Application of bifidobacterium breve synergistic lactoferrin in preparation of products for regulating intestinal flora or resisting bacterial infection

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of bifidobacterium breve and lactoferrin in preparation of products for regulating intestinal flora or resisting bacterial infection. The invention provides application of Bifidobacterium breve HH079 with the preservation number of GDMCC No: 64216 and lactoferrin in preparation of a product for regulating intestinal flora or a product for resisting bacterial infection, and the Bifidobacterium breve HH079 and the lactoferrin are combined and synergistically used for resisting the bacterial infection. The composition can reverse weight loss, colon shortening, bacterial load increase and colon epithelium injury caused by bacterial infection, can significantly inhibit the level of proinflammatory cytokines, promote the expression of anti-inflammatory cytokines, can significantly increase the concentration of acetic acid and propionic acid in excrement of infected mice, can generate positive regulation on intestinal flora, and can inhibit bacterial infection. Good application prospects are shown in regulation of intestinal flora and bacterial infection resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to the application of Bifidobacterium breve in combination with lactoferrin in the preparation of products for regulating intestinal flora or resisting bacterial infections. Background Art

[0002] In the process of maintaining human health, regulating intestinal flora and resisting bacterial infections are of crucial importance. At present, there are various related products on the market. However, these products have exposed many defects in actual applications.

[0003] Common products for regulating intestinal flora include probiotic preparations, prebiotic products, and some functional foods. Probiotic preparations mostly exist in the form of capsules, powders or oral liquids, aiming to supplement beneficial microorganisms into the intestine. Prebiotic products promote the growth and reproduction of beneficial bacteria in the intestine by providing "food" for them. Some fermented functional foods, such as yogurt, fermented soy products, etc., also claim to have the effect of regulating intestinal flora. However, although prebiotic products can promote the growth of beneficial bacteria, their effects are relatively single, and some people may experience discomfort symptoms such as abdominal distension due to the intake of prebiotics. The content of beneficial components in functional foods is unstable, greatly affected by processing techniques and raw material quality, and it is difficult to precisely regulate intestinal flora.

[0004] Products for resisting bacterial infections mainly include antibiotic drugs and some natural extract products with antibacterial effects. Antibiotics are widely used in clinical treatment and can quickly and effectively inhibit or kill bacteria. Natural extract products, such as products containing allicin, tea polyphenols, etc., also claim to have antibacterial effects. The defects of antibiotics are very prominent. Long-term use is likely to lead to enhanced bacterial drug resistance, disrupt the balance of the normal human flora, and cause a series of adverse reactions, such as diarrhea, fungal infections, etc. Diarrhea, as a common symptom of digestive system infectious diseases, has complex causes and diverse effects. Long-term or severe diarrhea will lead to dehydration, with a large amount of water and electrolytes lost. Mild dehydration shows symptoms such as thirst, dry skin, and reduced urine output. Moderate dehydration presents sunken eye sockets and listlessness. Severe dehydration can even lead to shock. At the same time, it will also cause electrolyte disorders, such as hypokalemia leading to muscle weakness and arrhythmia, hyponatremia causing headache and lethargy, etc., and cause malnutrition, affecting physical growth and development and immune function. The antibacterial effects of natural extract products are often weak, difficult to cope with severe bacterial infections, and the extraction and preservation technologies of their active ingredients are not yet perfect, and the product quality is uneven.

[0005] The emergence of probiotics and glycoproteins has brought new hope for solving problems. Patent CN119424485A encapsulates tannic acid and mucin in Escherichia coli Nissle 1917 and Lactobacillus plantarum NC8 to obtain a mucin-tannic acid encapsulated probiotic preparation. This probiotic preparation has excellent resistance to the harsh gastrointestinal environment and enhances its adhesion in the intestine, thereby strengthening the colonization and growth of probiotics in the mucus layer. At the same time, it can effectively treat bacterial enteritis, especially enteritis caused by enterotoxigenic Escherichia coli (ETEC).

