Bifidobacterium breve HH079, and product thereof and use thereof

Abstract

Bifidobacterium breve HH079, and a product thereof and the use thereof. Bifidobacterium breve HH079 was deposited at the Guangdong Microbial Culture Collection Center on 29 December 2023, with the deposit number GDMCC No: 64216. Bifidobacterium breve HH079 has good tolerance to gastric acid and bile salts, and can improve the structural integrity of intestinal epithelial cells, regulate the levels of pro-inflammatory factors and anti-inflammatory factors in the body after intestinal flora imbalance, promote M2 polarization of macrophages, and enhance the immunity of the body.

Description

Bifidobacterium breve HH079, products and applications thereof, and use thereof

[0001] The present application claims priority to the Chinese patent application No. 202411299552.7, filed on September 18, 2024, and entitled "Bifidobacterium breve HH079, products and applications thereof, and use thereof". The entire content of the aforementioned application is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application belongs to the field of microbial technology, and specifically relates to Bifidobacterium breve HH079, products and applications thereof. BACKGROUND

[0003] Immunity is the body's own defense mechanism, and is the ability of the body to recognize and eliminate any foreign invaders (viruses, bacteria, etc.), process aged, damaged, dead, and degenerated cells, and recognize and process mutant cells and virus-infected cells in the body. A body with low immunity is prone to infection or cancer, and children's weak resistance can lead to poor physical and intellectual development, and also easily induce major diseases, and can cause the recurrence of health problems that already exist. Therefore, modern people pay more and more attention to the improvement of their own immunity.

[0004] The gastrointestinal tract is the most important organ in the human digestive system, and is the place where the body exchanges substances with the outside world. Nutrients necessary for human life activities are absorbed by the gastrointestinal tract, and more than 90% of toxins in the body are excreted by the intestinal tract. The saying "Nine out of ten people have stomach problems" is common, and chronic colitis, old stomach disease, and gastrointestinal ulcers and other gastrointestinal diseases seriously endanger human health. Long-term ineffective treatment or chronic treatment of gastrointestinal diseases can cause erosion, ulceration, perforation, and even cancer of the gastrointestinal mucosa. At the same time, as a common and frequently-occurring disease, gastrointestinal diseases not only cause the body to suffer, but also accumulate toxins in the body, block the source of nutrients for the body, reduce the body's immunity, and cause serious complications. At present, the enhancement of immunity is mostly achieved by diet conditioning, enhanced exercise, and zinc supplementation, but the effects of these methods are relatively slow.

[0005] With the further development of the field of microbiology, more and more experiments have proved that probiotics can improve human immune health. The human intestinal tract is a complex ecosystem, and there are a large number of microorganisms in it, which together constitute the intestinal flora. These bacteria can participate in the physiological processes of material metabolism, nutrient absorption, immune regulation, and inflammation in the human body, and play an important role.

[0006] As a kind of active microorganism beneficial to host, probiotics play many important roles in maintaining human health. First, probiotics have the function of regulating intestinal flora balance. On the one hand, probiotics can inhibit the colonization and reproduction of harmful bacteria by competing for ecological niche and nutrients in the intestinal tract. On the other hand, probiotics help to strengthen the tight junction between intestinal epithelial cells, reduce intestinal permeability, and prevent harmful substances and pathogens from invading the body. Second, probiotics can enhance immunity. Studies have shown that probiotics can activate immune cells such as macrophages and natural killer cells, improve their phagocytosis and pathogen-killing ability. By regulating the balance of helper T cells (Th1 / Th2), probiotics can reduce excessive inflammatory response and prevent the occurrence of immune-related diseases. Many clinical studies have found that individuals who take probiotics regularly have relatively stable levels of immunoglobulin in their serum, indicating that the immune system is in a good state of regulation. Third, probiotics can improve digestive system health. For diarrhea and constipation caused by infection, improper medication or diet, probiotics have a certain regulating effect.

[0007] Although some researches on probiotics in the immune system have been made, there is still a need to develop more probiotics for enhancing immunity and improving intestinal function. SUMMARY

[0008] To solve the above problems, the present application provides a Bifidobacterium breve HH079 and its products and applications. The Bifidobacterium breve HH079 provided by the present application was preserved in the Guangdong Microbial Culture Collection Center on December 29, 2023, and the preservation number is GDMCC No:64216. The Bifidobacterium breve HH079 provided by the present application has good tolerance to gastric acid and bile salts, can effectively resist the extreme environment of the upstream digestive tract, and thus successfully reaches the downstream digestive tract (such as the colon) to exert health functions. The Bifidobacterium breve HH079 can improve the structural integrity of intestinal epithelial cells, regulate the levels of pro-inflammatory and anti-inflammatory factors in the body after intestinal flora imbalance, promote the polarization of macrophages M2, and regulate intestinal flora, thereby improving the immunity of the body. The Bifidobacterium breve HH079 provided by the present application provides a good application prospect for preparing products for enhancing the immunity of the body.

[0009] To achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0010] In one aspect, the present application provides a Bifidobacterium breve, which is Bifidobacterium breve HH079 with a preservation number of GDMCC No:64216.

