky white color, moist overall colonies, a soft and delicate texture, and short rod-shaped bacteria, most of which are forked "V"-shaped, and a few are short club-shaped with one end thick and the other end thin.

[0044] The present invention also provides a bacterial agent containing the Bifidobacterium breve HC2953 of claim 1. The viable cell count of Bifidobacterium breve HC2953 in the bacterial agent of the present invention is preferably 10 to 200 billion CFU / g, more preferably 20 billion CFU / g. The bacterial agent of the present invention preferably includes the cells of Bifidobacterium breve HC2953 and / or a fermentation broth of Bifidobacterium breve HC2953, more preferably includes the cells of Bifidobacterium breve HC2953 and a fermentation broth of Bifidobacterium breve HC2953.

[0045] The present invention also preferably provides a method for preparing the bacterial agent, comprising the following steps:

[0046] Fermenting Bifidobacterium breve HC2953 to obtain a fermentation liquid;

[0047] The fermentation liquid is centrifuged, and bacterial mud is taken for coating, freeze-drying and pulverization to obtain the bacterial agent.

[0048] Before the fermentation, the present invention preferably performs warming, primary seed culture and secondary seed culture on the Bifidobacterium breve HC2953.

[0049] In the present invention, the Bifidobacterium breve HC2953 is preferably subjected to a temperature regeneration step, wherein the temperature of the temperature regeneration step is preferably room temperature, and the time is preferably 0.5 to 1.5 hours, more preferably 1 hour.

[0050] After the rewarming, the present invention preferably performs a primary seed culture on the rewarmed Bifidobacterium breve HC2953. The volume ratio of the primary seed tank to the seed culture medium for the primary seed culture of the present invention is preferably 3-5:2-4, more preferably 5:3. The inoculum amount of the rewarmed Bifidobacterium breve HC2953 is preferably 2%-5%, more preferably 3%. The temperature of the primary seed culture of the present invention is preferably 35-37.5°C, more preferably 36.5°C; the time is preferably 16-20 hours, more preferably 18 hours. The seed culture medium of the present invention preferably comprises the following components: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, 0.5 g / L cysteine, and 1.0 g / L edible oil.

[0051] After the primary seed culture, the present invention preferably subjects the primary seed liquid obtained from the primary seed culture to secondary seed culture. The volume ratio of the secondary seed tank for the secondary seed culture of the present invention to the seed culture medium is preferably 80-100:55-75, more preferably 100:75. The inoculation amount of the primary seed liquid of the present invention is preferably 3-7% of the volume of the seed culture medium, more preferably 4%. The temperature of the secondary seed culture of the present invention is preferably 35-37°C, more preferably 36.5°C; the time is preferably 10-12h, more preferably 10h. The seed culture medium of the present invention is preferably the same as the seed culture medium used for the primary seed culture, and will not be described in detail.

[0052] In the present invention, the inoculum size during fermentation is preferably 8-12% of the volume of the fermentation medium, more preferably 10%. The volume ratio of the fermentation tank to the fermentation medium is preferably 1000-1200:750-1000, more preferably 1000:750. The fermentation temperature is preferably 35-37°C, more preferably 37°C; the fermentation time is preferably 8-11 hours, more preferably 10 hours. The fermentation medium of the present invention preferably comprises the following components: 20 g / L whey powder, 43 g / L glucose, 12 g / L yeast powder, 6 g / L peptone, 0.05 g / L manganese sulfate, 0.1 g / L magnesium sulfate, 1.0 g / L dipotassium hydrogen phosphate, 1.0 g / L triammonium citrate, 1.0 g / L sodium acetate, 0.2 g / L cysteine, and 1.0 g / L edible oil.

[0053] After the fermentation, the fermentation liquid is preferably centrifuged, and the bacterial sludge is coated, freeze-dried, and pulverized to obtain the raw powder, which is the bacterial agent. The centrifugation is not particularly limited in the present invention, and the bacterial sludge can be obtained by conventional centrifugation methods in the art.

[0054] The coating solution used in the present invention preferably comprises the following components by weight: 15% skim milk powder, 8% trehalose, 3% glycerol, 1% sodium ascorbate, and 73% distilled water. The mass ratio of the coating solution to the bacterial sludge is preferably 1-2:2-3, more preferably 1:2.

[0055] The coating method of the present invention is preferably freeze-drying microencapsulation coating.

[0056] The freeze-drying of the present invention is preferably vacuum freeze-drying. The present invention has no special limitation on the specific parameters of the vacuum freeze-drying, and conventional freeze-drying parameters in the art can be used to prepare the powder.

[0057] The particle size of the raw powder of the present invention is preferably 200-450 μm, more preferably 200 μm.

[0058] In order to obtain bacterial agents with different viable cell counts, the present invention preferably further comprises mixing the raw powder with auxiliary materials for preparation and dilution to obtain bacterial agents with different viable cell counts. The auxiliary materials of the present invention are preferably but not limited to oligofructose and / or oligomaltodextrose. When the auxiliary materials are oligofructose and oligomaltodextrose, the mass ratio of the oligofructose and oligomaltodextrose is preferably 0.5-1.5:0.5-1.5, more preferably 1:1. The present invention does not have strict regulations on the mass ratio of the raw powder and the auxiliary materials. As long as the raw powder is used as the active ingredient and the bacterial agent is prepared by mixing with the auxiliary materials, it falls within the scope of protection of the present invention.

[0059] The bacterial agent of the present invention is preferably used in the form of a bacterial suspension, preferably prepared by diluting the bacterial agent with physiological saline. The present invention does not specifically limit the concentration of viable bacteria of Bifidobacterium breve HC2953 in the bacterial suspension; the concentration of viable bacteria after dilution based on the properties of the product falls within the scope of protection of the present invention.

[0060] The present invention also provides the use of the Bifidobacterium breve HC2953 described in the above technical solution or the bacterial agent described in the above technical solution in preparing a product with weight loss function.

