Bifidobacterium animalis subsp. lactis strain, probiotic preparation, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of microorganisms, and in particular to a Bifidobacterium animalis subsp. Lactis strain, a probiotic preparation, and a preparation method therefor and the use thereof. The deposit number of the Bifidobacterium animalis subsp. Lactis strain is CGMCC No. 25680. The strain and the live bacteria preparation or dead bacteria preparation thereof can inhibit the differentiation of 3T3-L1 preadipocytes, thereby effectively suppressing fat deposition and possessing a weight-loss function, have an activity of inhibiting DPP-4 and α-glucosidase, and can stimulate NCI-H716 cells to secrete high levels of GLP-1, thereby possessing an effect of assisting in reducing blood glucose.

Description

Animal Bifidobacterium lactis subspecies strain, probiotic preparation, preparation method and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 202311630313.0 filed in China on November 30, 2023 and Chinese Patent Application No. 202311633450.X filed in China on November 30, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of microorganisms, and in particular to an animal Bifidobacterium lactis subspecies strain, a probiotic preparation, and a preparation method and use thereof. Background Art

[0004] In China, rapid economic development has led to changes in dietary habits and reduced physical activity, leading to the early onset of obesity-related diseases such as diabetes. Diabetes is a metabolic disease characterized by high blood sugar levels. High blood sugar levels are caused by impaired insulin secretion, impaired insulin action, or both. Diabetes can cause vascular and neurological changes, leading to various complications that seriously threaten patients' health and lives.

[0005] Currently, commercially available antidiabetic drugs have several common side effects. First, hypoglycemia. Excessive dosages of antidiabetic drugs or irregular diets can easily cause hypoglycemia. This is especially true for insulin secretagogues, including sulfonylureas and glinides. These drugs work by boosting insulin to lower blood sugar levels. Whether used alone or in combination, they can cause hypoglycemia, and in severe cases, hypoglycemic coma may occur. Second, gastrointestinal side effects. Oral antidiabetic drugs can cause gastrointestinal side effects, such as nausea, vomiting, diarrhea, and abdominal distension. Biguanides and glucosidase inhibitors, for example, can also cause gastrointestinal side effects. Third, liver and kidney damage. Oral antidiabetic drugs can aggravate liver and kidney damage, especially in patients with hepatic or renal insufficiency. Insulin has no effect on liver and kidney function. Fourth, drug allergies. Commonly used antidiabetic drugs have the potential to cause allergic reactions, such as rashes and itching. Fifth, other side effects. Drugs like thiazolidinediones can cause sodium and water retention, leading to edema. Sodium-glucose cotransporter 2 inhibitors can cause genitourinary infections and ketoacidosis.

[0006] Bifidobacterium animalis consists of two subspecies: Bifidobacterium animalis subsp. animalis and Bifidobacterium animalis subsp. lactis. These bacteria maintain intestinal microbial balance, regulate immune responses, reduce oxidative stress, and increase short-chain fatty acid secretion, thereby controlling blood sugar. They have a well-established safety profile and a long history of safe use, earning them inclusion in China's "List of Bacteria Suitable for Food Use" and the EU's Qualified Presumption of Safety (QPS) list. Ministry of Health Document No. 25 specifies nine strains approved for use in infants and young children, including Bifidobacterium animalis subsp. lactis Bb-12, Bi07, and HN019. Currently, no high-quality strains of Bifidobacterium animalis subsp. lactis have been identified in China.

[0007] Therefore, it is crucial to screen a superior strain of Bifidobacterium animalis lactis subspecies native to China that can help with weight loss and lowering blood sugar.

[0008] Summary of the Invention

[0009] The present invention aims to solve the problems existing in the prior art and provides a Bifidobacterium animalis subsp. lactis strain, food, health care product, pharmaceutical composition and probiotic preparation, as well as preparation methods and uses thereof.

[0010] In one aspect, the present invention provides a Bifidobacterium animalis subsp. lactis strain, the deposit number of which is CGMCC No. 25680.

[0011] Optionally, the Bifidobacterium animalis subsp. lactis strain comprises the 16S rRNA gene represented by SEQ ID NO: 1.

[0012] Optionally, the Bifidobacterium animalis subsp. lactis strain has high ability to produce lactic acid and short-chain fatty acids.

[0013] The present invention also provides use of the Bifidobacterium animalis subsp. lactis strain for producing lactic acid and short-chain fatty acids.

[0014] Optionally, the short-chain fatty acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid and isobutyric acid.

[0015] In addition, the animal Bifidobacterium lactis subspecies strain of the present invention can inhibit multiple pathogenic bacteria, can tolerate artificial gastrointestinal fluid, can adhere to intestinal epithelial cells, and is sensitive to erythromycin, chloramphenicol, vancomycin, ampicillin, clindamycin, and streptomycin.

[0016] Optionally, the Bifidobacterium animalis subsp. lactis strain has antioxidant activity.

[0017] Further optionally, the Bifidobacterium animalis subsp. lactis strain has hydroxyl radical scavenging and DPPH radical scavenging activities, and has SOD enzyme activity.

[0018] The present invention also provides use of the Bifidobacterium animalis subsp. lactis strain in preparing an anti-oxidative composition.

[0019] Optionally, the antioxidant is scavenging hydroxyl radicals and / or scavenging DPPH radicals.

[0020] Optionally, the Bifidobacterium animalis subsp. lactis strain has the ability to reduce oxidative stress.

[0021] The present invention also provides use of the animal Bifidobacterium lactis subsp. lactis strain in preparing a medicament for reducing oxidative stress.

[0022] In another aspect, the present invention provides a probiotic preparation for helping to lose weight or assist in lowering blood sugar, comprising the above-mentioned Bifidobacterium animalis subsp. lactis strain and excipients.

[0023] Preferably, the probiotic preparation is a live bacteria preparation, and the excipient is selected from one or more of starch, sucrose, trehalose and glycerol.

[0024] Preferably, the live bacteria preparation contains up to 5.0-7.0×10 11 CFU / g of viable bacteria.

[0025] On the other hand, the present invention provides a method for preparing the probiotic preparation as described above, comprising the following steps: expanding and culturing the above-mentioned Bifidobacterium animalis subsp. lactis strain in an optimized liquid culture medium; collecting the bacteria; adding a protective agent to resuspend, vacuum freeze-drying, and crushing to obtain an active bacterial agent.

[0026] In one embodiment, the Bifidobacterium animalis subsp. lactis strain of the present invention is derived from healthy infant feces.

[0027] In another aspect, the present invention provides use of the above-mentioned Bifidobacterium animalis subsp. lactis strain, a live bacteria preparation or a dead bacteria preparation thereof in the preparation of a medicament for weight loss.

[0028] Optionally, the Bifidobacterium animalis subsp. lactis strain, live bacteria preparation or killed bacteria preparation thereof is used to inhibit the differentiation of 3T3-L1 preadipocytes.

[0029] Optionally, the Bifidobacterium animalis subsp. lactis strain, its live bacteria preparation or killed bacteria preparation is used to reduce the triglyceride (TG) content in 3T3-L1 cells.

[0030] Optionally, the Bifidobacterium animalis subsp. lactis strain, live bacteria preparation or killed bacteria preparation thereof is used to inhibit fat production.

[0031] Optionally, the Bifidobacterium animalis subsp. lactis strain, its live bacteria preparation or killed bacteria preparation is used to treat metabolic diseases caused by obesity.

[0032] In another aspect, the present invention provides use of the above-mentioned Bifidobacterium animalis subsp. lactis strain, a live bacteria preparation or a dead bacteria preparation thereof in the preparation of a pharmaceutical composition for preventing or treating obesity and related diseases.

[0033] In another aspect, the present invention provides use of the above-mentioned Bifidobacterium animalis subsp. lactis strain, or a live bacteria preparation or a killed bacteria preparation thereof in the preparation of a medicament for assisting in lowering blood sugar.

[0034] Optionally, the Bifidobacterium animalis subsp. lactis strain, live bacteria preparation or killed bacteria preparation thereof is used to inhibit the activities of dipeptidyl peptidase 4 (DPP-4) and α-glucosidase.

[0035] Optionally, the Bifidobacterium animalis subsp. lactis strain, its live bacteria preparation or killed bacteria preparation is used to stimulate NCI-H716 cells to secrete GLP-1.

[0036] In another aspect, the present invention provides use of the above-mentioned Bifidobacterium animalis subsp. lactis strain, or a live or killed bacterial preparation thereof in a pharmaceutical composition for preventing or treating diabetes.

[0037] In yet another aspect, the present invention provides a food or health product comprising the above-mentioned Bifidobacterium animalis subsp. lactis strain.