[0011] In particular, the Bifidobacterium breve HH079 is classified as Bifidobacterium breve, and the accession number is GDMCC No: 64216, which was preserved in the Guangdong Microbial Culture Collection Center on December 29, 2023.

[0012] In another aspect, the present application provides a culture of Bifidobacterium breve HH079, and the accession number of the Bifidobacterium breve HH079 is GDMCC No: 64216.

[0013] In particular, the culture comprises live bacteria, inactivated bacteria, broken bacteria, secretions and / or metabolites of Bifidobacterium breve HH079.

[0014] Preferably, the metabolites comprise one or more of enzymes, exopolysaccharides or bacteriocins.

[0015] Preferably, the metabolites contain immunogenic components.

[0016] In particular, the preparation method of the culture comprises inoculating Bifidobacterium breve HH079 into a culture medium.

[0017] Preferably, the culture medium comprises a solid culture medium or a liquid culture medium.

[0018] Preferably, the culture medium can provide nutritional components for Bifidobacterium breve HH079.

[0019] Further preferably, the nutritional components comprise one or more of proteins, carbohydrates, fats, probiotics, enzymes, vitamins, minerals, amino acids, and milk substitutes.

[0020] In another aspect, the present application provides a probiotic composition comprising the above-mentioned Bifidobacterium breve or culture.

[0021] In particular, the probiotic composition further comprises food-scientifically acceptable nutrients.

[0022] Preferably, the food-scientifically acceptable nutrients comprise one or more of dietary fibers, prebiotics, proteins, lipid substances, minerals, and vitamins.

[0023] In another aspect, the present application provides the use of the above-mentioned Bifidobacterium breve or culture in the preparation of a product for enhancing immunity.

[0024] In particular, the product contains 1×10 6 -1×10 12 CFU / dose of Bifidobacterium breve HH079.

[0025] Preferably, the product contains 1 x 10 8 - 1 x 10 12 CFU / dose of Bifidobacterium breve HH079.

[0026] Further preferably, the product contains 1 x 10 9 CFU / dose of Bifidobacterium breve HH079.

[0027] Preferably, the CFU / dose includes, but is not limited to, CFU / g, CFU / mL or CFU / portion.

[0028] In particular, the product includes a food, a health product or a pharmaceutical product.

[0029] Preferably, the food or health product enhances the body's immunity by enhancing the gastrointestinal immune, regulating the intestinal flora, moistening the intestines to defecate, assisting in protecting the gastrointestinal mucosa or aiding digestion.

[0030] Preferably, the food or health product enhances the body's immunity by improving the structural integrity of intestinal epithelial cells, maintaining the intestinal barrier function, regulating the intestinal flora, regulating the levels of pro-inflammatory and anti-inflammatory factors in the body and / or promoting the polarization of macrophage M2.

[0031] In another aspect, the present application provides a health product comprising the above-mentioned Bifidobacterium breve or culture.

[0032] In particular, the health product further comprises a nutritionally acceptable nutritional additive.

[0033] Preferably, the nutritional additive includes one or more of dietary fiber, prebiotics, protein, lipid material, minerals, vitamins.

[0034] In particular, the dosage form of the health product includes tablets, capsules, soft capsules, granules, pills, gummy candies, powders, oral liquids or drops.

[0035] In another aspect, the present application provides a food comprising the above-mentioned Bifidobacterium breve or culture.

[0036] In particular, the food further comprises a nutritionally acceptable nutritional additive.

[0037] Preferably, the nutritional additive includes one or more of dietary fiber, prebiotics, protein, lipid material, minerals, vitamins.

[0038] In particular, the dosage form of the food includes a liquid dosage form, a solid dosage form or a semi-solid dosage form.

[0039] Preferably, the food product comprises a tablet candy, a soy milk, a yogurt, a canned food, a biscuit, a chocolate, a cake, a butter, a cheese, a cream, a milk powder, a formula milk powder, an ice cream, a jam, a puree, a preserved fruit, a preserved vegetable, a dried fruit, a bread, a roll, a protein beverage, a solid beverage, a lactic acid bacteria beverage, a plant protein beverage, a carbonated beverage, a coffee, or an extruded food.

[0040] In particular, the food product comprises a human food product or an animal food product.

[0041] In another aspect, the present application provides a pharmaceutical product comprising the Bifidobacterium breve or the culture as described above.

[0042] In particular, the pharmaceutical product further comprises one or more physiologically acceptable adjuvants or pharmaceutically acceptable excipients.

[0043] Preferably, the physiologically acceptable adjuvant comprises, but is not limited to, erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, betanin, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate, stearic acid, calcium stearate, magnesium stearate, or dextrin.

[0044] Preferably, the pharmaceutically acceptable excipient comprises, but is not limited to, a solvent, a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifying agent, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a coloring agent, a flavoring agent, a sweetening agent, an ion exchange agent, a release agent, a coating agent, a flavoring agent, or an antioxidant.

[0045] In another aspect, the present application provides the use of the health product, the food product, or the pharmaceutical product as described above, which comprises administering an effective amount of the health product, the food product, or the pharmaceutical product to a subject.

[0046] In particular, the subject is a mammal.

[0047] Preferably, the subject is a human.