[0006] In terms of antibacterial, glycoproteins can bind to bacterial surface receptors, interfere with bacterial physiological activities, and can also regulate the body's immune response and enhance the phagocytic ability of immune cells against bacteria. In terms of regulating the intestinal flora, glycoproteins can create a more favorable living environment for probiotics, promote the growth and metabolism of probiotics, and the synergistic effect between the two has great potential. Provide the application of Bifidobacterium breve ( Bifidobacterium breve , abbreviated as B . breve ) HH079 in combination with lactoferrin (Lactoferrin, abbreviated as LF) in regulating the intestinal flora or anti-bacterial infection, which has extremely high application value and significance. Summary of the Invention

[0007] In view of the above deficiencies, the present invention provides the application of Bifidobacterium breve in combination with lactoferrin in the preparation of products for regulating the intestinal flora or anti-bacterial infection. The present invention provides the application of Bifidobacterium breve ( Bifidobacterium breve ) HH079 with the preservation number of GDMCC No: 64216 in combination with lactoferrin in the preparation of products for regulating the intestinal flora or anti-bacterial infection. The present invention combines Bifidobacterium breve HH079 with lactoferrin (LF) and uses them synergistically to fight against bacterial infection, which can reverse the weight loss, colon shortening, increased bacterial load, and colon epithelial damage caused by bacterial infection, can significantly inhibit the level of pro-inflammatory cytokines, promote the expression of anti-inflammatory cytokines, can significantly increase the concentrations of acetic acid and propionic acid in the feces of infected mice, and has a positive regulation on the intestinal flora, showing good application prospects in regulating the intestinal flora and anti-bacterial infection.

[0008] The technical solution of the present invention is as follows: On the one hand, the present invention provides the application of Bifidobacterium breve ( Bifidobacterium breve ) in combination with lactoferrin in the preparation of products for regulating the intestinal flora or anti-bacterial infection. The Bifidobacterium breve is Bifidobacterium breve HH079 with the preservation number of GDMCC No: 64216, which was preserved in the Guangdong Provincial Microbial Culture Collection Center on December 29, 2023 and has been disclosed in patent CN118853502B.

[0009] Specifically, in the intestinal flora regulating product or anti-bacterial infection product, the ratio of the addition amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 -10 10 CFU: 1 - 1000 mg.

[0010] Preferably, in the intestinal flora regulating product or anti-bacterial infection product, the ratio of the addition amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 -10 10 CFU: 1 - 10 mg, 10 8 -10 10 CFU: 10 - 20 mg, 10 8 -10 10 CFU: 20 - 30 mg, 10 8 -10 10 CFU: 30 - 40 mg, 10 8 -10 10 CFU: 40 - 50 mg, 10 8 -10 10 CFU: 50 - 60 mg, 10 8 -10 10 CFU: 60 - 70 mg, 10 8 -10 10 CFU: 70 - 80 mg, 10 8 -10 10 CFU: 80 - 90 mg, 10 8 -10 10 CFU: 90 - 100 mg, 10 8 -10 10 CFU: 100 - 200 mg, 10 8 -10 10 CFU: 200 - 300 mg, 10 8 -10 10 CFU: 300 - 400 mg, 10 8 -10 10 CFU: 400 - 500 mg, 10 8 -10 10 CFU: 500 - 600 mg, 10 8 -10 10 CFU: 600 - 700 mg, 10 8 -10 10 CFU: 700 - 800 mg, 10 8 -10 10 CFU: 800 - 900 mg or 10 8 -10 10CFU: 900 - 1000 mg.

[0011] Further preferably, in the product for regulating intestinal flora or the product for anti - bacterial infection, the ratio of the addition amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 -10 10 CFU: 1 mg, 10 8 -10 10 CFU: 2.5 mg, 10 8 -10 10 CFU: 10 mg or 10 8 -10 10 CFU: 100 mg.

[0012] Even further preferably, in the product for regulating intestinal flora or the product for anti - bacterial infection, the ratio of the addition amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 CFU: 1 mg, 10 8 CFU: 10 mg, 10 8 CFU: 100 mg or 10 9 CFU: 2.5 mg.

[0013] Specifically, the Bifidobacterium breve HH079 includes viable bacteria, inactivated cells, cell - wall - broken cells, secretions or metabolites of Bifidobacterium breve HH079.

[0014] Preferably, the Bifidobacterium breve HH079 is viable bacteria of Bifidobacterium breve HH079.

[0015] Specifically, the product for regulating intestinal flora includes food or health products.

[0016] Preferably, the dosage form of the food includes liquid dosage form, solid dosage form or semi - solid dosage form.