[0038] In another aspect, the present invention provides a pharmaceutical composition comprising the Bifidobacterium animalis subsp. lactis strain as described above and a pharmaceutical excipient. Beneficial effects:

[0039] The 16S rRNA gene sequence of the animal Bifidobacterium lactis strain of the present invention is different from that of other strains of the same species and is unique; the strain and its live bacterial preparation or dead bacterial preparation can inhibit the differentiation of 3T3-L1 preadipocytes, thereby effectively inhibiting fat deposition and having a weight-loss function; the strain can inhibit the activities of DPP-4 and α-glucosidase by producing relatively high levels of DPP-4 inhibitors and α-glucosidase inhibitors, and can stimulate NCI-H716 cells to secrete a high content of GLP-1, thereby having an auxiliary effect of lowering blood sugar.

[0040] In addition, this strain also has the functions of high production of lactic acid and short-chain fatty acids, inhibiting a variety of pathogenic bacteria (with good antibacterial effects on Escherichia coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa and Clostridium difficile), and reducing oxidative stress.

[0041] The animal Bifidobacterium lactis subspecies strain of the present invention can be used as common food or health food, or as medicine for treating metabolic diseases caused by obesity, or preventing or treating diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG1 shows a biological evolutionary tree of Bifidobacterium animalis subsp. lactis HOM1119 strain.

[0043] FIG2 shows a RAPD cluster analysis diagram of the Bifidobacterium animalis subsp. lactis HOM1119 strain of the present invention constructed based on the UPGMA method.

[0044] FIG3 shows the results of the effect of the Bifidobacterium animalis subsp. lactis HOM1119 strain of the present invention on the viability of 3T3-L1 preadipocytes.

[0045] FIG4 shows the results of Oil Red staining of 3T3-L1 preadipocytes differentiated by the Bifidobacterium animalis subsp. lactis HOM1119 strain of the present invention.

[0046] Figure 5 shows the effect of the present invention's Bifidobacterium animalis subsp. lactis HOM1119 on lipid accumulation in differentiated 3T3-L preadipocytes. Note: *p<0.05 compared with the model control group, **p<0.01 compared with the model control group, ***p<0.001 compared with the model control group.

[0047] Figure 6 shows the effect of the present invention's Bifidobacterium animalis subsp. lactis HOM1119 on triglyceride content in differentiated 3T3-L preadipocytes. Note: *p<0.05 compared with the model control group, **p<0.01 compared with the model control group, ***p<0.001 compared with the model control group.

[0048] FIG7 shows the results of Oil Red staining of 3T3-L1 preadipocytes differentiated by the Bifidobacterium animalis subsp. lactis HOM1119 strain of the present invention.

[0049] Figure 8 shows the effect of the present invention's Bifidobacterium animalis subsp. lactis HOM1119 on lipid accumulation in differentiated 3T3-L preadipocytes. Note: *p<0.05 compared with the model control group, **p<0.01 compared with the model control group, ***p<0.001 compared with the model control group.

[0050] Figure 9 shows the effect of the present invention's Bifidobacterium animalis subsp. lactis HOM1119 on triglyceride content in differentiated 3T3-L preadipocytes. Note: *p<0.05 compared with the model control group, **p<0.01 compared with the model control group, ***p<0.001 compared with the model control group.

[0051] Figure 10 shows the effects of the present invention's Bifidobacterium animalis subsp. lactis HOM1119 on fasting blood glucose and glucose tolerance in rats with insulin resistance and glucose / lipid metabolism disorders. Note: *p<0.05 compared with the model control group, **p<0.01 compared with the model control group, ***p<0.001 compared with the model control group.

[0052] Microorganism Preservation Instructions

[0053] The Bifidobacterium animalis subsp. lactis HOM1119 strain of the present invention was deposited in the General Microbiology Center (CGMCC) of the China Culture Collection Administration on September 9, 2022. The address of the collection center is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the collection number is CGMCC No. 25680.

[0054] The Bifidobacterium animalis subsp. lactis HOM1119 strain of the present invention was sent to the Institute of Microbiology, Chinese Academy of Sciences for identification in May 2023.

[0055] The detection and identification conclusions are as follows: Under the conditions of this laboratory, based on a comprehensive analysis of experimental data such as the cell morphology, physiological and biochemical characteristics, 16S rRNA gene sequence, and tuf gene sequence of the tested bacteria, and with reference to relevant research papers in the "Bergey's Manual of Systematic Bacteriology" and the International Journal of Systematic and Evolutionary Microbiology, the identification result of the tested bacteria (strain number: HOM1119) is: Bifidobacterium animalis subsp. lactis (Bifidobacterium animalis subsp. lactis).

[0056] The cell morphology of the strain is polymorphic rod-shaped; the physiological and biochemical characteristics are Gram-positive, catalase-negative (-), and oxidase-negative (-); the 16S rRNA gene sequence is shown in SEQ ID NO: 1, and the tuf gene sequence is shown in SEQ ID NO: 9. DETAILED DESCRIPTION

[0057] The present invention discloses strains, characteristics, and applications. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0058] To further illustrate the technical means and effects of the present invention to achieve the intended purpose, the following technical solutions of the present invention are further described in conjunction with specific embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. Experimental methods for which specific conditions are not specified in the following implementations are carried out in accordance with conventional methods and conditions in the art.

[0059] definition

[0060] As used herein, the term DPPH radical refers to 1,1-diphenyl-2-trinitrophenylhydrazine, also known as 1,1-diphenyl-2-picrylhydrazyl (free radical); 1,2-dicryl-2-trinitrophenylhydrazine; 1,2-diphenyl-2-picrylhydrazyl. As a stable free radical, DPPH can stably exist in organic solvents. Its alcohol solution is purple and needs to be stored at low temperature and away from light. It has a single electron, so it can accept an electron or hydrogen ion and has maximum absorption at a wavelength of 517nm. In the presence of a free radical scavenger, the single electron of DPPH is captured, making its color lighter. The absorbance at the wavelength of maximum light absorption decreases, and the degree of decrease is linear. The decrease in the absorbance level indicates an increase in antioxidant activity, thereby evaluating the antioxidant capacity of the test sample. This antioxidant capacity is expressed by inhibition rate. The greater the inhibition rate, the stronger the antioxidant activity.

[0061] 3T3-L1 preadipocytes (mouse embryonic fibroblasts) are normal mouse cells, also known as 3T3L1 preadipocytes. 3T3-L1 preadipocytes, also known as mouse embryonic fibroblasts, exhibit contact inhibition and the ability to differentiate into adipocytes. High serum culture media promotes fat accumulation in these cells.

[0062] 3T3-L1 cells, a mouse embryonic fibroblast cell line, are widely used in the study of metabolic diseases such as obesity and diabetes due to their ability to differentiate into adipocyte-like cells in vitro. These cells undergo a transition from pre-adipocyte to adipocyte-like cell differentiation when they undergo contact inhibition, progressing from rapidly dividing cells to confluent cells. During culture, high serum levels promote fat accumulation.

[0063] It should be noted that the experimental methods used in the present invention are all conventional methods unless otherwise specified.

[0064] Unless otherwise specified, the reagents and materials used in the present invention were prepared using conventional methods or obtained from commercial channels.

[0065] Example

[0066] Example 1 Isolation and identification of Bifidobacterium animalis subsp. lactis HOM1119 strain

[0067] (1) Preparation of artificial gastrointestinal fluid

[0068] Artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid (1 mol / L), add 800 mL of water, adjust the pH to 3.0, add 10 g of pepsin, shake well, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter with 0.22 μm filter membrane for sterilization, and store at -20°C for later use.

[0069] Artificial intestinal fluid: Take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water, and adjust the pH to 6.8 with 0.4% (0.1 mol / L) sodium hydroxide solution. Take 10 g of pancreatic enzyme and 3 g of porcine bile salt, add appropriate amount of water to dissolve them, mix the two liquids, add water to 1000 mL, centrifuge at 5000 rpm for 5 min, take the supernatant, filter and sterilize with a 0.22 μm filter membrane, and store at -20°C for later use.

[0070] (2) Preparation of separation culture medium

[0071] MRS-Cys liquid culture medium: Add 0.5 g of L-cysteine ​​hydrochloride per liter of MRS broth medium (product number: CM1163, OXOID, UK), sterilize at 121°C for 15 min, set aside, and store at 2-8°C in the dark for one week.

[0072] MRS-Cys solid medium containing bromocresol purple: Add 0.04 g of bromocresol purple per liter of MRS solid medium (Cat. No. CM1175, OXOID, UK) and stir thoroughly. Stir at 121°C for 15 min. Pour the medium into a clean plate in a laminar flow hood, pour approximately 15 mL into each culture dish, and allow to solidify before use.