[0048] The present application has the following advantages:

[0049] (1) The Bifidobacterium breve HH079 provided by the application has good tolerance to gastric acid and bile salts, can effectively resist the extreme environment of the upstream digestive tract, and thus smoothly reaches the downstream digestive tract (such as the colon) to play a health function. It is indicated that the Bifidobacterium breve HH079 provided by the application can be used for the preparation of probiotic food, probiotic health products and gastrointestinal administration drugs.

[0050] (2) The Bifidobacterium breve HH079 provided by the application can improve the structural integrity of intestinal epithelial cells, regulate the levels of pro-inflammatory factors and anti-inflammatory factors in the body after intestinal flora imbalance, promote macrophage M2 polarization, and regulate intestinal flora, thereby improving the immunity of the body.

[0051] DEPOSIT DESCRIPTION

[0052] The name of the biological material is: Bifidobacterium breve HH079;

[0053] The classification name is: Bifidobacterium breve;

[0054] The deposit date is: December 29, 2023;

[0055] The deposit number is: GDMCC No: 64216;

[0056] The deposit unit is: Guangdong Microbial Culture Collection Center;

[0057] The deposit address is: 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100 Courtyard. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 is a gram staining diagram of Bifidobacterium breve HH079.

[0059] Figure 2 is an experimental design scheme flow.

[0060] Figure 3 is the effect of HH079 supplementation on the growth and development of mice; Figure A is the weight of mice; Figure B is the macrograph of colon; Figure C is the length of colon; ** in the figure represents p<0.01 compared with the Abx group.

[0061] Figure 4 is AB-PAS staining; Figure A is an AB-PAS staining diagram; Figure B is cup cell quantification data; ** in the figure represents p<0.01 compared with the Abx group; *** in the figure represents p<0.001 compared with the Abx group.

[0062] Figure 5 is MUC2 immunohistochemistry; Figure A is an immunohistochemical staining diagram; Figure B is Muc2 protein expression quantification data; ** in the figure represents p<0.01 compared with the Abx group

[0063] Figure 6 is the expression level of ZO-1, occludin, CLDN1 protein; A is the Western blotting figure; B is the gray value analysis; ** in the figure represents p<0.01 compared with the Abx group; * represents p<0.05 compared with the Abx group.

[0064] Figure 7 is the expression level of inflammatory factors; A-D in the figure are ELISA determination of serum inflammatory factor expression; E is the mRNA expression of inflammatory cytokines; ** in the figure represents p<0.01 compared with the Abx group; * represents p<0.05 compared with the Abx group.

[0065] Figure 8 is the macrophage determination result; A-B in the figure are flow cytometry determination of macrophage number; C is the mRNA expression of M1, M2 macrophage markers; ** in the figure represents p<0.01 compared with the Abx group; * represents p<0.05 compared with the Abx group.

[0066] Figure 9 is the effect of Bifidobacterium breve HH079 on the colon transcriptome after antibiotic exposure; A in the figure is PCA analysis; B is the number of up-regulated and down-regulated differentially expressed genes compared with the antibiotic exposure group; C is the volcano plot of differentially expressed genes in the Con group and the Abx group; D is the volcano plot of differentially expressed genes in the HH079 group and the Abx group.

[0067] Figure 10 is the expression of barrier function genes; ** in the figure represents p<0.01 compared with the Abx group; * represents p<0.05 compared with the Abx group.

[0068] Figure 11 is the expression of macrophage polarization-related genes; A is the expression of M1 macrophage-related genes; B is the expression of M2 macrophage-related genes.

[0069] Figure 12 is the pathway enrichment of differentially expressed genes in the HH079 group and the Abx group.

[0070] Figure 13 is the TLR4 / NF-κB determination; A in the figure is the WB figure of the TLR4 / NF-κB signaling pathway; B is the expression level of the TLR4 / NF-κB signaling pathway; ** in the figure represents p<0.01 compared with the Abx group; * represents p<0.05 compared with the Abx group.

[0071] Figure 14 is the regulation of intestinal microbial composition by Bifidobacterium breve HH079; A in the figure is the alpha-diversity index; B is the evaluation of beta-diversity by PCoA analysis, and each figure represents a single sample; C is the relative abundance of intestinal flora at the genus classification level of flora; ** in the figure represents p<0.01 compared with the Abx group; * represents p<0.05 compared with the Abx group. DETAILED DESCRIPTION

[0072] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following specific embodiments are further described to clarify the present application. However, the following embodiments are only preferred embodiments of the present application, not all. Based on the embodiments in the embodiments, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following embodiments, if not otherwise specified, the operation methods used are conventional operation methods, the equipment used is conventional equipment, and the equipment materials used in each embodiment are the same.

[0073] 1. Experimental reagents

[0074] All experimental reagents of the present application are shown in Table 1.

[0075] Table 1 Experimental reagents

[0076] 2. Experimental animals

[0077] The present application selects SPF healthy C57BL / 6J female mice for experiments.

[0078] 3. Experimental strains

[0079] The strain used in the present application is Bifidobacterium breve, classified as Bifidobacterium breve, with the preservation number GDMCC No:64216, preserved in Guangdong Microbial Culture Collection Center on December 29, 2023.