[0061] The products of the present invention preferably include products with lipid-lowering properties, and more preferably include products that meet one or more of the following requirements: I: reducing total cholesterol concentration in the body; II: reducing triglyceride concentration in the body; III: reducing low-density lipoprotein cholesterol concentration in the body; IV: reducing alanine aminotransferase levels in the body; V: increasing high-density lipoprotein cholesterol concentration in the body; and VI: increasing bile acid excretion in the body. The in vivo function of the present invention preferably includes serum or liver function.

[0062] The Bifidobacterium breve HC2953 of the present invention can reduce the concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in the body, increase the concentration of high-density lipoprotein cholesterol (HDL-C), increase the excretion of bile acid (TBA) in obese people, and improve the state of lipid metabolism disorder in the body. At the same time, it has extremely strong colonization and adhesion capabilities, thereby achieving the technical effect of lowering lipids. The strain can be used to prepare weight-loss drugs and has a good therapeutic effect on obesity.

[0063] The present invention also provides a product with weight loss function, comprising the Bifidobacterium breve HC2953 described in the above technical solution or the bacterial agent and auxiliary materials described in the above technical solution. The number of viable bacteria of Bifidobacterium breve HC2953 in the product of the present invention is preferably (100-2000)×10 8 CFU / g, more preferably 200×10 8CFU / g. The product of the present invention preferably comprises a pharmaceutical. When the product is a pharmaceutical, the excipient is preferably a pharmaceutically acceptable excipient.

[0064] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0065] In the embodiments of the present invention, some of the reagents used and their preparation methods are as follows:

[0066] Seed culture medium: water as solvent, peptone 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L, cysteine ​​0.5 g / L, and edible oil 1.0 g / L;

[0067] Fermentation medium: water as solvent, whey powder 20g / L, glucose 43g / L, yeast powder 12g / L, peptone 6g / L, manganese sulfate 0.05g / L, magnesium sulfate 0.1g / L, dipotassium hydrogen phosphate 1.0g / L, triammonium citrate 1.0g / L, sodium acetate 1.0g / L, cysteine ​​0.2g / L and edible oil 1.0g / L;

[0068] MRS solid medium formula: water as solvent, peptone 10.0 g / L, yeast extract powder 5.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.1 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L and agar powder 15 g / L;

[0069] TPY agar medium: water as solvent, hydrolyzed casein 10.0 g / L, yeast powder 2.0 g / L, phytol 5.0 g / L, glucose 5.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, magnesium chloride (6H2O) 0.5 g / L, zinc sulfate (7H2O) 0.25 g / L, calcium chloride 0.15 g / L, trace amount of ferric chloride, L-cysteine ​​0.5 g / L, Tween 80 1.0 g / L and agar 20.0 g / L;

[0070] M17 agar medium: water as solvent, soytone 5.0 g / L, peptone 2.5 g / L, casein peptone 2.5 g / L, yeast extract powder 2.5 g / L, lactose 5.0 g / L, sodium ascorbate 0.5 g / L, sodium β-glycerophosphate 19.5 g / L, magnesium sulfate 0.25 g / L, and agar 15.0 g / L;

[0071] MRS broth: peptone 10 g, beef powder 10 g, yeast powder 5 g, glucose 20 g, magnesium sulfate 0.1 g, sodium acetate 5 g, ammonium citrate 2 g, dipotassium hydrogen phosphate 2 g, manganese sulfate 0.05 g, Tween 80 1 mL, distilled water 1000 mL, pH 6.2 ± 0.2, sterilized at 121°C for 20 min;

[0072] MRS triglyceride medium: Heat and dissolve in a 1:1 ratio of Tween 80 to lard. Incubate for 15 minutes for ultrasonic emulsification to obtain triglyceride micelles. Sterilize at 121°C for 20 minutes. Aseptically add 10% by volume of triglyceride to MRS broth containing 0.2% sodium thioglycolate.

[0073] MRS glycerol medium: Sterilize an appropriate amount of glycerol at 121°C for 20 minutes. Aseptically add 2% by volume to MRS broth containing 0.2% sodium thioglycolate.

[0074] MRS Cholesterol Medium: Add 0.1 g cholesterol and 0.1 g sucrose ester to 1 mL of Tween 80, mix thoroughly, then add 1 mL of glacial acetic acid. Heat to dissolve. Incubate for 15 minutes of ultrasonic emulsification to obtain cholesterol micelles. Sterilize at 121°C for 20 minutes. Under sterile conditions, add 1% of the prepared triglyceride micelles to MRS broth containing 0.2% sodium thioglycolate.

[0075] Gastric fluid buffer (pH 3.0): Accurately weigh 0.5144 g KCl, 0.1225 g KH2PO4, 2.75085 g NaCl, 2.1002 g NaHCO3, 0.0203 g MgCl2·(H2O)6, 0.0480 g (NH4)2CO3, and 0.0083 g CaCl2, and dilute to 1000 mL.

[0076] Simulated gastric fluid: Dissolve 3 g of pepsin in 1000 mL of gastric fluid buffer, adjust the pH to 3.0 with 1 mol / L HCl, filter and sterilize with a 0.22 μm filter membrane, and prepare immediately before use.

[0077] Intestinal fluid buffer: Accurately weigh 0.5069 g KCl, 0.1089 g KH2PO4, 2.2442 g NaCl, 7.1408 g NaHCO3, 0.0067 g MgCl2·(H2O)6, 0.0333 g CaCl2, and dilute to 1000 mL;

[0078] Simulated intestinal fluid (0.1% bile salt): 1 g of trypsin and 1 g of bile salt were added to 1000 mL of intestinal fluid buffer and dissolved. The pH of the simulated intestinal fluid was adjusted to 8.0 with 1 mol / L NaOH and sterilized by filtration with a 0.22 μm filter membrane.