[0073] (3) Isolation and screening of Bifidobacterium animalis subsp. lactis HOM1119 strain

[0074] The Bifidobacterium animalis subsp. lactis of the present invention is isolated from the feces of healthy infants.

[0075] Use sterile anaerobic tubes to collect in vitro feces and store them at low temperature under anaerobic environment. The experiment started on the day of sampling. Take about 1g of fecal sample and add it to an anaerobic tube containing 9mL of MRS-Cys liquid culture medium. Incubate it anaerobically at 37℃ for 24h, centrifuge it at 8000rpm for 10min, discard the supernatant, add 10mL of artificial gastric juice, mix it, incubate it anaerobically at 37℃ for 3h, centrifuge it at 8000rpm for 10min, discard the supernatant, add 10mL of artificial intestinal juice, mix it, incubate it anaerobically at 37℃ for 3h, dilute the sample to 10 by 10-fold dilution method. -6 , from stock solution to 10 -6 100 μL of the solution was taken in sequence and spread on MRS-Cys solid culture medium plates containing bromocresol purple, and cultured anaerobically at 37° C. for 72 h.

[0076] Pick a single colony with yellowing edges, streak culture, and purify 3-4 times until the colony is single. Perform Gram staining and observe colony morphology under a microscope. Transfer the single colony to liquid culture medium for pure culture, preserve the seed in glycerol, and store at -80°C.

[0077] (4) Morphological observation of Bifidobacterium animalis subsp. lactis HOM1119 strain

[0078] Bifidobacterium animalis subsp. lactis HOM1119 was cultured anaerobically at 37°C on MRS agar for 48 hours, producing round, milky-white colonies with a smooth, moist surface, a raised center, and neat edges. The colonies were approximately 2.5 mm in diameter. Microscopic observation revealed rod-, club-, or Y-shaped rods with bulging ends. The bacteria measured approximately 0.5–0.9 μm by 3–6 μm, were arranged singly or in pairs, were Gram-positive, and did not form spores. Two other strains with the same morphology were also screened and designated Bifidobacterium animalis subsp. lactis BH13-25 and BH13-33.

[0079] (5) Identification of Bifidobacterium animalis subsp. lactis HOM1119 strain

[0080] 16S rDNA identification: DNA was extracted from the three preserved strains HOM1119, BH13-25, and BH13-33, and 16S rDNA was amplified using PCR using universal primers 27F: SEQ ID NO: 7; 1492R: SEQ ID NO: 8 (see Table 1). PCR amplification and agarose gel electrophoresis were then performed. The gel was then excised, recovered, and sequenced, and the isolated strains were subjected to 16S rDNA sequencing. Based on their 16S rDNA sequences, BLAST tools were used to compare the sequences in the NCBI database, and a biological evolutionary tree was drawn using Mega 7.0 software. As shown in FIG1 , the identification results of the three strains were all Bifidobacterium animalis subsp. lactis, and they were named Bifidobacterium animalis subsp. lactis HOM1119, BH13-25, and BH13-33, respectively. The sequence of the 16S rRNA gene of Bifidobacterium animalis subsp. lactis HOM1119 is shown in SEQ ID NO: 1:

[0081] In addition, animal Bifidobacterium lactis subspecies strains BH13-25 and BH13-33, which were isolated at the same time as the animal Bifidobacterium lactis subspecies HOM1119 strain of the present invention, are used as control strains in the present invention, and the sequences of their 16S rRNA genes are shown in SEQ ID NOs: 10 and 11, respectively. By comparing the 16S rRNA gene sequences of the animal Bifidobacterium lactis subspecies HOM1119 strain of the present invention and the animal Bifidobacterium lactis subspecies strains BH13-25 and BH13-33 used as control strains in the present invention, it can be seen that strain BH13-25 has 1401 bases identical with the strain HOM1119 of the present invention, with a sequence similarity of 98.87%; strain BH13-33 has 1396 bases identical with the strain HOM1119 of the present invention, with a sequence similarity of 98.52%.

[0082] (6) Random Amplified Polymorphic DNA (RAPD) Strain Identification: DNA was extracted from the preserved strains, and using the strain DNA as a template, five primers, OPA-02: SEQ ID NO: 2; OPA-18: SEQ ID NO: 3; OPL-07: SEQ ID NO: 4; OPL-16: SEQ ID NO: 5; OPM-05: SEQ ID NO: 6 (see Table 1), were used to amplify DNA fragments that can show polymorphism by PCR. After gel electrophoresis, different DNA differences were shown. Cluster analysis software was used for analysis, and the results are shown in Figure 2. As shown in Figure 2, the animal Bifidobacterium lactis subsp. lactis strain HOM1119 is different from some commercial animal Bifidobacterium lactis subsp. lactis strains and the other two strains BH13-25 and BH13-33 isolated at the same time as the present invention, and is unique.

[0083] Table 1

[0084] Example 2 Test on the ability to inhibit common pathogenic bacteria

[0085] (1) Activation of indicator bacteria

[0086] The indicator strains Escherichia coli ATCC8739, Staphylococcus aureus ATCC6538, Salmonella typhimurium ATCC14028, Pseudomonas aeruginosa ATCC9027, Listeria monocytogenes ATCC19111, and Clostridium difficile ATCC9689 were all purchased from the China Industrial Microorganism Collection Center. Indicator bacteria (Escherichia coli ATCC8739; Staphylococcus aureus ATCC6538; Salmonella typhimurium ATCC14028; Pseudomonas aeruginosa ATCC9027; Listeria monocytogenes ATCC19111) were inoculated at an inoculum size of 1% of the total culture volume into TSB medium (Tryptone Soy Broth, where T stands for tryptone, S stands for soy, and B stands for broth) and cultured aerobically at 37°C at 180 rpm for 24 hours before use. Clostridium difficile ATCC9689 was inoculated at an inoculum size of 1% of the total culture volume into brain heart infusion broth (BHI) medium and cultured anaerobically at 37°C for 24 hours before use.

[0087] (2) Activation of Bifidobacterium animalis subsp. lactis strains

[0088] The Bifidobacterium animalis subsp. lactis HOM1119 strain and a control strain prepared in Example 1 and stored frozen at -80°C were inoculated at 1% of the total culture medium into sterilized MRS-Cys liquid culture medium described in Example 1. The culture was anaerobically incubated at 37°C for 24 hours and activated twice to obtain a fermentation broth. The culture was then centrifuged at 8000 rpm for 10 minutes, and the supernatant was used for antibacterial testing.

[0089] (3) Plate preparation

[0090] Heat the sterilized TSA medium until completely melted, pour it into a petri dish, place it on a horizontal surface to form an even agar layer, and wait for it to solidify. Add the indicator bacteria to the TSA medium, shake it evenly, then pour it into a pre-prepared TSA blank agar plate and let it stand to solidify.

[0091] (4) Antibacterial experiment

[0092] Using sterile tweezers, gently place the Oxford cup on a flat plate, maintaining a certain distance between them. Add 0.2 mL of the lactic acid bacteria fermentation broth supernatant to each sample. After diffusion for 24 hours in a 4°C refrigerator, incubate in a 37°C incubator for at least 18 hours and observe for the appearance of an inhibition zone. Once the inhibition zone forms, measure it with a ruler. Using the liquid culture medium (MRS-Cys) described in Example 1 as a negative control, perform three replicates for each sample. The inhibition results are shown in Table 2.

[0093] Table 2 Inhibitory effect of Bifidobacterium animalis subsp. lactis HOM1119 strain on pathogenic bacteria

[0094] Note: “–” no antibacterial activity; “+” 11-16mm; “++” 17-22mm; “+++” ≥ 23mm

[0095] As shown in Table 2, the animal Bifidobacterium lactis subsp. lactis strain HOM1119 has an inhibitory effect on all six pathogenic bacteria. Compared with the other two strains of animal Bifidobacterium lactis subsp. lactis, it has a better antibacterial effect on Escherichia coli, Salmonella, Staphylococcus aureus, Pseudomonas aeruginosa and Clostridium difficile, indicating that it has a better ability to inhibit pathogenic bacteria.

[0096] Example 3 Gastrointestinal transit capacity test

[0097] (1) Activation of strains

[0098] The animal Bifidobacterium lactis subsp. lactis HOM1119 strain and the control strains BH13-25 and BH13-33 prepared in Example 1 and stored at -80°C were inoculated at an inoculum amount of 1% of the total culture medium into the sterilized MRS-Cys liquid culture medium described in Example 1, and cultured anaerobically at 37°C for 24 hours. After activation twice, the strain fermentation broth was obtained.

[0099] (2) Preparation of artificial gastric juice

[0100] Take 16.4 ml of dilute hydrochloric acid, add about 800 ml of water and 10 g of pepsin, shake well, adjust the pH to 3.0, add water to 1000 ml, filter with a 0.2 μm microporous membrane and set aside.