[0080] 4. Statistical analysis

[0081] The present experiment uses statistical software IBM SPSS 26.0, and the results are represented by mean ± standard deviation (x ± SD), and single factor variance analysis method is used for comparison between groups. p<0.05, considered to have significant difference.

[0082] Example 1 Gram staining

[0083] The Bifidobacterium breve HH079 used in the present application is subjected to Gram staining using a Gram staining kit, and the staining steps are as follows:

[0084] (1) Smear: Take the bacteria to be tested, smear it into a thin layer in the center of the slide or add a little sterile water on the slide, mix the bacteria and water evenly, and smear it into a thin layer.

[0085] (2) Dry: After smearing, dry naturally at room temperature, or slightly warm on an alcohol lamp to dry quickly.

[0086] (3) Fixation: Hold the slide by one end, with the specimen facing up, and move it quickly back and forth 3-5 times outside the flame of an alcohol lamp, each time for 1 s, the temperature should not be too high to prevent protein denaturation of the bacteria, and then stain after cooling. Alternatively, methanol or ethanol can be used for fixation.

[0087] (4) Primary staining: Add crystal violet staining solution dropwise and stain for 1-2 min, and then rinse with water to remove the staining solution.

[0088] (5) Mordanting: Add Gram iodine solution dropwise to cover the slide, and then place it at room temperature for about 1-2 min, and then wash with water.

[0089] (6) Decolorization: Add decolorizing solution dropwise, shake for 10-30 s, until the decolorizing solution flowing down is not purple, immediately rinse the decolorizing solution with water to terminate the reaction.

[0090] (7) Counterstaining: Add counterstaining solution dropwise and stain for 30-60 s, and then wash with water.

[0091] (8) Drying and microscopic examination: Place it under a microscope for observation.

[0092] The above experiment shows that the bacteria have strict anaerobic requirements for oxygen, the bacteria are gram-positive bacilli, the cells are rod-shaped or bifurcated rod-shaped, and no spores are present. It is preliminarily determined that the strain of the application is Bifidobacterium breve. As shown in Figure 1, the characteristics of Bifidobacterium breve HH079 in morphology and general properties are as follows: it is an anaerobic bacteria and a gram-positive bacteria; the colonies grown on MRS medium are white and solid round, and the bacterial morphology is short rod-shaped with bifurcated branches at both ends (Y-shaped or V-shaped).

[0093] Example 2: Acid and bile salt tolerance experiment of Bifidobacterium breve HH079

[0094] 1. Preparation of test bacterial suspension

[0095] Bifidobacterium breve HH079 was recovered by MRS solid medium, and after 48 h of culture, a single colony was picked and cultured in MRS broth, and then subcultured twice to adjust the concentration to 10 8 -10 9 CFU / mL for standby.

[0096] 2. Preparation of simulated gastric acid and bile salt culture solution

[0097] Preparation of simulated gastric acid culture solution: prepare MRS broth culture solution, sterilize, and then adjust the pH value to 3.0 with 1 mol / L HCL to prepare simulated gastric acid culture solution.

[0098] Preparation of simulated bile salt culture solution: MRS broth culture solution was prepared, sterilized, and then 0.1% pig bile salt was added to obtain a concentration of 0.1%, and the pH value was adjusted to 8.0 with 1 mol / L NaOH, and then sterilized by filtering through a 0.22 μm microporous filter to obtain the bile salt culture solution.

[0099] 3. Gastric acid and bile salt tolerance test

[0100] Short Bifidobacterium HH079 acid tolerance test: 0.9 mL of simulated gastric acid culture solution was dispensed into an EP tube, and then 0.1 mL of the prepared bacterial suspension was added, vortexed, and then placed in a 37°C anaerobic incubator for 2 hours. The test was performed at 0h and 2h, and three parallel samples were set at each time point. MRS broth without pH adjustment was used as a control to observe the growth of the bacteria and exclude bacterial death caused by other factors.

[0101] Short Bifidobacterium HH079 bile salt tolerance test: 0.9 mL of bile salt culture solution was dispensed into an EP tube, and then 0.1 mL of the prepared bacterial suspension was added, vortexed, and then placed in a 37°C anaerobic incubator for 24 hours. The test was performed at 0h and 24h, and three parallel samples were set at each time point. MRS broth without bile salt was used as a control to observe the growth of the bacteria and exclude bacterial death caused by other factors.

[0102] After the above culture was completed, it was immediately diluted by 10 times in gradient, and MRS plates were inoculated with appropriate dilution points, and then incubated anaerobically at 37°C for 24-48 hours for counting. The counting result of the 0-hour dilution inoculation was used as the initial bacterial concentration. The results were compared in terms of survival rate. The calculation formula is as follows:

[0103] Survival rate (%) = concentration of viable bacteria after culture (CFU / mL) / concentration of viable bacteria at 0h (CFU / mL) x 100%

[0104] The experimental results show that the survival rate of the short Bifidobacterium HH079 after 2 hours of digestion in the simulated gastric acid culture solution with pH = 3 is 84.5%, and the survival rate after 24 hours of culture in the bile salt culture solution is 84.9%. Therefore, it can be known that the short Bifidobacterium HH079 has good tolerance to gastric acid and bile salt, can effectively resist the extreme environment of the upstream digestive tract, and thus successfully reaches the downstream digestive tract (such as the colon) to exert health functions. It is worth noting that the short Bifidobacterium HH079 provided by the present application is a non-frozen bacterial suspension. In this case, the short Bifidobacterium HH079 has good tolerance to gastric acid and bile salt. If it is prepared into a commercial freeze-dried bacterial powder, the short Bifidobacterium HH079 has better tolerance to gastric acid and bile salt. Therefore, the short Bifidobacterium HH079 meets the basic requirements as a probiotic.