[0017] Preferably, the food includes but is not limited to: candies, soy milk, yogurt, canned food, biscuits, chocolates, pastries, cream, cheese, milk powder, formula milk powder, ice cream, jams, purees, candied fruits, preserved fruits, bread, egg rolls, protein drinks, solid drinks, lactic acid bacteria drinks, plant protein drinks, carbonated drinks, coffee or puffed foods.

[0018] Preferably, the dosage form of the health products includes but is not limited to: tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops.

[0019] Preferably, the food or health products further include nutritionally acceptable nutritional additives.

[0020] Further preferably, the nutritional additive includes, but is not limited to, one or more of dietary fiber, prebiotics, protein, lipid substances, minerals, and vitamins.

[0021] Specifically, the anti-bacterial infection product includes a food additive or a drug.

[0022] Preferably, the food additive is added to human food or animal food.

[0023] Preferably, the dosage form of the drug includes, but is not limited to: tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays or injections.

[0024] Preferably, the drug also includes one or more physiologically acceptable excipients.

[0025] Further preferably, the excipients include, but are not limited to: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, demolding agents, coating agents, flavor correctors or antioxidants.

[0026] The beneficial effects of the present invention are: The present invention combines Bifidobacterium breve HH079 with lactoferrin and synergistically uses them to combat bacterial infections. It can reverse weight loss, colon shortening, increased bacterial load, and colon epithelial damage caused by bacterial infections, can significantly inhibit the levels of pro-inflammatory factors, promote the expression of anti-inflammatory factors, can significantly increase the concentrations of acetic acid and propionic acid in the feces of infected mice, and has a positive regulation on the intestinal flora, showing good application prospects in regulating the intestinal flora and anti-bacterial infections. Description of the Drawings

[0027] Figure 1 To verify the destruction effect of the composition preparation on the biofilm in vitro; A in the figure shows the effect of the composition preparation on the biofilm biomass during the adhesion of E. coli biofilm to epithelial cells; B shows the effect of the composition preparation on the supernatant biomass during the adhesion of E. coli biofilm to epithelial cells; C shows the destruction effect of the composition preparation on the biofilm after E. coli adheres to the Caco-2 cell biofilm.

[0028] Figure 2 It is a flow chart of the in vivo experimental design plan.

[0029] Figure 3 It is a graph of the body weight changes of mice in each group; * in the figure represents a significant difference from the CR group, p <0.05.

[0030] Figure 4 For the colon length of each group of mice; ** in the figure represents a significant difference from the CR group, p <0.01.

[0031] Figure 5 For the bacterial load in mouse feces; * in the figure represents a significant difference from the CR group, p <0.05.

[0032] Figure 6 is EspB the result of gene quantitative detection; * in the figure represents a significant difference from the CR group, p <0.05.

[0033] Figure 7 For the results of pathological examination of colon tissues of each group of mice.

[0034] Figure 8 For the results of determination of serum inflammatory factors in each group of mice; A in the figure is the determination result of TNF-α; B is the determination result of IL-6; C is the determination result of IL-10; different lowercase letters in the figure represent significant differences between groups, p <0.05.

[0035] Figure 9 For the changes in mouse fecal SCFA; A in the figure is the determination result of acetic acid; B is the determination result of propionic acid; C is the determination result of butyric acid; different lowercase letters in the figure represent significant differences between groups, p <0.05.

[0036] Figure 10 For the results of α-diversity analysis.

[0037] Figure 11 For the results of principal coordinate analysis.

[0038] Figure 12 For the analysis of intestinal microbiota composition; A in the figure is the classification at the family level; B, the right figure is the classification at the genus level.

[0039] Figure 13 For the results of α-diversity analysis before infection.

[0040] Figure 14 For the results of principal coordinate analysis before infection.

[0041] Figure 15 For the analysis of intestinal microbiota composition before infection; A in the figure is the classification of microorganisms at the phylum level; B is the classification of microorganisms at the family level; C is the classification of microorganisms at the genus level (average within the group); D is the classification of microorganisms at the genus level (5 parallels in each group). Specific implementation manners

[0042] The present invention will be further clearly and completely described below through examples. The following examples are only a part of the examples of the present invention, and are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following examples are all conventional experiments unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0043] Example 1 In vitro experiment 1. Effects on biofilm biomass and supernatant biomass during the adhesion of E. coli biofilm to epithelial cells (1) 1×10 4 Caco-2 cells per well were inoculated into DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin, and incubated at 37 °C for 24 hours. Before adding the bacterial culture, the medium was replaced with DMEM without antibiotics.