[0079] Example 1

[0080] The isolation and identification of Bifidobacterium breve HC2953 were carried out as follows:

[0081] 1. Sampling: Take stool samples from healthy people, store them in a refrigerator, and record them by number.

[0082] 2. Enrichment: Take a small amount of the sample from step 1 and dissolve it in MRS liquid medium and TPY liquid medium respectively. MRS liquid medium is a selective enrichment medium for lactic acid bacteria, and TPY liquid medium is a culture medium for the enrichment of bifidobacteria. The sample dissolved in MRS liquid medium is cultured under aerobic conditions at 37°C for 24 hours, and the sample dissolved in TPY liquid medium is cultured under anaerobic conditions at 37°C for 24 hours for selective enrichment to obtain the enriched sample. Aerobic means naturally placed in an aerobic environment in the air, and anaerobic needs to be placed in an anaerobic bag with anaerobic conditions.

[0083] 3. Isolation: Under sterile conditions, perform a 10-fold gradient dilution of the sample enriched in step 2 with sterile physiological saline. Based on the growth of the culture medium, select 100 μL of each of three appropriate gradients and apply them to MRS solid medium (Lactic Acid Bacteria Isolation Medium) and TPY agar medium (Bifidobacterium Identification Medium). Incubate the cells under aerobic and anaerobic conditions at 37°C for 48 hours. Select different colonies and repeatedly streak them for isolation and purification. The colonies are named and stored for future use. After selective isolation using selective medium, anaerobic bacillus HC2953 was initially isolated.

[0084] 1) Activate the HC2953 isolated and purified in step 3 and inoculate it into MRS broth at an inoculum rate of 2%. Cultivate at 37°C until the stationary phase. Collect the cells by centrifugation at 6000 rpm for 8 minutes. Wash twice with sterile PBS buffer and resuspend. Adjust the cell suspension to about 10 9 CFU / mL is reserved.

[0085] 2) The bacterial suspension obtained in step 1) was inoculated into MRS triglyceride medium, MRS glycerol medium, and MRS cholesterol medium at an inoculum size of 2% of the culture medium volume, and cultured at 37°C for 24 hours. Using basal MRS broth as a blank, the degradation capacity (degradation ability) of triglycerides, glycerol, and cholesterol by the strain was measured every 6 hours. The method for determining the degradation capacity of triglycerides, glycerol, and cholesterol by the strain is shown in 2.1 to 2.3.

[0086] 2.1 Determination of triglyceride degradation ability

[0087] Determination of triglyceride content by acetylacetone colorimetric method:

[0088] (1) Reagents: ① Extraction agent: n-heptane: isopropanol in a ratio of 2:3.5; ② 0.04 mol / L sulfuric acid; ③ Saponifying agent: 2.5 g analytical grade potassium hydroxide dissolved in 50 mL distilled water, and the volume was adjusted to 500 mL with isopropanol; ④ Oxidizing agent: 77.0 g anhydrous ammonium acetate was weighed and dissolved in distilled water, 60 mL glacial acetic acid and 1.0 g periodic acid were added, and the volume was adjusted to 1000 mL with distilled water; ⑤ Color developer: 2 mL acetylacetone was adjusted to 500 mL with isopropanol; ⑥ Standard solution: 1.76 mmol / L (150 mg / dL) triglyceride;

[0089] (2) Test process:

[0090] The fermentation broth of HC2953 was sampled at 0 h, 6 h, 12 h, 18 h, and 24 h. 4 mL of the fermentation broth was centrifuged at 4000 rpm for 5 min. The supernatant was collected for later use and then the following procedures were performed according to Table 1:

[0091] Table 1 Triglyceride degradation ability test process

[0092]

[0093] After adding the above reagents, mix the tubes thoroughly, cool in a 65°C water bath for 15 minutes, adjust to zero with a blank tube, and measure the absorbance at 420 nm.

[0094] (3) Calculate the triglyceride degradation rate of the strain according to the formula:

[0095] ①Triglyceride concentration at each sampling point (mmol / L) = A 测样管 / A 标准管 ×C 标准管

[0096] Where:

[0097] A 测样管 : Absorbance of sample tube

[0098] A 标准管 : Standard tube absorbance

[0099] C 标准管 :Standard tube concentration (mmol / L)

[0100] ② Triglyceride degradation rate (%) = (AB) / A×100%

[0101] Where:

[0102] A: Triglyceride concentration at 0h (mmol / L) i.e. total triglyceride concentration in the culture medium (mmol / L)

[0103] B: Triglyceride concentration at each sampling point (mmol / L)

[0104] AB: reduced triglyceride concentration (mmol / L).

[0105] 2.2 Glycerol degradation ability determination

[0106] Determination of glycerol content by periodic acid oxidation method:

[0107] Sample the fermentation broth of strain HC2953 at 0, 6, 12, 18, and 24 hours. Centrifuge 4 mL of the fermentation broth at 4000 rpm for 5 minutes, and remove the supernatant for later use. Accurately pipette 2 mL of the supernatant, add 10 mL of distilled water and 2 drops of phenolphthalein, and titrate with 0.05 mol / L sodium hydroxide standard solution until color changes. Add 10 mL of 0.1 mol / L sodium periodate solution, incubate in the dark for 5 minutes, then add 5 mL of 25% ethylene glycol solution, incubate in the dark for 5 minutes, and titrate again with the same sodium hydroxide solution until color changes, ensuring consistent color change. Record the amount of sodium hydroxide used.

[0108] The degradation rate of glycerol by the strain was calculated according to the formula:

[0109] ① Glycerol concentration at each sampling point (g / L) = 92.1×C NaOH ×V NaOH / n

[0110] Where:

[0111] C NaOH :NaOH concentration (mol / L)

[0112] V NaOH :NaOH consumption volume (mL)

[0113] n: sampling volume (mL)

[0114] ② Glycerol degradation rate (%) = (AB) / A×100%

[0115] Where:

[0116] A: 0h glycerol concentration (g / L) i.e. total glycerol concentration in the culture medium (g / L)

[0117] B: Glycerol concentration at each sampling point (g / L)

[0118] AB: reduced glycerol concentration (g / L).