[0101] (3) Preparation of artificial intestinal fluid

[0102] Take 6.8g of potassium dihydrogen phosphate, add 500mL of water to dissolve, adjust the pH to 6.8, add 10g of trypsin and 3g of porcine bile salt, mix the two solutions after dissolution, add water to 1000mL, filter with a 0.22um sterile filter membrane under sterile environment, and set aside.

[0103] (4) Evaluation of the survival ability of strains in the simulated gastrointestinal tract

[0104] Take 1 mL of activated bacterial suspension of the test strain and add the cells to 9 mL of artificial gastric fluid (pH 3.0). Mix thoroughly and count the viable cells. Incubate in a 37°C incubator for 3 hours and then count the viable cells. After 3 hours of incubation in artificial gastric fluid, transfer the entire cell to an equal volume of artificial intestinal fluid (pH 6.8), mix thoroughly, and incubate at 37°C.

[0105] The viable bacteria were counted on the plate using MRS medium at 3 h and 24 h, and the survival rate was calculated using the following formula:

[0106] Gastric juice 3-hour survival rate (%) = [logCFUN1 / logCFUN0] × 100%

[0107] 3-hour survival rate in intestinal fluid (%) = [logCFUN2 / logCFUN0] × 100%

[0108] 24-hour survival rate in intestinal fluid (%) = [logCFUN3 / logCFUN0] × 100%

[0109] N0 = viable cell count of the untreated Bifidobacterium animalis subsp. lactis strain, N1 = viable cell count of the Bifidobacterium animalis subsp. lactis strain after 3 hours of gastric juice treatment, N2 = viable cell count of the Bifidobacterium animalis subsp. lactis strain after 3 hours of intestinal juice treatment, and N3 = viable cell count of the Bifidobacterium animalis subsp. lactis strain after 24 hours of intestinal juice treatment. Data were analyzed using one-way ANOVA with Duncan's multiple comparisons using SPSS 25.0 software. The results are shown in Table 3.

[0110] Table 3 Survival rate of Bifidobacterium animalis subsp. lactis HOM1119 strain in simulated gastrointestinal fluid

[0111] Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0112] As shown in Table 3, the survival rate of the Bifidobacterium animalis subsp. lactis HOM1119 strain exceeded 98% after 3 hours of treatment with simulated gastric fluid. Even after 24 hours of treatment with simulated intestinal fluid, its survival rate remained above 85%. Its intestinal fluid tolerance was significantly (p<0.01) superior to that of the other two strains, BH13-25 and BH13-33. This indicates that the Bifidobacterium animalis subsp. lactis HOM1119 strain has a high survival rate in the intestine, laying the foundation for its colonization and effective function in the intestine.

[0113] Example 4 Intestinal epithelial cell adhesion ability test

[0114] (1) Preparation of cell culture medium

[0115] Complete culture medium: high-glucose DMEM medium supplemented with 10% inactivated fetal bovine serum (FBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin), mixed and stored at 4°C.

[0116] Incomplete culture medium: high-glucose DMEM medium to which 10% inactivated fetal bovine serum (FBS) was added, mixed, and stored at 4°C.

[0117] (2) Cell recovery and culture

[0118] Human colorectal adenocarcinoma Caco-2 cells were purchased from the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. The cells were resuspended in fresh culture medium and evenly dispersed in a culture flask. The cells were cultured at 37°C in an atmosphere of 5% CO2 and 95% air. The culture medium was replaced every 48 hours during recovery. When the cells were well grown (80% confluence), the Caco-2 cells were digested with trypsin-EDTA solution at 37°C. After digestion, the cell concentration was adjusted to 2 × 105 The cells were seeded into 24-well plates and cultured until the confluence reached 80%.

[0119] (3) Activation of Bifidobacterium animalis subsp. lactis strains

[0120] The animal Bifidobacterium lactis subsp. lactis HOM1119 strain prepared in Example 1 and stored at -80°C was inoculated into the sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum amount of 1% of the total culture medium, and anaerobically cultured at 37°C for 24 hours. After activation twice, the fermentation liquid of the strain was obtained.

[0121] (4) Adhesion test

[0122] Centrifuge at 8000 rpm for 10 min, collect the bacteria grown in the corresponding culture medium; wash the bacteria three times with DPBS, resuspend the bacteria in incomplete culture medium, and adjust the concentration of the bacteria to 10 8 cfu / mL; 1 mL of the above bacterial suspension was added to a 24-well plate containing Caco-2 cells grown to a monolayer and incubated at 37°C in a 5% CO2 incubator for 2 h; after incubation, the cells were washed three times with sterile DPBS; the Caco-2 cells were digested with trypsin-EDTA solution at 37°C, and the cell number and the number of viable bacteria before and after adhesion were counted. Three parallels were made for each sample, and the data were analyzed by one-way ANOVA and Duncan's multiple comparisons using SPSS25.0 software. The results are shown in Table 4.

[0123] Table 4 Adhesion ability of Bifidobacterium animalis subsp. lactis HOM1119 strain to Caco-2 cells

[0124] Note: The numbers with different capital letters in the same column are extremely significantly different (p<0.01), and the numbers with different lowercase letters are significantly different (p<0.05)

[0125] The results in Table 4 show that the adhesion rate of animal Bifidobacterium lactis subsp. lactis strain HOM1119 to human colon cancer cells Caco-2 was 5.14%, and the adhesion index was 4.56, which was better than the other two strains of animal Bifidobacterium lactis subsp. lactis strains and had a better ability to adhere to intestinal epithelial cells.

[0126] Adhesion index = number of bacteria after adhesion / number of cells per dish

[0127] Adhesion rate = number of bacteria after adhesion / number of bacteria before adhesion

[0128] Example 5 Lactic acid and short-chain fatty acid production capacity test

[0129] The animal bifidobacterium lactis subspecies HOM1119 bacterial strain of-80 ℃ of freezing preservations prepared among the embodiment 1 is accessed in the sterilized MRS-Cys liquid nutrient medium described in the embodiment 1 with the inoculum size accounting for culture medium total amount 1%, 37 ℃ were left to stand anaerobic culture 24 hours, activated bacterial strain fermentation liquid after twice.Then under the 8000rpm condition, centrifugal 10min, get the content of supernatant and gas chromatograph detection lactic acid and short-chain fatty acids.The MRS-Cys liquid nutrient medium described in embodiment 1 is a negative control, and other 2 strains animal bifidobacterium lactis subspecies bacterial strains are as positive controls, and each sample is done 3 parallels, and data adopt SPSS25.0 software to carry out one-way variance analysis (ANOVA) and Duncan's multiple comparison, and the results are shown in Table 5.

[0130] Table 5 Lactic acid and short-chain fatty acid production capacity of Bifidobacterium animalis subsp. lactis HOM1119 strain

[0131] Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0132] As shown in Table 5, compared with other Bifidobacterium animalis subsp. lactis strains, the Bifidobacterium animalis subsp. lactis HOM1119 strain has a stronger ability to produce lactic acid and short-chain fatty acids (acetic acid, formic acid, propionic acid, butyric acid and isobutyric acid).

[0133] Example 6 Antibiotic Sensitivity Test

[0134] The antimicrobial susceptibility test was conducted according to ISO10932-2010 Milk and dairy products developed by the International Organization for Standardization, using the antibiotic minimum inhibitory concentration (MIC) determination method applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB).

[0135] (1) Culture medium preparation

[0136] MRS-Cys liquid culture medium: same as Example 1

[0137] LSM-Cys liquid medium: Weigh 21.06 g of Iso-Sensitest medium (Cat. No. CM0473B, OXOID, UK), 5.2 g of MRS Broth, and 0.3 g of L-cysteine ​​hydrochloride, add water to 0.5 L, adjust the pH to 6.85 ± 0.1, sterilize at 121°C for 15 min (the pH should be 6.7 ± 0.1), set aside, and store in the dark at 2-8°C for one week.

[0138] (2) Activation and proliferation of Bifidobacterium animalis subsp. lactis

[0139] The -80°C frozen Bifidobacterium animalis subsp. lactis HOM1119 strain prepared in Example 1 was inoculated into the sterilized liquid culture medium (MRS-Cys) described in Example 1 at an inoculum size of 1% of the total culture medium, and anaerobically cultured at 37°C for 24 hours. After activation twice, a fermentation broth was obtained. The activated Bifidobacterium animalis subsp. lactis HOM1119 strain was propagated on MRS-Cys agar medium and anaerobically cultured at 37°C for 48 hours.