[0044] (2) 1×10 5 CFU / mL E. coli O157:H7 was inoculated into the medium. At the same time, different composition preparations were inoculated into the medium and incubated at 37 °C for 8 h. The preparations were as follows: Control group: Treated with sterile PBS; HH-8 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) alone; HH-10 group: Treated with Bifidobacterium breve HH079 (1×10 10 CFU) alone; LF-1 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (1 mg, 12.5 μM); LF-10 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (10 mg, 125 μM); LF-100 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (100 mg, 1250 μM);

[0045] LF-1000 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (1000 mg, 12500 μM);

[0046] (3)Biofilm biomass in cell adhesion: After the incubation was completed, the culture supernatant was removed, and the biofilm was washed with PBS. Finally, the biofilm was resuspended in sterile PBS for serial dilution, then inoculated onto BHI agar plates, and after incubation at 37 °C for 24 hours, the measurement was carried out.

[0047] 2. Effects of E. coli on biofilm after adhesion to Caco-2 cells (1)1×10 4 Caco-2 cells per well were inoculated into DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin, and incubated at 37 °C for 24 hours. Before adding the bacterial culture, the medium was replaced with DMEM without antibiotics.

[0048] (2)1×10 5 CFU / mL E. coli O157:H7 was inoculated into the medium and incubated for 24 h to form a biofilm; (3)After biofilm formation, different composition preparations were inoculated into the medium and incubated at 37 °C for 8 h. The preparations were as follows: Control group: Treated with sterile PBS; LF-0 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) alone; LF-1 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (1 mg, 12.5 μM); LF-10 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (10 mg, 125 μM); LF-100 group: Treated with Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (100 mg, 1250 μM).

[0049] (4)Biofilm biomass in cell adhesion: After the incubation was completed, the culture supernatant was removed, and the biofilm was washed with PBS. Finally, the biofilm was resuspended in sterile PBS for serial dilution, then inoculated onto BHI agar plates, and after incubation at 37 °C for 24 hours, the measurement was carried out.

[0050] The measurement results are as Figure 1 shown. The results show that compared with the untreated group, 1×10 8After treatment with Bifidobacterium breve HH079 alone or in combination with different concentrations of LF (12.5, 125, 1250 μM), more than 95% of biofilm formation was inhibited, and the higher the concentration of LF, the worse the biofilm formation.

[0051] Example 2 Experimental grouping and administration method In this invention, neonatal C57BL / 6J mice with an average body weight of about 12.5 g were used as experimental animals. The mice were randomly divided into a blank control group (Con, n = 8), a model control group (CR, n = 8), an HH079 group (n = 8), an LF group (n = 8), an LF+HH079 group (n = 8), and a WPC+HH079 group (n = 8).

[0052] From the 10th day to the 29th day after the mice were born, the mice in the HH079 group were given 1×10 9 CFU of Bifidobacterium breve HH079 by oral gavage every day; the mice in the LF group were given 2.5 mg of lactoferrin (LF) by oral gavage every day; the mice in the LF+HH079 group were given the LF+HH079 synergistic composition (2.5 mg of lactoferrin and 1×10 9 CFU of Bifidobacterium breve HH079) by oral gavage every day; the mice in the WPC+HH079 group were given the WPC+HH079 composition (2.5 mg of whey protein (WPC) and 1×10 9 CFU of Bifidobacterium breve HH079) by oral gavage every day; the mice in the Con group and the CR group were given an equal volume of the carrier solution PBS by oral gavage every day.

[0053] On the 22nd day after the mice were born, the mice in the CR group, the HH079 group, the LF group, the LF+HH079 group, and the WPC+HH079 group were given 1×10 9 CFU of Citrobacter rodentium ( C.rodentium , CR) by gavage; the mice in the Con group were given an equal volume of the carrier solution PBS by oral gavage. The experimental design scheme process is shown in Figure 2 .

[0054] Example 2 Mouse physical signs and phenotypes The body weight changes of each group of mice were recorded at 0, 1, 3, 5, and 7 days after CR bacterial infection (i.e., on the 22nd, 23rd, 25th, 27th, and 29th days after birth), as Figure 3As shown, significant weight loss was observed in mice infected with Citrobacter rodentium, while the weight loss in mice was alleviated after treatment with LF or Bifidobacterium breve HH079. Bifidobacterium breve HH079 combined with WPC supplementation (WPC+HH079 group) also showed a tendency to promote weight gain. In contrast, infected mice treated with a combination of Bifidobacterium breve HH079 and LF (LF+HH079 group) showed significant weight gain.