[0105] Experimental grouping and administration of test samples in Example 3

[0106] The C57BL / 6J mother mice were continuously given the drinking water supplemented with antibiotics (ampicillin 1 g / L, neomycin sulfate 0.5 g / L) to lactate the offspring until weaning, to establish the mouse model of early life intestinal dysbiosis. At the same time, the offspring mice without antibiotic drinking water were taken as the control group (Con, n = 8). The mice born for 10 days were randomly divided into two groups, the Bifidobacterium breve HH079 group (HH079, n = 10) and the model group (Abx, n = 8) after antibiotic exposure; the mice in the HH079 group were given 1 x 10 9 CFU Bifidobacterium breve HH079 was orally gavaged, and the mice in the Abx group were orally gavaged with the same volume of carrier solution PBS until weaning. At the same time, the mice in the control group were orally gavaged with the same volume of carrier solution PBS until weaning. Eight hours after the last gavage, the mice were humanely euthanized by CO2 asphyxiation, and serum and tissues were immediately harvested. The intestinal contents were squeezed into sterile vials, immediately transferred to -80°C storage after rapid freezing in liquid nitrogen. The changes of intestinal barrier and immune cells in the colon tissue of mice were determined. The experimental design scheme is shown in Figure 2.

[0107] Example 4 Effect of Bifidobacterium breve HH079 supplementation on the growth and development of mice

[0108] 4.1 Effect of Bifidobacterium breve HH079 supplementation on the body weight and colon length of mice

[0109] After the last gavage, the body weight data of the mice in the control (Con) group, the model (Abx) group, and the Bifidobacterium breve HH079 (HH079) group were measured and recorded, and after the mice were humanely euthanized by CO2 asphyxiation, the intestinal tissues of the mice were collected, and the colon length was measured and recorded.

[0110] The determination results are shown in Figure 3. Compared with the control group, antibiotic intervention significantly reduced the body weight and colon length of the mice (p < 0.05), and the average body weight and colon length of the mice after Bifidobacterium breve HH079 intervention were significantly increased (p < 0.05). In summary, early life antibiotic exposure inhibited the growth and development of mice, and Bifidobacterium breve HH079 could reverse these changes. These results suggest that the growth retardation caused by early life antibiotic exposure may be related to its disturbance of the balance of intestinal microbiota.

[0111] 4.2 AB-PAS staining to detect the number of goblet cells

[0112] The colon tissue was fixed in 4% buffered paraformaldehyde solution for 48 hours, then embedded in paraffin, and the sections were deparaffinated to water. Alcian blue staining solution was immersed or dropped for 10 min, and then washed slightly with water. Oxidation with 0.5% aqueous solution of high iodine for 10 min. Washed with running water for several minutes and then washed twice with distilled water. Schiff reagent was immersed or dropped in the dark for 30 min. Washed with running water for 10 min. Nuclei were counterstained with hematoxylin for about 1 min, differentiated with hydrochloric alcohol, and then blued with ammonia water. Dehydrated with 95% alcohol (5 min), 95% alcohol (5 min), anhydrous ethanol (5 min), and anhydrous ethanol (5 min), transparentized with xylene, and then sealed with neutral resin. The sections were observed under an optical microscope and representative photographs were taken to observe the number of goblet cells.

[0113] It was found by AB-PAS staining (Figure 4) that, compared with the healthy control group, the number of viscous secretions in the model group was significantly reduced, and the number of goblet cells was significantly reduced. On the contrary, after being treated with Bifidobacterium breve HH079, the secretion of mucus protein secreted by colon epithelial cells was increased. Therefore, to some extent, Bifidobacterium breve HH079 effectively protects the goblet cells in the intestinal tract and stimulates the secretion of viscous protein substances.

[0114] 4.3 Immunohistochemical experiment

[0115] After the colon tissue was fixed in 4% buffered paraformaldehyde solution for 48 hours, it was embedded in paraffin, and the sections were deparaffinated to water. Immunofluorescence staining was performed on the Muc2 protein in the colon tissue.

[0116] The immunohistochemical results are shown in Figure 5. Compared with the Con group, antibiotic exposure (Abx group) reduced the expression of Muc2 protein. Bifidobacterium breve HH079 promoted the expression of Muc2 protein in the colon and protected the integrity of the intestinal mucus barrier.