[0140] (3) Preparation of antibiotic microdilution plates

[0141] Weigh 0.0512 g of antibiotics and add 10 mL of solvent. Dissolve chloramphenicol and erythromycin in ethanol (without filtration), ampicillin in phosphate buffer (pH 8.0, 0.1 mol / L), and other antibiotics in water. After shaking, filter through a 0.22 μm filter and aliquot into EP tubes at a concentration of 5120 μg / mL (5.12 mg / mL). Store at -20°C until needed. Dilute the antibiotic stock solution with water (phosphate buffer for ampicillin) to the appropriate concentration range. Add 50 μL of the dilution to the wells of a microdilution plate.

[0142] (4) Preparation of bacterial suspension

[0143] Pick up a single colony from the agar plate. Suspend the colony in a sterile culture tube filled with 2 mL to 5 mL of sterile saline. Suspend the colony in pre-reduced LSM-Cys liquid medium. Suspend the colony until the turbidity of the solution reaches McFarland standard 1 or the optical density at 625 nm is 0.16 to 0.2 using a spectrophotometer. The suspension is approximately equivalent to 3 × 10 8 CFU / mL. Dilute the bacterial suspension with the recommended culture medium. Dilute the bacterial suspension 500-fold with MRS-Cys liquid medium because the antibiotic solution will dilute the culture medium twice. Dispense the diluted bacterial suspension within 30 minutes of preparation. When using frozen plates, thaw the frozen antibiotic solution under anaerobic conditions immediately before use. Dispense 50 μL of the diluted suspension into each well of the microdilution plate (approximately 3×10 4 CFU / well). Incubate the plates at 37°C under static anaerobic conditions for 48 hours. When using anaerobic jars, cover the plates with lids between each plate to create a uniform environment within the jar. Each experiment is repeated three times, with both positive and negative control groups. Positive control wells contain no antibiotic but instead contain the test strain and culture medium containing the solvent containing the highest concentration of antibiotic. Negative control wells contain no test strain or antibiotic but instead contain culture medium.

[0144] (5) Read the MIC result

[0145] After 48 hours of incubation, read the MIC visually. After incubation, check the negative control wells for visible bacterial growth. If contamination is found, reject all data generated for the strain. Note: If there is no growth in the positive control wells, it means that the strain being tested is sensitive to the solvent used to dissolve the antibiotic. In this case, reading the MIC for that particular antibiotic is meaningless. If the negative and positive controls test normal, determine the bacterial growth visually for each antibiotic by comparison with the positive control. It is best to place the microdilution plate on top of a rack containing a magnifying glass and a workbench lamp that provides indirect light for easy reading. Bacterial growth is easily detected under a magnifying glass as a pellet at the bottom of the well. Discard any series of wells where discontinuous growth is observed (for example, growth at 16 μg / mL and 64 μg / mL, but no growth at 32 μg / mL). The endpoint is defined as the lowest antibiotic concentration at which no growth is observed visually. This concentration is the MIC for that antibiotic for that particular strain. Each sample was replicated three times, with Lactobacillus plantarum ATCC14917 and Bifidobacterium animalis subsp. lactis BH13-25 and BH13-33 strains serving as positive control strains. The antibiotic susceptibility results of Bifidobacterium animalis subsp. lactis HOM1119 are shown in Table 6. The MIC results of Lactobacillus plantarum ATCC14917 are consistent with those shown in the appendix of ISO10932-2010, indicating that the experimental method is accurate (Lactobacillus plantarum is the recommended control strain for the MIC method, and this strain was used to demonstrate the accuracy of the method). According to the antibiotic resistance standards for bacterial species used in food established by the European Food Safety Authority (EFSA) in 2012, it is known that Bifidobacterium animalis subsp. lactis HOM1119 is sensitive to erythromycin, chloramphenicol, vancomycin, ampicillin, clindamycin, and streptomycin, and the results are consistent with those of the control strains Bifidobacterium animalis subsp. lactis BH13-25 and BH13-33. Therefore, the animal Bifidobacterium lactis subsp. lactis HOM1119 strain is relatively safe.

[0146] Table 6 Antibiotic sensitivity results of Bifidobacterium animalis subsp. lactis HOM1119 strain

[0147] Example 7 Antioxidant Activity Detection Test

[0148] (1) Activation of strains

[0149] The commercial strain Lactobacillus rhamnosus GG (ATCC53103) was selected (purchased from the China Industrial Microorganism Collection Center) (GG is a commercial strain and is a strain with antioxidant effects in the literature) and Bifidobacterium lactis subspecies BH13-25 and BH13-33 strains as control strains. Animal Bifidobacterium lactis subspecies HOM1119, BH13-25 and BH13-33 strains and Lactobacillus rhamnosus GG strain were inoculated into the sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum amount of 1% of the total culture medium, and were placed in anaerobically at 37°C for 24 hours. After activation twice, the fermentation liquid was obtained, centrifuged at 8000rpm for 10min, and the concentration of the strain was adjusted to 1×10 10 CFU / mL, to be determined.

[0150] (2) Antioxidant activity assay

[0151] The oxidative capacity of the cells was determined using the total antioxidant capacity (T-AOC) assay kit, total superoxide dismutase (SOD) assay kit, DPPH free radical scavenging capacity kit, and hydroxyl free radical assay kit, all manufactured by the Nanjing Jiancheng Bioengineering Institute. Three replicates were used for each treatment, and data were analyzed using one-way analysis of variance (ANOVA) with Duncan's multiple comparisons using SPSS 25.0 software. The results are shown in Table 7.

[0152] Table 7. In vitro antioxidant activity results of Bifidobacterium animalis subsp. lactis HOM1119 strain

[0153] Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0154] As shown in Table 7, compared with the control strain, the hydroxyl radical scavenging ability, DPPH free radical scavenging ability, and SOD enzyme activity of the animal Bifidobacterium lactis subsp. lactis HOM1119 strain were significantly higher (p < 0.01) than those of Lactobacillus rhamnosus GG; the total antioxidant capacity (T-AOC), hydroxyl radical scavenging ability, DPPH free radical scavenging ability, and SOD enzyme activity were significantly higher (p < 0.01) than those of the animal Bifidobacterium lactis subsp. lactis BH13-25 and BH13-33 strains. This indicates that the animal Bifidobacterium lactis subsp. lactis HOM1119 strain can alleviate the damage caused by excessive reactive oxygen species in the body, thereby protecting insulin-producing β cells.

[0155] Example 8 DPP-4 Inhibitory and α-Glucosidase Inhibitory Tests

[0156] (1) Activation of strains

[0157] The commercial strain Lactobacillus rhamnosus GG (ATCC53103) was purchased from the China Industrial Microorganism Collection Center and the Bifidobacterium lactis subspecies BH13-25 and BH13-33 strains were selected as control strains. Animal Bifidobacterium lactis subspecies HOM1119, BH13-25 and BH13-33 strains and Lactobacillus rhamnosus GG strain were inoculated into the sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum rate of 1% of the total culture medium, and were cultured anaerobically at 37°C for 24 hours. After activation twice, the fermentation broth was obtained, centrifuged at 8000 rpm for 10 minutes, and the strain concentration was adjusted to 4×10 10 CFU / mL, cultured anaerobically for 8 h, centrifuged at 8000 rpm for 10 min, and the supernatant was taken for later use.

[0158] (2) DPP-4 inhibition and α-glucosidase inhibition assays

[0159] In vitro screening for hypoglycemic activity was performed using a DPP-4 inhibitor screening kit (Abnova #KA1311) and an α-glucosidase inhibitor screening kit (Biovision #K938). The inhibitory rates of the strain samples against these two enzymes were calculated. Three replicates were obtained for each treatment. Data were analyzed using one-way analysis of variance (ANOVA) with Duncan's multiple comparisons using SPSS 25.0 software. The results are shown in Table 8.

[0160] Table 8 Results of DPP-4 inhibition and α-glucosidase inhibition by animal Bifidobacterium lactis subsp.

[0161] Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0162] As shown in Table 8, compared with the control strains Bifidobacterium animalis subsp. lactis BH13-25, BH13-33, and Lactobacillus rhamnosus GG, the Bifidobacterium animalis subsp. lactis HOM1119 strain had higher inhibition rates against both DPP-4 and α-glucosidase, with extremely significant differences (p < 0.01). This suggests that the Bifidobacterium animalis subsp. lactis HOM1119 strain can achieve the function of lowering blood sugar levels by producing higher levels of DPP-4 inhibitors and α-glucosidase inhibitors.

[0163] Example 9 Bifidobacterium animalis subsp. lactis HOM1119 strain stimulates NCI-H716 cells to secrete GLP-1

[0164] (1) Culture of NCI-H716 cells

[0165] In this experiment, NCI-H716 cells (purchased from the Cell Bank of the Chinese Academy of Sciences) were selected and grown in RPMI-1640 (Gibco) medium containing 10% fetal bovine serum (Hyclone) and 1% double antibiotics (penicillin and streptomycin, Hyclone). Cells were grown in suspension in an incubator at 37°C and 5% CO2.