[0055] Twenty-four hours after the last gavage, the mice were humanely euthanized by CO2 asphyxiation, and the colon tissues of the mice in each group were immediately collected. The colon length of the mice was measured, and the results are as Figure 4 shown. CR bacterial infection significantly shortened the colon length of mice, while intervention with LF, Bifidobacterium breve HH079, and the LF+HH079 combination promoted the reduction of colon length in mice, indicating that the combination of Bifidobacterium breve HH079 and LF had a positive effect on colon inflammation caused by CR bacterial infection.

[0056] Example 3 Bacterial load / virulence factors in mouse feces Mouse feces of the CR group, HH079 group, LF group, LF+HH079 group, and WPC+HH079 group were collected at 0, 1, 3, 5, and 7 days after CR bacterial infection (i.e., on the 22nd, 23rd, 25th, 27th, and 29th days after birth). The collected fresh mouse feces (0.1 g) were resuspended in PBS (1 mL) and vortexed to disperse the fecal precipitate. The fecal suspension was serially diluted to 10 -8 dilutions, and the number of live bacteria in the fecal samples was counted by plating on LB agar plates.

[0057] The bacterial load in feces is as Figure 5 shown. The C.rodentium count in the feces of mice in the CR group increased significantly during the entire infection period. Five days after CR bacterial infection, C.rodentium reached 10 9 CFU. Seven days after CR bacterial infection, compared with the CR group, the C.rodentium load in the feces of mice in the HH079 group, LF group, LF+HH079 group, and WPC+HH079 group was significantly reduced, indicating that treatment with LF alone, Bifidobacterium breve HH079 alone, the LF+HH079 combination, and the WPC+HH079 combination had resistance to C.rodentium infection.

[0058] For EspBFor gene quantitative detection, fecal DNA extraction and purity determination, see Example 7. The SYBR kit was used to quantify the target gene on a PCR detection system (CFX384, BioRad, USA) according to the following procedure: 95°C for 30 seconds; 40 cycles of 95°C for 10 s and 60°C for 30 s. For EspB the genes, the primers designed (as shown in Table 1) were used for quantification C.rodentium of the load, and the cycle threshold (Ct) value was normalized to the total number of bacteria.

[0059] Table 1

[0060] Note: In the table, "F" represents the forward primer; "R" represents the reverse primer.

[0061] EspB The results of gene quantitative detection are as Figure 6 shown, C.rodentium infection led to an increase in the expression of virulence factors in the intestinal flora of mice. The HH079 group and the WPC+HH079 group showed a tendency to reduce the expression of virulence factors, while the LF group and the LF+HH079 group showed a significant decrease EspB in the ability of gene expression, indicating that the intervention of Bifidobacterium breve HH079 in combination with LF has a positive effect on the expression of bacterial virulence factors after CR infection.

[0062] Example 4 Mouse colon pathology The colon tissues of each group of mice were fixed in 4% buffered paraformaldehyde solution for 48 hours, then embedded in paraffin, sectioned, dewaxed to water, and stained with H&E. The sections were observed under an optical microscope and representative photos were taken, see Figure 7 .

[0063] H&E staining revealed that after mice were infected with Citrobacter rodentium, there was a large area of inflammatory cell infiltration in the colon, accompanied by epithelial cell shedding and crypt structure damage, indicating that the mouse colon had inflammatory lesions.

[0064] The individual supplementation of LF and Bifidobacterium breve HH079 reduced the inflammatory lesions of the colon to a certain extent. Among them, the epithelial cells in the LF treatment group were arranged tightly, and the inflammatory cell infiltration in the Bifidobacterium breve HH079 intervention group was reduced. In the WPC+HH079 group, the inflammatory infiltration of the colon epithelium was partially reduced.

[0065] The LF+HH079 group showed a more complete intestinal epithelial cell structure, reduced infiltration of inflammatory granulocytes, and increased goblet cells. This indicates that the intervention of Bifidobacterium breve HH079 in combination with LF has an obvious effect on resisting Citrobacter rodentium infection in mice.