[0117] 4.4 Western blot experiment

[0118] Western blot experiment was performed on the mouse colon tissue for ZO-1, occludin, and CLDN1: The ileum was cut into 2-3 cm and ground into powder with liquid nitrogen. The ratio of tissue to buffer was 1:10, and the protein loading buffer was added for dissolution. The dissolution solution was boiled for 5 min, and then the supernatant was taken after cooling to room temperature. The prepared 10% protein gel was loaded, and then the membrane was transferred. The 10% non-fat milk was blocked for 30 min, the first antibody was combined for 30 min, the second antibody was combined for 30 min, DAB was developed, and then the photograph was recorded.

[0119] The integrity of the colon epithelial structure was evaluated by measuring the expression of ZO-1, occludin, CLDN1 protein. Compared with the Con group, the Abx group showed lower expression levels of ZO-1, occludin, CLDN1 genes and proteins, indicating that early-life antibiotic exposure can lead to the destruction of the intestinal epithelial cell structure and increase the intestinal permeability. However, the relative expression levels of ZO-1, occludin, CLDN1 in the HH079 group were significantly increased compared with the Abx group (p<0.05). This indicates that probiotic supplementation has a positive effect on intestinal damage caused by early-life antibiotic exposure (Figure 6).

[0120] Example 5 Effect of Bifidobacterium breve HH079 supplementation on inflammatory response in mice

[0121] 5.1 Effect of Bifidobacterium breve HH079 supplementation on inflammatory factors

[0122] 5.1.1 Elisa determination of inflammatory factor levels

[0123] The TNF-a, IL-1b, LBP, IL-10 indicators in the mouse serum samples were determined using an ELISA kit. The operation was performed according to the instructions provided in the ELISA kit.

[0124] 5.1.2 mRNA determination of inflammatory factor levels

[0125] (1) Total RNA was extracted from the tissue using an RNA rapid extraction kit, and according to the instructions of the cDNA synthesis kit, the RNA was reverse transcribed into cDNA.

[0126] (2) qPCR amplification: according to the manufacturer's instructions, RNA reverse transcription reaction was performed using a synthesis kit. The following reaction system was configured under ice bath conditions: 8 μL of RNA template (about 0.2 μg of total RNA), 15 μL of 2x IM-MLV RT Mix, and 7.5 μL of RNase-free water. Then set the PCR program parameters and run the reverse transcription reaction: 25°C for 5 min, 42°C for 30 min, 85°C for 5 min. After the reaction, the concentration of the product cDNA was determined using a Nanodrop ND-2000 spectrophotometer, the OD values at 260 nm and 280 nm were read, and the OD260 / OD280 ratio was calculated. If the ratio is between 1.8 and 2.2, it indicates high purity. At this time, the cDNA stock solution can be diluted and the concentration adjusted to <150 ng / μL, and stored at -80°C after aliquoting for subsequent experiments.

[0127] (3) GAPDH was used as an internal reference, and the mRNA relative expression amount of each target gene was calculated according to formula 2 -ΔΔCT Table 2 is the primer sequence used in this experiment.

[0128] Table 2 Primer sequences

[0129] The results are shown in Figure 7. In the Abx group, the secretion expression of proinflammatory cytokines (TNF-a, IL-1 b and IL-6) was significantly increased compared with the control group, while the level of anti-inflammatory cytokine (IL-10) was significantly decreased compared with the control group. In contrast, the levels of TNF-a and LBP in the HH079 group were significantly lower than those in the Abx group. At the mRNA level, the HH079 group significantly down-regulated the level of IL-1 b and up-regulated the level of IL-10 compared with the Abx group. LBP is an acute inflammatory protein produced by the body when the body is stimulated by lipopolysaccharide, which can cause host inflammatory response and immune disorders. The above results show that the supplementation of B. breve HH079 can reduce and increase the levels of proinflammatory and anti-inflammatory cytokines in the body after Abx-induced intestinal dysbiosis. Therefore, the signaling pathways and metabolites of B. breve HH079 are further studied.

[0130] 5.2 Effect of B. breve HH079 supplementation on macrophages

[0131] 5.2.1 Flow cytometry for determining the number of macrophages

[0132] To further confirm the effect of B. breve HH079 supplementation on macrophages, flow cytometry was used to determine the number of macrophages.

[0133] 5.2.2 mRNA expression of M1 and M2 macrophage markers

[0134] qRT-PCR for detecting gene expression was determined according to 5.1.2, and the primer sequences are shown in Table 3.

[0135] Table 3 Primer sequences

[0136] The results of flow cytometry determination are shown in Figure 8A, where antibiotic treatment significantly affected the balance of macrophages in the colon, while B. breve HH079 intervention significantly reduced the number of macrophages. The type of macrophages was further detected by RT-qPCR, and CD86 is a costimulatory molecule commonly considered as one of the markers of M1 macrophages (proinflammatory macrophages). As shown in Figures 8B-8D, early-life antibiotic exposure significantly stimulated the expression of CD86 gene, that is, antibiotics promoted macrophages to be dominated by M1 proinflammatory macrophages. In contrast, B. breve HH079 promoted the expression of CD206 in the colon tissue and significantly inhibited the expression of CD86. HH079 can reverse the polarization of antibiotic-stimulated macrophages from M1 to M2.

