Application of Bifidobacterium breve in synergistic effect with lactoferrin in the preparation of products for regulating intestinal flora or preventing bacterial infection

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the application of Bifidobacterium breve in cooperating with lactoferrin in the preparation of products for regulating intestinal flora or resisting bacterial infection. The present invention provides Bifidobacterium breve ( Bifidobacterium breve ) The application of HH079 in conjunction with lactoferrin in the preparation of products for regulating intestinal flora or anti-bacterial infection products. The present invention combines Bifidobacterium breve HH079 with lactoferrin, which are synergistically used to fight bacterial infection. It can reverse the weight loss, colon shortening, increased bacterial load and colon epithelial damage caused by bacterial infection, significantly inhibit the level of pro-inflammatory cytokines, promote the expression of anti-inflammatory cytokines, significantly increase the concentration of acetic acid and propionic acid in the feces of infected mice, and produce positive regulation on intestinal flora, showing good application prospects in regulating intestinal flora and anti-bacterial infection.

Description

Technical Field

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

[0002] In the process of maintaining human health, regulating intestinal flora and resisting bacterial infections are crucial. Currently, there are many related products on the market, however, these products have exposed many defects in practical applications.

[0003] Common products for regulating intestinal flora include probiotic preparations, prebiotic products, and some functional foods. Probiotic preparations are mostly in the form of capsules, powders, or oral liquids, and are intended to supplement beneficial microorganisms in the intestines. Prebiotic products promote the growth and reproduction of beneficial bacteria in the intestines by providing "food" for them. Some fermented functional foods, such as yogurt and fermented soy products, 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 simple, and some people may experience discomfort symptoms such as bloating due to the intake of prebiotics. The content of beneficial ingredients in functional foods is unstable and is greatly affected by the processing technology and the quality of raw materials, making it difficult to accurately regulate the intestinal flora.

[0004] Antibacterial products primarily include antibiotics and some natural extracts with antibacterial properties. Antibiotics are widely used in clinical treatments and can quickly and effectively inhibit or kill bacteria. Natural extracts, such as those containing ingredients like allicin and tea polyphenols, also claim to have antibacterial properties. However, antibiotics have significant drawbacks. Long-term use can easily lead to bacterial resistance, disrupt the body's normal bacterial balance, and trigger a series of adverse reactions, such as diarrhea and fungal infections. Diarrhea, a common symptom of digestive system infections, has complex causes and diverse effects. Prolonged or severe diarrhea can lead to dehydration, resulting in significant water and electrolyte loss. Mild dehydration manifests as thirst, dry skin, and decreased urine output, while moderate dehydration can cause sunken eyes and lethargy. Severe dehydration can even lead to shock. It can also cause electrolyte imbalances, such as hypokalemia leading to muscle weakness and cardiac arrhythmias, and hyponatremia leading to headaches and lethargy. It can also lead to malnutrition, impacting growth and immune function. The antibacterial effect of natural extract products is often weak and cannot cope with serious bacterial infections. In addition, the extraction and preservation technology of their active ingredients is still imperfect, and the product quality is uneven.

[0005] The emergence of probiotics and glycoproteins has brought new hope for resolving this problem. Patent CN119424485A encapsulates tannins and mucins in Escherichia coli Nissle 1917 and Lactobacillus plantarum NC8, resulting in a mucin-tannin encapsulated probiotic formulation. This probiotic formulation exhibits excellent resistance to the harsh gastrointestinal environment and enhances its adhesion to the intestine, thereby strengthening the colonization and growth of the probiotics within the mucus layer. Furthermore, it is effective in treating bacterial enteritis, particularly that caused by enterotoxigenic Escherichia coli (ETEC).