[0066] Example 5 Mouse serum inflammatory factors At 24 hours after the last gavage, mouse serum samples were immediately collected. The TNF-α, IL-6, and IL-10 indices of the mouse serum samples were measured using an ELISA kit. The operation was carried out according to the instructions provided in the ELISA kit. The results are as Figure 8 shown.

[0067] Seven days after CR bacterial infection, it was found that in the mouse serum, compared with the blank control group, C.rodentium infection significantly increased the production of serum pro-inflammatory factors (IL-6, TNF-α) and decreased the expression of serum anti-inflammatory factor (IL-10).

[0068] Supplementation with LF alone, Bifidobacterium breve HH079 alone, or the LF+HH079 synergistic composition could significantly inhibit the expression of pro-inflammatory cytokines in mouse serum and promote the expression of anti-inflammatory factors. Among them, the LF+HH079 group showed a more obvious downward trend in TNF-α, indicating that the combination of LF and Bifidobacterium breve HH079 has a more positive effect on regulating the inflammatory imbalance caused by Citrobacter rodentium infection in mice. Although the WPC+HH079 group also showed a trend of reducing pro-inflammatory cytokines and increasing anti-inflammatory factors, the overall effect of the LF+HH079 group was the best.

[0069] Example 6 Changes in mouse fecal SCFAs The SCFA content in the feces of mice on the 7th day after infection was measured using a gas chromatograph. An internal standard mixture was prepared with 4-methylvaleric acid, and 30 μL of the internal standard mixture was added to the ground fecal supernatant (120 μL), mixed evenly, and injected into the gas chromatograph. The short-chain fatty acids in the sample were separated by a capillary column (Zebron, ZB-FFAP, 30 m × 0.25 mm × 0.25 μm) and a hydrogen flame detector, and the concentration was measured in nitrogen with a flow rate of 1 mL / min. The initial temperature of the oven was maintained at 60 °C, heated to 220 °C at a rate of 20 °C / min, and held at 220 °C for 3 min. The temperatures of the syringe and detector were set at 250 °C and 280 °C, respectively. After the program ended, the peak area was recorded, and the target short-chain fatty acids in the sample were quantitatively calculated by correcting the peak area of the internal standard.

[0070] The measurement results are as Figure 9 shown. After Citrobacter rodentium infection, the concentrations of short-chain fatty acids in the feces of mice, including acetic acid and propionic acid concentrations, decreased significantly.

[0071] The HH079 group, LF group, LF+HH079 group, and WPC+HH079 group all significantly increased the acetic acid content in the feces of mice. Supplementary LF alone showed a certain increasing trend in propionic acid and butyric acid. Supplementary HH079 alone showed an increasing trend in propionic acid and significantly increased the concentration of butyric acid. Co-supplementation of WPC and HH079 promoted acetic acid, propionic acid, and butyric acid. The synergistic supplementation of LF and Bifidobacterium breve HH079 significantly increased the propionic acid concentration in the feces of infected mice, indicating that the synergistic supplementation had a positive effect on restoring the short-chain fatty acid concentration in the feces of mice.

[0072] Example 7 Changes in the intestinal flora of mice The changes in the intestinal flora in the feces of mice were measured on the 7th day after infection.

[0073] The total DNA of mouse fecal samples was extracted using the QIAGEN DNA Mini-Kit. At the same time, the DNA was quantified using Nanodrop, and the quality of DNA extraction was evaluated by 0.8% agarose gel electrophoresis. The V3-V4 region of the fecal 16S rRNA gene was amplified by PCR using the universal forward primer SEQ ID NO.6 (5′-ACTCCTACGGGAGGCAGCA-3′) and the reverse primer SEQ ID NO.7 (5′-GGACTACHVGGGTWTCTAAT-3′), and the amplified products were purified and recovered by magnetic beads. The sequencing library was prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit. The MiSeq sequencer was used for on-machine sequencing. After screening, resequencing, library and sample partitioning, and removing chimeras and barcode sequences, the obtained original high-throughput sequencing data was denoised and ASV clustered. The composition of each sample at different species taxonomic levels was explored in combination with existing databases. Based on the different OUT distributions, the Alpha diversity level of the samples and the beta diversity differences between different groups were evaluated to further measure the community structure differences between different groups.