[0137] Example 6 Impact of B. breve HH079 on colon transcriptome after antibiotic exposure

[0138] 6.1 Transcriptome analysis

[0139] Total RNA was extracted from colon tissue and RNA library was constructed. Quantitative library was analyzed by single-end sequencing on Illumina gene analyzer. Data analysis was performed using R software. Raw data of counts and FPKM were available (https: / / bioinfogo.org / ResKsumo / data). Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on differentially expressed genes using the "clusterProfiler", "GOplot" packages in R software, with a standard differential fold change less than or equal to 2 and a P value less than 0.05.

[0140] 6.2 WB assay TLR4 / NF-κB signaling pathway

[0141] WB assay was performed on TLR4 / NF-κB signaling pathway according to the method described in 4.4 Western blotting experiment in Example 4.

[0142] 6.3 Assay results

[0143] The impact of B. breve HH079 supplementation on antibiotic exposure was studied based on RNAseq transcriptome analysis. As shown in the tight clustering of each group from each other and different from the control group in the PCA plot (A in Figure 9), this indicates that the transcription results between different treatments are not the same. Volcano plot found that antibiotic exposure significantly down-regulated 688 genes, and B. breve HH079 supplementation up-regulated 605 genes, indicating that B. breve HH079 induced transcriptome changes were not identical to Abx.

[0144] Through KEGG enrichment analysis, compared with genes after antibiotic exposure, B. breve HH079 supplementation can inhibit the activation of NFkb signaling pathway. Among the significantly related genes, the cytokine-cytokine receptor interaction, Th17 chemokine, extracellular matrix receptor interaction and focal adhesion related signaling pathways were significantly enriched (FDR < 0.001). These indicate that early antibiotic exposure will cause tissue cell damage and cause inflammatory signaling pathway expression.

[0145] For intestinal epithelial cell proliferation, protective protein peptide secretion genes, B. breve HH079 significantly promoted the up-regulation of IgA, Reg3b, Reg3g and Lyz2 genes, among which secretory IgA is the main component of the body's mucosal defense system and is the first line of defense against pathogen invasion. This shows that early life antibiotic exposure can lead to impaired intestinal epithelial cell integrity and function, and the supplementation of B. breve HH079 reverses this situation, as shown in Figure 10.

[0146] The expression of macrophage polarization-related genes is shown in Figure 11, Abx up-regulated multiple M1 macrophage marker genes such as IL6ra, Nos1, IL1, Cxcl12, etc., while HH079 treatment reversed the expression of these pro-inflammatory M1 genes. Similarly, HH079 restored the expression of M2 macrophage markers suppressed by early life antibiotic intervention to normal levels. These findings suggest that B. breve HH079 can promote M1 to M2 polarization in macrophages.

[0147] In combination with the transcriptome findings, B. breve HH079 may modulate the expression of the Toll-NF-κB signaling pathway, which is the main signaling pathway for regulating inflammatory changes (Figure 12). The protein expression of the Toll-NF-κB signaling pathway after different interventions was determined by WB method. TLR4, as the main receptor of LPS, TLR4 was significantly expressed in the Abx group. At the same time, downstream proteins Myd88 and P65 are activated after receiving signals. However, B. breve HH079 treatment significantly attenuated the expression of Myd88 and P65, indicating that B. breve HH079 can promote the transformation of M1 type to M2 type by regulating the activation of NF-κB signaling pathway in epithelial tissue cells, further inhibiting the occurrence of inflammation (Figure 13).

[0148] Example 7 Effect of B. breve HH079 on intestinal flora

[0149] Total DNA of mouse fecal samples was extracted using QIAGEN DNA Mini-Kit. Meanwhile, DNA was quantified by Nanodrop and the quality of DNA extraction was evaluated by 0.8% agarose gel electrophoresis. The V3-V4 region of fecal 16S rRNA gene was amplified by PCR using universal forward primer SEQ ID NO. 15 (5'-ACTCCTACGGGAGGCAGCA-3') and reverse primer SEQ ID NO. 16 (5'-GGACTACHVGGGTWTCTAAT-3'), and the amplification product was recovered by magnetic bead purification. Sequencing library was prepared using TruSeq Nano DNA LT Library Prep Kit of Illumina. The raw high-throughput sequencing data obtained was screened, supplemented, and subjected to library and sample division, and chimeras, barcode sequences were removed, followed by denoising and ASV clustering. The composition of each sample at different taxonomic levels of species was explored in combination with existing databases. Based on different OUT distribution, the Alpha diversity level of the sample and the beta diversity difference between different groups were evaluated to further measure the difference in community structure between different groups.

[0150] The results of the determination are shown in Figure 14. Compared with the control group, antibiotic treatment significantly reduced the observed species, Chao 1, Shannon and Simpson indices (A in Figure 14, p<0.05), indicating that early-life antibiotic exposure reduced the species richness and evenness of the mouse gut microbiota. In contrast, the addition of B. breve HH079 restored the Chao 1, Observed species, Shannon and Simpson indices by 12.17%, 12.51%, 59.51% and 92.78%, respectively, relative to the Abx group. Principal coordinate analysis (PCoA) showed that the gut microbiota clusters in the Abx group were further away from those in the B. breve HH079 group compared with the Con group (B in Figure 14).