[0119] 2.3 Cholesterol degradation capacity determination

[0120] Determination of cholesterol content by o-phthalaldehyde colorimetric method:

[0121] (1) Draw a standard curve: Use n-hexane as solvent to prepare a cholesterol standard solution with a concentration of 1 mg / mL. Accurately pipette 0.1 mL, 0.2 mL, 0.3 mL, 0.5 mL, 0.7 mL, and 0.9 mL of the cholesterol standard solution into a colorimetric tube. After drying with nitrogen in a 60°C water bath, add 4 mL of freshly prepared 0.5 mg / mL o-phthalaldehyde solution. After standing at room temperature for 10 minutes, slowly add 2 mL of concentrated sulfuric acid along the wall, mix well, and let stand for 20 minutes to develop color. Measure the absorbance at a wavelength of 560 nm and draw a standard curve, as shown in the following example. Figure 1 shown.

[0122] (2) Determination of cholesterol content in fermentation broth: Sample the fermentation broth at 0 h, 6 h, 12 h, 18 h, and 24 h, take 4 mL of fermentation broth, centrifuge at 4000 rpm for 5 min, take 1 mL of supernatant, add 3.0 mL of anhydrous ethanol, 2.0 mL of 50% potassium hydroxide, keep constant temperature at 60 ° C in a water bath for 10 min, cool, add 5 mL of n-hexane, 1 mL of distilled water, shake and extract for 2 min, let stand and separate, take 3 mL of the upper n-hexane layer, blow dry with nitrogen in a 60 ° C water bath, add 4 mL of freshly prepared 0.5 mg / mL o-phthalaldehyde solution, let stand at room temperature for 10 min, slowly add 2 mL of concentrated sulfuric acid along the wall, mix well and let stand for 20 min to develop color, and measure the absorbance at a wavelength of 560 nm.

[0123] (3) Calculate the cholesterol degradation rate of the strain according to the formula:

[0124] Cholesterol degradation rate (%) = (AB) / A×100%

[0125] Where:

[0126] A: Cholesterol content at 0 h (mg / mL) i.e. total cholesterol content in the culture medium

[0127] B: Cholesterol content at each sampling point (mg / mL)

[0128] AB: reduced cholesterol amount (mg / mL).

[0129] The results showed that the degradation rates of cholesterol, triglyceride and glycerol in the blank group CK were all 0, while the degradation rates of cholesterol, triglyceride and glycerol by HC2953 were 73.55%, 68.35% and 52.10% respectively.

[0130] 3 Determination of the colonization ability of strains under simulated digestion conditions of gastrointestinal fluid

[0131] 3.1 Gastric juice simulated digestion

[0132] Take 1 mL of enriched bacteria and add 9 mL of simulated gastric fluid. Set up three replicates for each strain, incubate in a 37°C constant temperature water bath at 100 rpm, and oscillate for 0, 1, 2, 3, and 4 hours, respectively. Count the number of viable bacteria using the dilution plate method. Spread three replicates for each sample. Calculate the colonization survival rate of the strain according to the formula:

[0133] Gastric fluid colonization survival rate = number of colonized and surviving bacteria / initial bacterial number × 100%.

[0134] 3.2 Intestinal fluid simulated digestion

[0135] Take 1 mL of enriched bacteria and add 9 mL of simulated intestinal fluid. Set up three replicates for each strain, incubate in a 37°C constant temperature water bath, rotate at 100 rpm, and oscillate for 0 h, 1 h, 2 h, 3 h, and 4 h. Count the number of viable bacteria using the dilution plate method. Spread three replicates for each sample. Calculate the colonization survival rate of the strain according to the formula:

[0136] Intestinal fluid colonization survival rate = number of colonized and surviving bacteria / initial bacterial number × 100%.

[0137] 3.3 Gastric juice-intestinal juice two-step method to simulate digestion

[0138] Take 1 mL of enriched bacteria and add 9 mL of simulated gastric fluid. Set up three replicates for each strain. Incubate in a 37°C water bath at 100 rpm for 3 hours, then remove and rapidly centrifuge. Add 9 mL of simulated intestinal fluid and incubate under the same shaking conditions for 2 hours. Finally, count the cells using the dilution plate method, with three replicates for each sample. Calculate the colonization survival rate of the strain according to the formula:

[0139] Colonization survival rate in gastrointestinal fluid = number of surviving colonized bacteria / initial bacterial number × 100%.

[0140] The results of the colonization ability test of the strains under the conditions of simulated digestion of gastrointestinal fluid are as follows Figure 3 ~5 shown.

[0141] Gastric acid and bile salts have a certain inhibitory effect on bacteria. To enter the human body, probiotics must be able to pass smoothly through gastric juice and resist bile salts. Therefore, the strain's tolerance to gastric acid and bile salts is an important indicator of probiotic performance. Probiotic colonization and adhesion in the host's gastrointestinal tract can prolong their residence time in the body, thereby affecting host health by improving the local microbiome or its metabolites.

[0142] After 0h, 1h, 2h, 3h, and 4h of simulated gastric fluid digestion, the survival rates of HC2953 were 100%, 99.11%, 96.30%, 93.03%, and 87.42%, respectively. It can be seen that the survival rate of HC2953 decreased to varying degrees with the extension of time. As time changes in simulated gastric fluid, the colonization ability of HC2953 strain is stronger.

[0143] After 0h, 1h, 2h, 3h, and 4h of digestion in simulated intestinal fluid, the survival rates of HC2953 were 100%, 93.93%, 86.00%, 77.95%, and 70.38%, respectively. It can be seen that the survival rate of HC2953 decreased to varying degrees with the extension of time. As time changes in simulated intestinal fluid, the colonization ability of HC2953 strain is stronger.