[0166] (2) Culture of strains

[0167] The commercial strain Lactobacillus rhamnosus GG (ATCC53103) was purchased from the China Industrial Microorganism Collection Center and the Bifidobacterium lactis subspecies BH13-25 and BH13-33 strains were selected as control strains. Animal Bifidobacterium lactis subspecies HOM1119, BH13-25 and BH13-33 strains and Lactobacillus rhamnosus GG strain were inoculated into the sterilized MRS-Cys liquid culture medium described in Example 1 at an inoculum amount of 1% of the total culture medium, and were placed in an anaerobic culture at 37°C for 24 hours. After activation twice, the fermentation liquid was obtained, centrifuged at 8000 rpm for 10 minutes, and the concentration of the strain was adjusted to 1×10 10 CFU / mL, cultured anaerobically for 8 h, centrifuged at 8000 rpm for 10 min, and the supernatant was taken for later use.

[0168] (3) GLP-1 endocrine test

[0169] NCI-H716 cells were cultured at 1.5×10 6 Cells were seeded at a density of 100 cells / well in a 24-well plate coated with Matrigel. Endocrine differentiation medium was added and cultured in a 37°C, 5% CO2 incubator for 2 days for endocrine differentiation experiments. Endocrine differentiation medium is high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum and 1% double-stranded antibody.

[0170] After 2 days, the DMEM medium was replaced with Krebs-Ringer buffer containing 1×10 10 After culturing for 2 hours with a probiotic strain containing 100 CFU / mL, the cells were centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected. 50 μg / mL of phenylmethylsulfonyl fluoride and 10 μg / mL of sitagliptin were added to the supernatant, and GLP-1 concentrations were measured using an ELISA kit (Ray Biotech). Three replicates were performed for each treatment, and data were analyzed using one-way analysis of variance (ANOVA) with Duncan's multiple comparisons using SPSS 25.0 software. The results are shown in Table 9.

[0171] Table 9 Bifidobacterium animalis subsp. lactis stimulates NCI-H716 cells to secrete GLP-1 production

[0172] Note: The numbers with different capital letters in the same column are significantly different (p<0.01).

[0173] As shown in Table 9, compared with the control strains Bifidobacterium animalis subsp. lactis BH13-25, BH13-33, and Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis HOM1119 stimulated NCI-H716 cells to secrete high levels of GLP-1, reaching a concentration of 2413.02 pg / mL, with a highly significant difference (p < 0.01). This suggests that Bifidobacterium animalis subsp. lactis HOM1119 has the effect of lowering blood sugar, and the hypoglycemic mechanism may be through stimulating intestinal L cells to produce high levels of GLP-1.

[0174] Example 10 In vitro lipogenesis inhibition test of animal Bifidobacterium lactis subspecies HOM1119 strain 1

[0175] (1) Preparation of cell culture medium

[0176] Complete medium 1: high-glucose DMEM medium, supplemented with 10% inactivated fetal bovine serum (NBS) and 1% (v / v) antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin), mixed and stored at 4°C.

[0177] Complete medium 2: high-glucose DMEM medium to which 10% inactivated fetal bovine serum (FBS) was added and stored at 4°C.

[0178] Cell differentiation medium 1: Add 3-isobutyl 1-methylxanthine (IBMX) to complete medium 2 to a final concentration of 0.5 mol / L, dexamethasone (DEX) to a final concentration of 1 μmol / L, and insulin to a final concentration of 10 μg / mL.

[0179] Cell differentiation medium 2: Add insulin to complete medium 2 to a final concentration of 10 μg / mL and store at 4°C until ready for use.

[0180] (2) Preparation of test bacterial samples

[0181] With animal Bifidobacterium lactis subspecies HOM1119 bacterial strain, be accessed in the described MRS-Cys liquid nutrient medium of sterilized embodiment 1 with the inoculum size of some culture medium total amount 1%, 37 ℃ left standstill aerobic culture 24 hours, after continuous activation two generations, 8000r / min centrifugal 10min collection thalline, use PBS washing 2 times again, and be resuspended in (0.1mol / L, pH7.2~7.4) in the PBS solution, become bacterial suspension.Bacterial suspension is placed in a water-bath, and 70 ℃ of processing are the heat-inactivated thalline after 30 minutes, in-80 ℃ of storages, standby.

[0182] (3) Culture of 3T3-L1 preadipocytes

[0183] 3T3-L1 preadipocytes (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were inoculated into complete medium 1 and passaged at a ratio of 1:3. Cells of passages 3 to 10 were selected for the experiment.

[0184] (4) Cell viability assay

[0185] Adjust the cell number to 2 × 10 cells using complete medium 2. 5 Cells / mL were cultured, and 0.1 mL of cell fluid was aspirated from each well and plated in a 96-well culture plate. The cells were cultured for 2 days and then used for later use. The cultured cells in the 96-well plate were taken, the cell culture medium was aspirated, and 0.1 mL of test bacteria of different concentrations adjusted with complete medium 2 was added to each well. The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. Cell viability was tested using the MTS cell proliferation and cytotoxicity detection kit produced by Promega. The brief steps were to add 20 μL of MTS to each well, incubate in an incubator in the dark for 1 hour, and read the absorbance at 490 nm using a microplate reader. Each treatment used cell culture medium 2 + test bacteria + MTS as a blank control. Complete medium 2 + cells + MTS was used as a negative control. The test results are shown in Figure 3.

[0186] FIG3 shows the effect of Bifidobacterium animalis subsp. lactis HOM1119 strain on the viability of 3T3-L1 preadipocytes.

[0187] As shown in Figure 3, when the concentration of dead bacteria is not greater than 1×10 8 TFU / mL, the viability of 3T3-L1 preadipocytes was higher than 100%. Therefore, the experiment selected 1×10 5 TFU / mL, 1×10 6 TFU / mL, 5×10 6 TFU / mL, 1×10 7 TFU / mL, 5×10 7 TFU / mL, 1×10 8 TFU / mL and 1×10 9 TFU / mL, and conduct cell lipid-lowering effect evaluation test.

[0188] (5) 3T3-L1 preadipocyte differentiation

[0189] The cultured cells were washed with complete medium at 2 × 10 5Cells / mL, 1 mL per well was inoculated into a 24-well culture plate coated with 0.1% gelatin, and 3T3-L1 preadipocytes were allowed to grow and cultured to 100% confluence. After 3 days of fusion, it was recorded as day 0. Cell differentiation medium 1 was added and cultured for 4 days, which was recorded as day 4. Then, cell differentiation medium 2 was changed for induction and cultured for 2 days, which was recorded as day 6. After induction of differentiation, complete medium 2 was used for 2 days of culture, which was recorded as day 8. On day 8, 3T3-L1 preadipocytes differentiated into round mature adipocytes with round triglyceride particles generated inside. The culture medium was changed every 2 days during this period. Bacterial samples were added on day 0, and bacterial samples of different concentrations with cell viability higher than 100% were added and co-cultured with 3T3-L1 cells from day 0 to day 8 of induction of differentiation.

[0190] (6) Oil Red O staining

[0191] Wash the differentiated mature adipocytes 3 times with an appropriate amount of PBS, add 0.5mL 4% paraformaldehyde to fix for 30 minutes, and wash 3 times with ultrapure water; add 0.5mL Oil Red O working solution to each well, stain for 30 minutes in the dark at 37℃, discard the staining solution, and wash 3 times with ultrapure water; place the culture plate under an inverted microscope to observe the results and take pictures. After the photo is taken, add an equal amount of 1mL 100% isopropanol to each well to extract the Oil Red O stain in the lipid droplets, let it stand at room temperature for 20 minutes, aspirate the extracted staining solution and transfer it to a clean 96-well culture plate, and measure the absorbance at 500nm. Use 100% isopropanol as a blank and measure the absorbance at 500nm. The relative lipid content is calculated as follows: Relative lipid content (%) = (OD 样品 / OD 空白 ) × 100%. Each treatment was performed in triplicate, and data were analyzed using GraphPad Prism 9 software for one-way analysis of variance (ANOVA) and Dunnett's t-test (pairwise comparisons of means between multiple experimental groups and a control group). The results of oil staining and lipid content are shown in Figures 4 and 5.