[0151] Next, the intestinal microbiota composition of the three groups was evaluated at the taxonomic level. It was found from Figure C that at the genus taxonomic level, the relative abundance of Parabacteroides and Prevotella dominated in the Con group, while the relative abundance of Pseudomonadaceae_Pseudomonas and Morganella was significantly enriched in the Abx group. A novel genotoxin produced by Morganella, a type of Enterobacteriaceae bacteria, was reported to cause colorectal cancer risk, which is considered a marker of intestinal dysbiosis. A significant shift in the microbiota to a community dominated by Morganella and Pseudomonadaceae_Pseudomonas was observed in the Bifidobacterium breve HH079 group, accompanied by an increase in Bifidobacterium, Bacteroides and Proteus. In summary, the significantly changed intestinal bacterial community after antibiotic exposure can bring the risk of inflammation, while the enrichment of more Bifidobacterium in Bifidobacterium breve HH079 helps to restore the intestinal flora environment.

[0152] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application. It should be noted that any equivalent implementation or change made without departing from the present application shall be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims.

Claims

1. Bifidobacterium breve characterized in that, The short bifidobacterium is short bifidobacterium HH079, and the preservation number is GDMCC No: 64216.

2. A culture of Bifidobacterium breve HH079, characterized in that, The preservation number of the short bifidobacterium HH079 is GDMCC No: 64216.

3. The culture of claim 2, wherein, The culture includes live bacteria, inactivated bacteria, broken bacteria, secretions and / or metabolites of the short bifidobacterium HH079.

4. The culture of claim 3, wherein, The metabolites include one or more of enzymes, extracellular polysaccharides or bacteriocins.

5. The culture of claim 3, wherein, The metabolites contain immunogenic components.

6. The culture of claim 2, wherein, The preparation method of the culture includes inoculating the short bifidobacterium HH079 into a culture medium.

7. The culture of claim 6, wherein, The culture medium can provide nutritional components for the short bifidobacterium HH079.

8. The culture of claim 7, wherein, The nutritional components include one or more of proteins, carbohydrates, fats, probiotics, enzymes, vitamins, minerals, amino acids, milk substitutes.

9. A probiotic composition, characterized in that, The probiotic composition contains the short bifidobacterium of claim 1 or the culture of any one of claims 2-8.

10. The probiotic composition according to claim 9, characterized in that, The probiotic composition further includes food-scientifically acceptable nutrients.

11. The probiotic composition according to claim 10, characterized in that, The food-scientifically acceptable nutrients include one or more of dietary fiber, protein, lipid material, mineral, vitamin.

12. Use of the short bifidobacterium of claim 1 or the culture of any one of claims 2-8 in the preparation of a product for enhancing immunity of an organism.

13. Use according to claim 12, characterized in that, The enhanced immunity of the organism is enhanced immunity of a mammal.

14. Use according to claim 13, characterized in that, The mammal is a human.

15. The use according to claim 12, characterized in that, The product contains 1 x 10 6 -1 x 10 12 CFU / dose of Bifidobacterium breve HH079.

16. The use according to claim 12, characterized in that, The product includes food, health product or pharmaceutical product.

17. Use according to claim 16, characterized in that, The food or health product enhances immunity of an organism by enhancing gastrointestinal immunity, regulating intestinal flora, moistening the intestines, protecting the gastrointestinal mucosa or aiding digestion.

18. The use according to claim 17, characterized in that, The short bifidobacterium HH079 in the food or health product enhances immunity of an organism by improving the structural integrity of intestinal epithelial cells, maintaining the intestinal barrier function, regulating intestinal flora, regulating the levels of pro-inflammatory and anti-inflammatory factors and / or promoting the polarization of macrophage M2.

19. A health product containing the short bifidobacterium of claim 1 or the culture of any one of claims 2-8.

20. The health care product of claim 19, wherein, The health product further includes nutritionally acceptable nutrients.

21. The health care product of claim 20, wherein, The nutrients include one or more of dietary fiber, protein, lipid material, mineral, vitamin.

22. The health care product of claim 19, wherein, The dosage form of the health product includes tablets, capsules, granules, pills, gummy candies, powders, oral liquids or drops.

23. A food containing the short bifidobacterium of claim 1 or the culture of any one of claims 2-8.

24. The food product of claim 23, wherein, The dosage form of the food includes liquid, solid or semi-solid dosage form.

25. The food product of claim 24, wherein, The food includes candies, soy milk, yogurt, cans, biscuits, chocolates, pastries, butter, cheese, cream cheese, milk powder, formula milk powder, ice cream, jam, puree, preserved fruits, bread, egg rolls, protein drinks, solid beverages, lactic acid bacteria beverages, carbonated beverages, coffee or puffed foods.

26. The food product of claim 23, wherein, The food includes human food or pet food.

27. A pharmaceutical product containing the short bifidobacterium of claim 1 or the culture of any one of claims 2-8.

28. The pharmaceutical product according to claim 27, characterized in that The pharmaceutical product also includes one or more physiologically acceptable adjuvants. The pharmaceutical product also includes one or more physiologically acceptable adjuvants.

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