[0144] In the simulated gastric juice-intestinal juice two-step experiment, the survival rate of HC2953 was 80.15% after 3 hours of gastric juice digestion and 2 hours of intestinal juice digestion.

[0145] 4. Determination of strain adhesion ability

[0146] 4.1 Determination of the ability of strains to adhere to mucin:

[0147] Mucin model establishment: Weigh 10 mg of mucin and prepare a 1 mg / mL mucin solution in sterile PBS buffer. Store at -20°C. Immobilize 500 μL of the mucin solution on a 24-well cell culture plate for 1 hour and incubate overnight at 4°C. Add an equal volume of mucin and continue incubation at 37°C for 2 hours to fill in blank sites. Wash twice with sterile PBS buffer.

[0148] Adhesion ability assay: The three strains of bacteria cultured overnight were collected and resuspended in sterile PBS, and the bacterial count was adjusted to 10 8 CFU / mL, spread plate, count, and record. Add 500 μL of bacterial suspension to the mucin model, incubate at 37°C for 1 hour, wash five times with sterile citric acid buffer to remove unbound bacteria, and add 1 mL of 0.5% Tween 80 (v / v) to collect adherent bacteria. Finally, count using the dilution plate method, with three replicates for each sample. Calculate the strain adhesion rate according to the formula:

[0149] Mucin adhesion rate = number of adherent bacteria / initial bacterial number × 100%.

[0150] 4.2 Determination of the Adhesion Ability of Strain to Caco-2 Cells:

[0151] 0.5 mL of Caco-2 cells were seeded into a 24-well cell culture plate, and the Caco-2 cells were adjusted to 2 × 10 5 cell / mL, cultured to a monolayer, washed twice with PBS, and then added with 500 μL of bacterial suspension (10 8 CFU / mL), culture at 37°C for 2 hours, wash three times with PBS to remove non-adherent bacteria, add 150 μL of trypsin cell digestion solution, and add 350 μL of MEM complete culture medium to terminate digestion after the cells are completely detached. Finally, count the cells using the dilution plate method, with three replicates for each sample. Calculate the strain adhesion rate according to the formula:

[0152] Caco-2 cell adhesion rate = number of adherent bacteria / initial number of bacteria × 100%.

[0153] The colonization and adhesion of probiotics in the host's gastrointestinal tract can prolong their residence time in the body, thereby affecting the host's health by improving the local microbiota or its metabolites.

[0154] In the test of the strain's adhesion to mucin, the adhesion rate of HC2953 to mucin was 47.20%. In the test of the strain's adhesion to Caco-2 cells, the adhesion rate of HC2953 to mucin was 57.24%.

[0155] Combining the two results, the adhesion rate of HC2953 to mucin and Caco-2 cells was relatively good, so this strain was selected as the target strain of this experiment for subsequent strain identification and animal testing.

[0156] 5.1) Physiological and biochemical identification of strain HC2953 was performed. The identification results are shown in Table 2:

[0157] According to the methods in Bergey's Manual of Bacterial Identification and Manual of Identification of Common Bacterial Systems, some indicators were screened for physiological and biochemical identification of the strains.

[0158] Table 2 Physiological and biochemical identification results of HC2953 strain

[0159]

[0160] 2) A single colony of the purified HC2953 strain was selected and added to sterile water to prepare a bacterial suspension as a PCR template for colony PCR amplification of the strain's 16S rDNA. The PCR primers used were universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') as shown in SEQ ID NO.2 and 1492R (5'-GGTTACCTTGTTACGACTT-3') as shown in SEQ ID NO.3, synthesized and sequenced by Shanghai Sangon Biotechnology Co., Ltd. The sequenced sequence is shown in SEQ ID NO.1. The sequence was aligned with the strain's 16S rDNA sequence in the National Center for Biotechnology Information (NCBI) database of the United States. MEGA 5.0 software was used for analysis and a phylogenetic tree was constructed. The results are shown in FIG. Figure 2 shown.

[0161] Depend on Figure 2It can be concluded that the homology between the HC2953 strain and Bifidobacterium breve ATCC15700 in the NCBI database is as high as 99%, indicating that this strain is Bifidobacterium breve, and is named HC2953.

[0162] Example 2

[0163] The preparation steps of the bacterial agent containing Bifidobacterium breve HC2953 are as follows:

[0164] The preserved Bifidobacterium breve HC2953 was warmed up at room temperature for 1 hour, inoculated into a 3.5 L seed tank containing seed culture medium and cultured for 16-20 hours to produce primary seeds, which were then transferred to a 100 L seed tank containing seed culture medium and cultured for 6-8 hours to obtain secondary seeds. Subsequently, the seeds were transferred to a 1000 L fermenter at an inoculum rate of 10% by volume for fermentation to obtain a fermentation broth.

[0165] The fermentation liquid is centrifuged to obtain bacterial sludge, the bacterial sludge is coated and then freeze-dried to obtain freeze-dried blocks, and the freeze-dried blocks are crushed to obtain Bifidobacterium breve raw powder;

[0166] The raw powder of Bifidobacterium breve is fully mixed with a mixture of oligofructose and oligoisomaltose, and then diluted to obtain bacterial agents with different specifications of viable cell counts, wherein the mass ratio of oligofructose to oligoisomaltose is 1:1.

[0167] 1.0 g of Bifidobacterium breve powder was mixed with 39.0 g of a mixture of oligofructose and oligoisomaltose, so that the viable count of Bifidobacterium breve HC2953 in the obtained bacterial powder was ≥ 10 billion CFU / g;

[0168] 1.0 g of Bifidobacterium breve powder was mixed with 19.0 g of a mixture of oligofructose and oligoisomaltose, so that the viable count of Bifidobacterium breve HC2953 in the obtained bacterial powder was ≥ 20 billion CFU / g;

[0169] 1.0 g of Bifidobacterium breve powder was mixed with 7.0 g of a mixture of oligofructose and oligoisomaltose, so that the viable count of Bifidobacterium breve HC2953 in the obtained bacterial powder was ≥ 50 billion CFU / g;

[0170] 1.0 g of Bifidobacterium breve powder was mixed with 3.0 g of a mixture of oligofructose and oligoisomaltose, so that the viable count of Bifidobacterium breve HC2953 in the obtained bacterial powder was ≥ 100 billion CFU / g;

[0171] 1.0 g of Bifidobacterium breve raw powder is mixed with 1.0 g of a mixture of oligofructose and oligoisomaltose, so that the number of viable bacteria of Bifidobacterium breve HC2953 in the obtained bacterial powder is ≥200 billion CFU / g.