[0192] After differentiation and maturation, 3T3-L1 cells will accumulate a large number of lipid droplets in the cells. Oil red O staining can intuitively reflect the amount of lipid accumulation after fat differentiation. As shown in Figure 4, 90% of the cells in the model group differentiated into mature adipocytes on the 8th day of induction. The cells were further enlarged and rounded, and the cells were covered with red lipid droplets, which were densely distributed around the nucleus, forming a "finger-like" structure, showing the typical morphology of mature fat. Compared with the model control group, after 8 days of induction and differentiation, 1×10 7 TFU / mL, 5×10 6 TFU / mL, 1×10 6 TFU / mL and 1×10 5The four concentrations of TFU / mL can significantly reduce the number of intracellular lipid droplets, and there is a dose effect. 6 TFU / mL has the best effect. The quantitative results of oil red O dye of adipocytes (Figure 5) show that the cell-free extracts of animal Bifidobacterium lactis subspecies HOM1119 can significantly (p < 0.01) reduce the intracellular lipid content, and there is a dose-dependent effect. As the concentration of the cell-free extract of animal Bifidobacterium lactis subspecies HOM1119 increases, its ability to inhibit the differentiation of 3T3-L1 preadipocytes first increases and then decreases. When the concentration of the cell-free extract of animal Bifidobacterium lactis subspecies HOM1119 is 1×10 6 When TFU / mL was 0.1%, the relative intracellular lipid content was the lowest and the weight loss effect was the best.

[0193] (6) Determination of triglyceride (TG) content in 3T3-L1 cells

[0194] Intracellular triglyceride content was determined according to the instructions of the triglyceride (TG) detection kit produced by Nanjing Jiancheng Bioengineering Research Institute. The steps were as follows: add 2.5 μL of sample and 250 μL of working solution to each well, mix, incubate at 37°C for 10 minutes, and read the absorbance at 510 nm using a microplate reader. Protein concentration was determined using a total protein quantification kit (BCA method). The steps were as follows: add 10 μL of sample and 250 μL of working solution to each well, mix, incubate at 37°C for 10 minutes, and read the absorbance at 562 nm using a microplate reader. Calculate the glyceride content per gram of protein according to the formula.

[0195] Calculation formula:

[0196] Each treatment was repeated three times, and the data were analyzed using GraphPad Prism9 software for one-way analysis of variance (ANOVA) and Dunnett's t-test, i.e., pairwise comparisons of means between multiple experimental groups and a control group. The triglyceride (TG) content in 3T3-L1 cells is shown in Figure 6. As the concentration of the cell-free extract of Bifidobacterium animalis subsp. lactis HOM1119 increased, the triglyceride (TG) content in 3T3-L1 preadipocytes first decreased and then increased. The dead cell concentration of Bifidobacterium animalis subsp. lactis HOM1119 was 1×10 6 TFU / mL, it can significantly (p<0.01) reduce the intracellular lipid content, and the concentration is 5×10 6 TFU / mL, significantly (p<0.05) reduced the triglyceride (TG) content in 3T3-L1 cells, indicating that Bifidobacterium animalis subsp. lactis HOM1119 has the function of reducing weight.

[0197] Example 11 In vitro lipogenesis inhibition test 2 of animal Bifidobacterium lactis subspecies HOM1119

[0198] According to the method of Example 10, the strains of animal Bifidobacterium lactis subsp. lactis BH13-25 and BH13-33 were used as control strains, and the dead bacteria concentration was selected as 1×10 6 TFU / mL was used to evaluate the lipid-lowering effect of cells. The results of oil staining and lipid content are shown in Figures 7 and 8, and the triglyceride (TG) content in 3T3-L1 cells is shown in Figure 9. This shows that compared with the control strains Bifidobacterium animalis subsp. lactis BH13-25 and BH13-33, the Bifidobacterium animalis subsp. lactis HOM1119 strain can significantly (p < 0.01) reduce intracellular lipid and triglyceride (TG) content.

[0199] Example 12 Preparation process of active bacterial powder of animal Bifidobacterium lactis subspecies HOM1119 strain

[0200] (1) Culture of bacterial strains

[0201] The animal bifidobacterium lactis subspecies HOM1119 strain frozen at -80°C was inoculated in a sterile MRS-Cys liquid medium with an inoculum size of 1%, cultivated at 37°C for 16 to 24 hours, and the activated seed culture liquid was obtained by passage culture twice. The seed culture liquid was inoculated in a fermentation medium M473 with an inoculum size of 1%, and the culture was carried out at a constant temperature of 35 to 37°C under an anaerobic condition with a rotation speed of 100 rpm and a nitrogen pressure of 200 to 500 mbar. During the fermentation process, sodium hydroxide solution was added automatically to keep the pH constant at 5.0 to 5.5. The fermentation was continued for 16 to 24 hours to obtain a high-density culture liquid of the animal bifidobacterium lactis subspecies HOM1119 strain, with a viable count of 15 to 20 billion CFU / mL. Table 10 shows the formula of the fermentation medium M473.

[0202] Table 10 Fermentation medium M473 formula

[0203] (2) Preparation of freeze-drying protective agent

[0204] Sterile water is mixed with protective agent raw materials to prepare a protective agent containing 100-150 g / L modified starch, 20-50 g / L trehalose, 0.2-1 g / L vitamin C and 0.2-1 g / L glycerol.

[0205] (3) Freeze-drying

[0206] The fermented bacterial liquid of Bifidobacterium animalis subsp. lactis HOM1119 strain after culturing was centrifuged at 2-8°C and 6500 rpm for 15 min, the supernatant was discarded, the bacterial sludge was collected, and the bacterial sludge was washed 1-2 times with 0.9% sterile saline, and the washed bacterial sludge was mixed with the above protective agent so that the strain concentration in the mixed bacterial liquid reached 10 10 CFU / mL or more, freeze-dry in a freeze dryer, pre-freeze at -50 degrees Celsius for 4 hours, vacuumize, raise the temperature to -20 degrees Celsius, dry for 40 hours, raise the temperature to 35 degrees Celsius, and dry for 6 hours. After freeze-drying, crush the cake with a fine grinder to obtain the freeze-dried powder. The number of viable bacteria in the freeze-dried powder reaches 5 to 7×10 11 CFU / g.

[0207] Example 13 Effects of Bifidobacterium animalis subsp. lactis HOM1119 on blood glucose in rats with insulin resistance and glucose / lipid metabolism disorder induced by alloxan

[0208] (1) 55 healthy SPF-grade SD male rats weighing 140g-160g were bred by Beijing Huafukang Biotechnology Co., Ltd. [License No.: SCXK-(Beijing) 2019-0008]. The experimental animals were kept in the SPF-grade animal room of the Health Food Function Testing Center of the School of Applied Arts and Sciences of Beijing Union University. The experimental animal use license No.: SYXK(Beijing) 2017-0038. The experiments were conducted in two batches. The first batch of 10 rats was used to test the effect of fasting blood glucose on normal rats; the second batch of 45 rats was used to test the fasting blood glucose of rats with alloxan-induced insulin resistance and sugar / lipid metabolism disorder; glucose tolerance test; serum triglyceride and total cholesterol test; serum insulin test. The high-energy feed formula was as follows: 52.6% basic feed, 15% sucrose, 15% egg yolk powder, 10% lard, 5% casein, 1.2% cholesterol, 0.2% sodium cholate, 0.6% calcium bicarbonate, and 0.4% stone powder.

[0209] (2) Experiment on the effect of fasting blood glucose in normal rats: The rats were acclimated to a normal diet for 3 days, fasted for 4 hours, and blood was collected. After standing for 10 minutes, the serum was separated by centrifugation at 3000 r / min for 10 minutes. The blood glucose value before glucose administration (i.e., 0 hour) and the blood glucose value 0.5 and 2 hours after glucose administration at 2.5 g / kg body weight (BW) were measured using a biochemical analyzer as the basal value for this batch of animals. A normal control group (0 CFU / rat) and a HOM1119 group (5×10 10 CFU / kg BW); 5 rats in each group. At the end of the experiment, the rats in each group were fasted for 4 hours, and blood was collected from the epicanthal venous plexus. The fasting blood glucose value was detected using a biochemical analyzer, and the initial and final body weights of the rats were measured at the same time.

[0210] (3) Alloxan-induced fasting blood glucose experiment in rats with insulin resistance and glucose / lipid metabolism disorder: a blank control group, a model group, and a HOM1119 strain group were set up, with 15 rats in each group. The oral gavage dose of the HOM1119 group was 5×10 10 CFU / kg BW. The test substance was administered orally at a volume of 0.5 mL per animal, while the blank control group and the model control group were given the same volume of normal saline once daily. After each group was fed with a basic diet for one week, the model control group and the HOM1119 group were switched to a high-energy diet. After three weeks of continued feeding, the model control group and the HOM1119 group were fasted for 24 hours and given 105 mg / kg BW of alloxan by intraperitoneal injection (1 mL / 100 g body weight). After the injection, the high-energy diet was continued for 5 days. At the end of the experiment, the rats in each group were fasted for 4 hours, and blood was collected to test fasting blood glucose, glucose tolerance, serum insulin, total cholesterol, and triglyceride levels.