[0074] The α-diversity (Chao1, Pielou, Shannon, and Simpson indices) showed that compared with the Con group, the richness and evenness of the fecal microbial community of the CR group of young mice were significantly reduced. Different intervention methods reversed the α-diversity index, indicating that the individual or synergistic intervention of LF / Bifidobacterium breve HH079 affected the intestinal microbial diversity of the cubs ( Figure 10 )

[0075] Principal coordinate analysis (PCoA) further showed obvious grouping of microbiota in different experimental groups, and the LF+HH079 group was closer to the control group cluster than the CR group, indicating that the synergistic supplementation of LF and Bifidobacterium breve HH079 was more beneficial to restore the diversity of intestinal microbiota after CR infection ( Figure 11 ).

[0076] Next, the composition of the gut microbiota of each group was evaluated at the taxonomic level. After infection with C. rodentium, the diversity of pathogenic bacteria in the intestines of mice increased, including Staphylococcus , Citrobacter_A and Enterococcusus_H Different interventions reduced the abundance of intestinal pathogens, among which LF supplementation reduced the abundance of Lactobacillus and significantly promoted Alistipes_A abundance (reduced inflammation, produced small amounts of acetic acid and propionic acid), supplemented with Bifidobacterium breve HH079 enrichment Bifidobacterium and Muribaculum (Potential probiotics, cross-feeding relationship with probiotics such as Bifidobacterium and Lactobacillus), synergistic supplementation of LF and Bifidobacterium breve HH079 reduced intestinal pathogens and promoted the enrichment of more beneficial bacteria ( Figure 12 ).

[0077] Example 8 Changes in intestinal flora of mice before infection 21 days after birth, feces of mice in the Con group, HH079 group, LF group and LF+HH079 group were collected. Alpha diversity analysis and principal coordinate analysis were performed according to the method described in Example 7, and the composition of the intestinal microbiota of each group was evaluated at the taxonomic level.

[0078] α-diversity analysis ( Figure 13 ) and principal coordinates analysis ( Figure 14 ) The results showed that single / synergistic supplementation of LF and Bifidobacterium breve HH079 in early life had little effect on the α-diversity composition of the mouse intestinal flora; PCOA analysis confirmed that single / synergistic supplementation of LF and Bifidobacterium breve HH079 made the composition of the mouse intestinal flora tend to be consistent.

[0079] The results of taxonomic evaluation of the intestinal microbiota composition of each group showed that LF supplementation alone promoted Alistipes_ A abundance (reduced inflammation, produced small amounts of acetic acid and propionic acid), and supplementation with Bifidobacterium breve HH079 alone enriched Bifidobacterium and UBA3282 (Lachnospiraceae) , synergistic supplementation of LF and Bifidobacterium breve HH079 promoted the enrichment of more beneficial bacteria ( Figure 15 ).

[0080] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Application of Bifidobacterium breve ( Bifidobacterium breve ) in combination with lactoferrin in the preparation of products for regulating intestinal flora or anti-bacterial infection products, characterized in that, The Bifidobacterium breve is Bifidobacterium breve HH079, and the preservation number is GDMCC No: 64216.

2. The application according to claim 1, wherein In the described intestinal flora regulating product or anti-bacterial infection product, the ratio of the addition amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 -10 10 CFU: 1 - 1000 mg.

3. The application according to claim 1, wherein The Bifidobacterium breve HH079 includes viable bacteria, inactivated cells, cell wall-broken cells, secretions or metabolites of Bifidobacterium breve HH079.

4. The application according to claim 1, characterized in that The product for regulating intestinal flora includes food or health products.

5. The application according to claim 4, characterized in that, The dosage form of the food includes liquid dosage form, solid dosage form or semi-solid dosage form.

6. The application according to claim 4, characterized in that, The dosage form of the health product includes tablets, capsules, granules, pills, gel candies, powders, oral liquids or drops.

7. The application according to claim 4, wherein The food or health product also includes nutritionally acceptable nutritional additives.

8. The application according to claim 7, wherein The nutritional additives include one or more of dietary fiber, prebiotics, proteins, lipid substances, minerals, vitamins.

9. The application according to claim 1, wherein The product for anti-bacterial infection includes food additives or drugs.

10. The application according to claim 9, wherein The food additives are added to human food or animal food.

11. The application according to claim 9, wherein The drug is used for preventing, treating or adjuvant treating diarrhea.

12. The application according to claim 9, characterized in that, The dosage form of the drug includes tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays or injections.

13. The application according to claim 9, characterized in that, The drug also includes one or more physiologically acceptable excipients.

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