[0172] Application Example 1

[0173] Animal experiments confirm the lipid-lowering function of Bifidobacterium breve HC2953

[0174] Animal test feed, grouping and feeding:

[0175] (1) Basic feed: Calculated by mass percentage, corn flour 25%, bran 20%, flour 18%, soybean meal 20%, rice flour 8%, fish meal 2%, calcium powder 2%, bone meal 2%, yeast powder 1.9%, salt 0.9%, multivitamins 0.1%, trace elements 0.1%, mix well and granulate, and sterilize before use.

[0176] (2) High-fat feed: Calculated by mass percentage, 12% egg yolk powder, 12% lard, 5% soybean oil, 6% sucrose, 0.2% bile salt, 64.8% basic feed, mix well and granulate, and sterilize before use.

[0177] (3) Grouping and feeding:

[0178] 60 5-week-old SPF mice with an initial weight of 20±2g were fed with free water and food, and the bedding was changed regularly. After one week of adaptive feeding, abnormal mice were eliminated and divided into 4 groups using the randomized block grouping method: blank control group (NCD), high-fat model group (HFD), high-fat probiotic group A1 (HFD-A1) and high-fat probiotic group A2 (HFD-A2). HFD and HFD-A2 were cultured together before the modeling was successful. After the modeling was successful, they were randomly divided into 2 groups, namely HFD and HFD-A2. They were fed according to the table, and feed and water were freely available. The source of live bacteria was prepared by diluting the original powder prepared in Example 2 with physiological saline. The number of live bacteria of Bifidobacterium breve HC2953 in the prepared bacterial solution was 1×10 9 CFU / mL. The feeding methods of each experimental group are shown in Table 3.

[0179] Table 3 Feeding methods of Bifidobacterium breve HC2953 verified by animal experiments

[0180]

[0181] During the experiment, the feed intake and body weight of the mice were recorded and their feces were collected at fixed times every week. In the fourth week of the experimental cycle, blood was collected from the eye sockets of the grouped mice and various indicators were measured to test whether the high-fat model was successfully established. In the tenth week of the experimental cycle, the mice were fasted for 12 hours but not water. The final body weight and body length of the mice were recorded, and blood was collected from the eyeballs before being killed by cervical dislocation. During the dissection process, the organs and fat were quickly removed, weighed and recorded, and temporarily stored in liquid nitrogen and then stored at -80°C for subsequent sampling.

[0182] High-fat model establishment:

[0183] The body weights of the four groups of mice were recorded in the fourth week of the experimental period. Figure 5-1 As shown, orbital blood was collected, and the blood samples were kept at room temperature for 1 hour and then centrifuged at 8000 rpm for 10 minutes at 4°C to separate the serum for later use. The total cholesterol (TC) and triglyceride (TG) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute to determine the total cholesterol (TC) and triglyceride (TG) levels in the serum. The results Figure 5-2 shown.

[0184] According to the analysis of mouse body weight in the fourth week in Figure 5, the weight differences between the groups of mice were already significant. The HFD group gained 20.15% more weight than the NCD group. Combined with the significant differences in serum TC and TG between the groups, it can be concluded that the high-fat model was successfully established.

[0185] Mouse body weight and food utilization:

[0186] The mice were weighed at 20:00 every Sunday, the weight of feed consumed was recorded, and the feed utilization rate of the mice was calculated according to the formula.

[0187] Formula: Feed utilization (%) = final weight gain (g) / final feed consumption (g) × 100%, the result is as follows Figures 6-7 shown.

[0188] Depend on Figure 6 It can be concluded that after the second week of the experimental cycle, the weight difference of mice in each group became more and more obvious. Taking the weight of the blank control group (NCD) as the reference, the weight of the high-fat model group (HFD) was 20.15% higher in the fourth week. The weight excess in each week was higher than that in the previous week. The HFD-A1 group was gavaged with 1×10 9 CFU / mL live bacteria, while the HFD-A2 group was gavaged with 1×10 9 CFU / mL of live bacteria. Weekly weight changes indicate that the HFD-A1 group, which began oral administration at the beginning of the experiment, showed significant weight loss compared to the HFD-A2 group, which began oral administration after model establishment. Furthermore, the HFD-A1 and HFD-A2 groups also showed significant weight loss differences compared to the HFD group. Therefore, it can be concluded that Bifidobacterium breve HC2953 bacterial powder can significantly reduce the weight of high-fat mice, and the earlier it is administered, the greater the effect.

[0189] Depend on Figure 7It was concluded that the feed utilization rates of the four groups were HFD > HFD-A2 > HFD-A1 > NCD. The HFD, HFD-A2, and HFD-A1 groups were all fed a high-fat diet, whose calorie and fat content was much higher than that of the basal diet. Analysis of the data revealed that the feed utilization rate of the HFD group was 1.97 times that of the NCD group; the feed utilization rate of HFD-A2 was 1.55 times that of the NCD group, a 27.70% decrease compared to the HFD group; and the feed utilization rate of HFD-A1 was 1.34 times that of the NCD group, a 47.77% decrease compared to the HFD group. These results suggest that the probiotic Bifidobacterium breve can inhibit weight gain in obese mice by reducing feed utilization.