[0211] (4) Data processing: SPSS software was used for data processing. Independent sample t-test was used to compare the blank control group with the model control group, and the model control group with the test substance group.

[0212] Table 11. Body weight of normal rats before and after the experiment

[0213] As shown in Table 11, there was no significant difference in the body weight of normal rats before and after the experiment between the two groups (p>0.05).

[0214] Table 12. Effects of Bifidobacterium animalis subsp. lactis HOM1119 on fasting blood glucose in normal rats

[0215] As shown in Table 12, there were no significant differences in the blood glucose levels and percentages of blood glucose reduction in normal rats before the experiment and after administration of the test substance between the normal animal dose groups and the normal control group (p>0.05).

[0216] Table 13. Body weight of rats in blank and model control groups

[0217] As shown in Table 13, there was no significant difference in the body weight of rats before and after the experiment between the model control group and the blank control group (p>0.05).

[0218] Table 14. Blood glucose and glucose tolerance in rat lipid metabolism disorder model

[0219] **: There is a significant difference compared with the blank control group (p<0.01)

[0220] Table 15. Blood lipids and insulin resistance index in rat models of glucose and lipid metabolism disorders

[0221] *: There is a significant difference compared with the blank control group (p<0.05)

[0222] As shown in Tables 14 and 15, the model control group showed significant differences in the blood glucose levels and area under the blood glucose curve at 0 and 0.5 hours after glucose administration (p < 0.01). Furthermore, the blood glucose level in the model control group at 0.5 hours was ≥ 10 mmol / L, indicating that the model had established glucose metabolism disorder. The model control group also showed a significant difference in the increase in serum total cholesterol compared to the blank control group (p < 0.05), confirming that the model had established lipid metabolism disorder. The insulin resistance index was significantly decreased compared to the blank control group (p < 0.05), indicating that the insulin resistance and glucose / lipid metabolism disorder model was successful.

[0223] Table 16. Effects of Bifidobacterium animalis subsp. lactis HOM1119 on body weight in rats with insulin resistance and glucose / lipid metabolism disorder model

[0224] As shown in Table 16, after the model rats were orally administered with different probiotic powders, there was no significant difference in the body weight of the rats between each group and the model control group (p>0.05).

[0225] Table 17. Effects of Bifidobacterium animalis subsp. lactis HOM1119 on fasting blood glucose in rats with insulin resistance and lipid metabolism disorder model

[0226] *: There is a significant difference compared with the model control group (p<0.05)

[0227] As shown in Table 17 and Figure 10, oral administration of Bifidobacterium lactis subsp. HOM1119 to rats with insulin resistance and glucose / lipid metabolism disorder model showed a significant decrease in fasting blood glucose compared with the model control group (p<0.05).

[0228] Table 18. Effects of Bifidobacterium animalis subsp. lactis HOM1119 on glucose tolerance in rats with insulin resistance and lipid metabolism disorder model

[0229] As shown in Table 18 and Figure 10, after oral administration of Bifidobacterium lactis subsp. HOM1119 strain to rats with insulin resistance and glucose / lipid metabolism disorder model, the blood glucose levels at 0.5h and 2h after glucose administration were not significantly different from those of the model control group (p>0.05).

[0230] Table 19. Effects of Bifidobacterium animalis subsp. lactis HOM1119 on the area under the blood glucose curve in rats with insulin resistance and lipid metabolism disorder model

[0231] As shown in Table 19, after oral administration of Bifidobacterium animalis subsp. lactis HOM1119 to rats, the area under the blood glucose curve 0-2 hours after glucose administration was not significantly different from that of the model control group (p>0.05).

[0232] Table 20. Effects of Bifidobacterium animalis subsp. lactis HOM1119 on serum cholesterol and triglycerides in rats with insulin resistance and lipid metabolism disorder model

[0233] As shown in Table 20, after oral administration of Bifidobacterium animalis subsp. lactis HOM1119 to rats, there were no significant differences in serum total cholesterol and serum triglycerides compared with the model control group (p>0.05).

[0234] Table 21. Effects of Bifidobacterium animalis subsp. lactis HOM1119 on serum insulin resistance index in rats with insulin resistance and lipid metabolism disorder model

[0235] As shown in Table 21, after oral administration of Bifidobacterium animalis subsp. lactis HOM1119 to rats, there was no significant difference in serum insulin resistance index compared with the control group (p>0.05).

[0236] In summary, oral administration of Bifidobacterium animalis subsp. lactis HOM1119 to rats with insulin resistance and glucose / lipid metabolism disorders showed a significant decrease in fasting blood glucose (FBG) compared to the model control group (0 g / kg BW) (p < 0.05). There was no significant difference in glucose tolerance (p > 0.05), and no significant increase in blood lipids (total cholesterol and triglycerides) in the rats. The test substance, Bifidobacterium animalis subsp. lactis HOM1119, had no adverse effects on FBG or body weight in normal rats. Therefore, it is believed that Bifidobacterium animalis subsp. lactis HOM1119 has an auxiliary blood glucose-lowering function.

[0237] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

[0238] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the details shown and described herein.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis, wherein: The deposit number of the Bifidobacterium animalis subsp. lactis strain is CGMCC No.25680.

2. The Bifidobacterium animalis subsp. lactis strain according to claim 1, wherein The Bifidobacterium animalis subsp. lactis strain comprises the 16S rRNA gene represented by SEQ ID NO:

1.

3. The animal Bifidobacterium lactis subsp. lactis strain according to claim 1 or 2, which has the ability to produce high amounts of lactic acid and short-chain fatty acids.

4. The Bifidobacterium animalis subsp. lactis strain according to claim 3, wherein The short-chain fatty acid is selected from one or more of formic acid, acetic acid, propionic acid, butyric acid and isobutyric acid.

5. The Bifidobacterium animalis subsp. lactis strain according to claim 1 or 2, which has antioxidant activity.

6. The Bifidobacterium animalis subsp. lactis strain according to claim 5, wherein The animal Bifidobacterium lactis subsp. lactis strain has hydroxyl radical scavenging and DPPH radical scavenging activities, and has SOD enzyme activity.

7. A probiotic preparation that helps to lose weight or assists in lowering blood sugar, comprising the animal Bifidobacterium lactis subsp. lactis strain according to claim 1 or 2 and auxiliary materials.

8. The probiotic preparation according to claim 7, wherein The probiotic preparation is a live bacteria preparation, and the auxiliary material is selected from one or more of starch, sucrose, trehalose and glycerol.

9. The probiotic preparation according to claim 7, wherein The live bacterial preparation contains up to 5.0-7.0×10 11 CFU / g of live bacteria.

10. A method for preparing a probiotic preparation according to any one of claims 7 to 9, comprising the following steps: The animal Bifidobacterium lactis subsp. lactis strain according to claim 1 is expanded and cultured in an optimized liquid culture medium; the bacterial bodies are collected; a protective agent is added to resuspend the cells, vacuum freeze-dried, and crushed to obtain an active bacterial agent.

11. Use of the Bifidobacterium animalis subsp. lactis strain according to claim 1 or 2 in the preparation of a medicament for weight loss.

12. The use according to claim 11, wherein The Bifidobacterium animalis subsp. lactis strain is used to inhibit the differentiation of 3T3-L1 preadipocytes.

13. The use according to claim 11, wherein The Bifidobacterium animalis subsp. lactis strain is used to reduce the triglyceride (TG) content in 3T3-L1 cells.

14. The use according to claim 11, wherein The Bifidobacterium animalis subsp. lactis strain is used to inhibit fat production.

15. The use according to claim 11, wherein The animal Bifidobacterium lactis subsp. lactis strain is used for treating metabolic diseases caused by obesity.

16. Use of the Bifidobacterium animalis subsp. lactis strain according to claim 1 or 2 in the preparation of a pharmaceutical composition for preventing or treating obesity and related diseases.

17. Use of the Bifidobacterium animalis subsp. lactis strain according to claim 1 or 2 in the preparation of a medicament for assisting in lowering blood sugar.

18. The use according to claim 17, wherein The Bifidobacterium animalis subsp. lactis strain is used to inhibit the activities of dipeptidyl peptidase 4 (DPP-4) and alpha-glucosidase.

19. The use according to claim 17, wherein The Bifidobacterium animalis subsp. lactis strain is used to stimulate NCI-H716 cells to secrete GLP-1.

20. Use of the Bifidobacterium animalis subsp. lactis strain according to claim 1 or 2 in the preparation of a pharmaceutical composition for preventing or treating diabetes.

21. A food or health product comprising the Bifidobacterium animalis subsp. lactis strain according to claim 1 or 2.

22. A pharmaceutical composition comprising the Bifidobacterium animalis subsp. lactis strain according to claim 1 or 2 and a pharmaceutical excipient.

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