[0190] Body fat percentage of mice:

[0191] During the 10th week of the experimental period, mice were fasted for 12 hours but not water. Following sacrifice, inguinal subcutaneous fat, epididymal fat, and perirenal fat were precisely and rapidly separated during dissection, and their wet weights were measured and recorded. Body fat percentage was calculated using the formula.

[0192] Formula: Body fat percentage (%) = [Inguinal subcutaneous fat (g) + Epididymal fat (g) + Perinephric fat (g)] / Body weight (g) × 100%. The result is as follows: Figure 8 shown.

[0193] Depend on Figure 8 It can be concluded that compared to the NCD group, the HFD group had a 174.31% higher body fat percentage. Although the HFD-A1 and HFD-A2 groups showed a significant increase in body fat percentage compared to the NCD group, they were significantly lower than the HFD group by 99.77% and 27.79%, respectively. Body fat percentage is the ratio of abdominal fat to body weight, primarily reflecting fat content and thus used to determine whether a person is normal, underweight, or obese. These results indicate that the probiotic Bifidobacterium breve has a significant effect on reducing body fat percentage in obese mice, and the earlier the administration, the better.

[0194] Determination of Lee's index and BMI index of mice:

[0195] In the 10th week of the experimental period, mice were fasted for 12 hours without food or water. They were weighed and the length from the nose to the anus was measured (recorded as body length). The Lee's index and BMI of the mice were calculated according to the formula.

[0196] Formula: Lee's index = [weight (g) × 10 3 ] 1 / 3 / body length (cm);

[0197] BMI index = weight (kg) / body length (m) 2 , the results are as follows Figure 9 shown.

[0198] The BMI index can reflect the relationship between the weight and body length of mice and is an important indicator for judging the obesity level of mice. The Lee's index is also an effective index for evaluating the obesity level. Figure 9 Analysis of the Lee's index and BMI index in each group showed that compared with the data of the HFD group, the Lee's index and BMI index of the HFD-A1 and HFD-A2 groups were reduced to varying degrees, indicating that the obesity level of mice was significantly reduced after using Bifidobacterium breve HC2953 powder.

[0199] Organ index measurement:

[0200] During the 10th week of the experimental period, mice were fasted for 12 hours without food or water. Following sacrifice, their organs (heart, kidneys, spleen, liver, and thymus) were precisely and rapidly removed during dissection, weighed, and the data recorded. The organ index of each mouse was calculated using the formula.

[0201] Liver index = liver weight (g) / body weight (g) × 100%;

[0202] Spleen index = spleen weight (g) / body weight (g) × 100%;

[0203] Cardiac index = heart weight (g) / body weight (g) × 100%;

[0204] Renal index = kidney weight (g) / body weight (g) × 100%;

[0205] Thymus index = thymus weight (g) / body weight (g) × 100%.

[0206] The results are as follows Figures 10-11 shown.

[0207] Obesity may cause differences in organ indexes compared to normal indexes, and is also an important reference for detecting health indicators in obese mice. Figures 10-11 It can be concluded that, with the exception of the heart, the indices of several organs in the obese mice in the HFD group were significantly higher than those in the normal NCD group. The indices of these organs in the HFD-A1 and HFD-A2 groups, which received oral administration of Bifidobacterium breve powder, were significantly lower than those in the HFD group and approached those of the NCD group, with the HFD-A1 group showing the most significant effect. In summary, consuming the probiotic Bifidobacterium breve HC2953 powder can help obese mice maintain relatively normal liver, spleen, heart, kidney, and thymus indices, thereby maintaining their health.

[0208] Blood collection and related index determination:

[0209] In the 10th week of the experimental cycle, the mice were fasted but not watered for 12 h, and their eyeballs were removed to collect blood. The collected blood samples were allowed to stand at room temperature for 1 h and then centrifuged at 8000 rpm for 10 min at 4°C to separate the serum for later use. The total cholesterol (TC) detection kit, triglyceride (TG) detection kit, high-density lipoprotein cholesterol (HDL-C) kit, low-density lipoprotein cholesterol (LDL-C) kit and alanine aminotransferase (ALT) kit (the kits were purchased from Nanjing Jiancheng Bioengineering Research Institute) were used to determine the levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in the serum, and the arteriosclerosis index (AI) was calculated according to the formula.

[0210] Arteriosclerosis index (AI) = [total cholesterol (TC) (mmol / L) - high-density lipoprotein cholesterol (HDL-C) (mmol / L)] / high-density lipoprotein cholesterol (HDL-C) (mmol / L). The results are as follows Figure 12 shown.

[0211] A high-fat diet leads to a significant decrease in HDL levels and a significant increase in TC, TG and LDL levels in mice, causing lipid metabolism disorders in the body.

[0212] Depend on Figure 12 As can be seen in this experiment, the serum HDL-C levels of mice in the NCD group fed a basal diet were significantly higher than those in the HFD group (p < 0.05), while the TC, TG, LDL-C, ALT, and AI indices were significantly lower than those in the obese mice in the HFD group (p < 0.05). This indicates that obese mice fed a high-fat diet have lipid imbalances in their serum. After intervention with the probiotic Bifidobacterium breve, the TC, TG, LDL-C, ALT, and AI indices of the HFD-A1 and HFD-A2 groups were significantly lower than those in the HFD group. HDL-C was significantly higher than that in the obese group, with the HFD-A1 group showing the most significant effect, approaching normal levels. These results indicate that the probiotic Bifidobacterium breve can maintain lipid metabolism balance in mouse serum by reducing TC, TG, LDL-C, and ALT levels and increasing HDL-C levels.

[0213] Liver tissue related index determination:

[0214] In the 10th week of the experimental cycle, mice were fasted but not watered for 12 h. After being killed, the mouse livers were dissected and stored in liquid nitrogen. 0.5 g of liver tissue was accurately weighed and added with 10 mL of a mixture of chloroform and methanol (2:1, v / v) and shaken to mix. The mixture was kept at 37°C for 30 min and centrifuged at 8000 rpm for 10 min at 4°C. The chloroform layer was collected in a sterile tube, and 6 mL of normal saline was added and centrifuged twice. The bottom chloroform layer was collected and dried with a nitrogen blower. 0.8 mL of a mixture of isopropanol and Triton-100 (9:1, v / v) was added and shaken to mix. 1.2 mL of distilled water was added and mixed thoroughly to obtain a total lipid solution of liver tissue.

[0215] The total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) kits were used to measure the levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and alanine aminotransferase (ALT) in liver homogenate. The results were as follows: Figure 13-1 and 13-2 .

[0216] The liver is the center of lipid metabolism and is closely related to metabolic diseases. Under normal circumstances, lipid metabolism in the liver remains balanced. However, obesity caused by a long-term high-fat diet can lead to disorders in liver lipid metabolism, resulting in abnormally high levels of TC and TG in the liver.

[0217] As shown in Figure 13, compared to the NCD group, the HFD group showed significantly lower liver TC, TG, LDL-C, and ALT levels (p < 0.05), and significantly higher HDL-C levels (p < 0.05). After intervention with the probiotic Bifidobacterium breve, TC, TG, LDL-C, ALT, and HDL-C in the HFD-A1 and HFD-A2 groups returned to normal levels, with the HFD-A1 group showing the most significant effect. These results indicate that the probiotic Bifidobacterium breve reduces TC, TG, LDL-C, and ALT levels in the mouse livers and increases HDL-C levels, thereby maintaining lipid balance in the mouse livers.

[0218] Liver probiotic test:

[0219] In the 10th week of the experimental cycle, the mice were fasted but not watered for 12 hours. After being killed, the mouse livers were dissected and quickly placed on a sterile plate. 1 g of liver was cut out and suspended in sterile PBS buffer and homogenized. 1 mL of the solution was spread on an MRS solid plate and cultured aerobically and anaerobically at 37°C for 48 hours to detect whether probiotics grew in the plate.

[0220] Studies have shown that when high-fat model mice are fed certain probiotics, strains of bacteria will grow in the liver, leading to liver failure.

[0221] In this experiment, liver tissues from mice in each group were aseptically plated on MRS plates and incubated aerobically and anaerobically for 48 hours. No colonies were observed, indicating that the probiotic does not undergo enterohepatic translocation in animals.

[0222] Mouse fecal bile acid detection:

[0223] Mouse feces collected weekly were dried to constant weight. 1 g of dry feces was extracted three times with 10 mL of anhydrous ethanol at 80°C to obtain the extract. After evaporation to dryness in a water bath at 90°C, the extract was dissolved in 10 mL of petroleum ether and the supernatant was discarded. The extract was thoroughly shaken and mixed with ethanol containing 2% Triton X-100, evaporated to dryness again, and dissolved in 10 mL of distilled water. The resulting solution was used to determine the total bile acid (TBA) content in the feces using a kit. The results are as follows: Figure 14 shown.

[0224] Cholesterol is a precursor for the synthesis of bile acids. Promoting the excretion of bile acids in the intestine is the main way to reduce cholesterol metabolism in the body.

[0225] Depend on Figure 14 It can be concluded that the fecal bile acid levels of mice in the probiotic-treated HFD-A1 and HFD-A2 groups were 1.92 and 1.56 times higher than those in the HFD group, respectively. This suggests that oral administration of Bifidobacterium breve HC2953 powder can effectively increase TBA excretion in mice, thereby reducing cholesterol levels in the body.

[0226] The above examples demonstrate that the Bifidobacterium breve HC2953 probiotic strain of the present invention is highly efficient in degrading cholesterol, triglycerides, and glycerol, and exhibits strong colonization and adhesion abilities. Furthermore, HC2953 can reduce the feed utilization rate of hyperlipidemia obese mice, thereby controlling their weight. By improving organ indices and reducing fat synthesis in the mice, the hyperlipidemia obese mice achieve a healthy, balanced body fat state. HC2953 also reduces serum and liver levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and alanine aminotransferase (ALT), while increasing high-density lipoprotein cholesterol (HDL-C) and bile acid (TBA) excretion in the obese mice, thereby improving lipid metabolism disorders in the hyperlipidemia obese mice, thereby achieving both lipid-lowering and weight-loss effects.

[0227] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A Bifidobacterium breve HC2953, wherein the preservation number of the Bifidobacterium breve HC2953 is CCTCC NO: M 20222007.

2. A bacterial agent containing the Bifidobacterium breve HC2953 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The number of live bacteria of Bifidobacterium breve HC2953 in the bacterial agent is 10 to 200 billion CFU / g.

4. The bacterial agent according to claim 2 or 3, characterized in that: The bacterial agent includes the Bifidobacterium breve HC2953 bacterial body and / or the Bifidobacterium breve HC2953 fermentation liquid.

5. Use of the Bifidobacterium breve HC2953 according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in the preparation of a product with weight loss function.

6. The use according to claim 5, characterized in that: The products include products with lipid-lowering function.

7. The use according to claim 6, characterized in that: The products with lipid-lowering function include products that meet one or more of the following requirements: Ⅰ: Reduce the total cholesterol concentration in the body; Ⅱ: Reduce the concentration of triglycerides in the body; III: Reduce the concentration of low-density lipoprotein cholesterol in the body; IV: Reduce the level of alanine aminotransferase in the body; Ⅴ: Increase the concentration of high-density lipoprotein cholesterol in the body; Ⅵ: Increase the excretion of bile acid in the body.

8. A product with weight loss function, characterized in that: The invention comprises the Bifidobacterium breve HC2953 as claimed in claim 1 or the bacterial agent as claimed in any one of claims 2 to 4 and auxiliary materials.

9. The product according to claim 8, characterized in that The viable bacterial count of Bifidobacterium breve HC2953 in the product is 10 to 200 billion CFU / g.

10. The product according to claim 8 or 9, characterized in that The products include pharmaceutical products.

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