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

[0007] In view of the above shortcomings, the present invention provides the use of Bifidobacterium breve in conjunction with lactoferrin in the preparation of products for regulating intestinal flora or resisting bacterial infection. The present invention provides Bifidobacterium breve ( Bifidobacterium breve ) The application of HH079 in conjunction with lactoferrin in the preparation of products for regulating intestinal flora or anti-bacterial infection products. The present invention combines Bifidobacterium breve HH079 with lactoferrin (LF) to synergistically fight bacterial infection, which can reverse weight loss, colon shortening, increased bacterial load and colon epithelial damage caused by bacterial infection, significantly inhibit the level of pro-inflammatory cytokines, promote the expression of anti-inflammatory cytokines, significantly increase the concentration of acetic acid and propionic acid in the feces of infected mice, and produce positive regulation on intestinal flora, showing good application prospects in regulating intestinal flora and anti-bacterial infection.

[0008] The technical solution of the present invention is:

[0009] In one aspect, the present invention provides Bifidobacterium breve ( Bifidobacterium breve ) The use of synergistic lactoferrin in the preparation of products for regulating intestinal flora or anti-bacterial infection products, the Bifidobacterium breve is Bifidobacterium breve HH079, with a preservation number of GDMCC No: 64216, deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 29, 2023, and has been disclosed in patent CN118853502B.

[0010] Specifically, in the product for regulating intestinal flora or anti-bacterial infection, the ratio of the added amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 -10 10 CFU: 1-1000mg.

[0011] Preferably, in the product for regulating intestinal flora or anti-bacterial infection, the ratio of the added amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 -10 10 CFU: 1-10mg, 10 8 -10 10 CFU: 10-20mg, 10 8 -10 10 CFU: 20-30mg, 10 8 -10 10 CFU: 30-40mg, 10 8 -10 10 CFU: 40-50mg, 10 8 -10 10 CFU: 50-60mg, 10 8 -10 10 CFU: 60-70mg, 10 8 -10 10 CFU: 70-80mg, 10 8 -10 10 CFU: 80-90mg, 10 8 -10 10 CFU: 90-100mg, 10 8 -10 10 CFU: 100-200mg, 10 8 -10 10 CFU: 200-300mg, 10 8 -10 10 CFU: 300-400mg, 10 8 -10 10 CFU: 400-500mg, 10 8 -10 10 CFU: 500-600mg, 10 8 -10 10 CFU: 600-700mg, 10 8 -10 10 CFU: 700-800mg, 10 8 -10 10 CFU: 800-900mg or 10 8 -10 10CFU:900-1000mg.

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

[0013] Still further preferably, in the product for regulating intestinal flora or anti-bacterial infection, the ratio of the added amount of Bifidobacterium breve HH079 to lactoferrin is 10 8 CFU: 1mg, 10 8 CFU: 10mg, 10 8 CFU: 100mg or 10 9 CFU: 2.5mg.

[0014] Specifically, the Bifidobacterium breve HH079 includes live bacteria, inactivated bacteria, broken bacteria, secretions or metabolites of Bifidobacterium breve HH079.

[0015] Preferably, the Bifidobacterium breve HH079 is a live bacterium of Bifidobacterium breve HH079.

[0016] Specifically, the anti-bacterial infection products include medicines.

[0017] 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.

[0018] Preferably, the medicine further comprises one or more physiologically acceptable excipients.

[0019] Further preferably, the excipients include but are not limited to: solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, adhesives, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0020] The beneficial effects of the present invention are:

[0021] The present invention combines Bifidobacterium breve HH079 with lactoferrin, which are synergistically used to combat bacterial infections. The combination can reverse weight loss, colon shortening, increased bacterial load, and colon epithelial damage caused by bacterial infections, significantly inhibit the levels of pro-inflammatory factors, promote the expression of anti-inflammatory factors, and significantly increase the concentrations of acetic acid and propionic acid in the feces of infected mice, and positively regulate the intestinal flora. The combination shows good application prospects in regulating intestinal flora and combating bacterial infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is an in vitro experiment verifying the destructive effect of the composition preparation on biofilms; Figure A shows the effect of the composition preparation on biofilm biomass during the process of E. coli biofilm adhesion to epithelial cells; Figure B shows the effect of the composition preparation on supernatant biomass during the process of E. coli biofilm adhesion to epithelial cells; and Figure C shows the destructive effect of the composition preparation on biofilms after E. coli adheres to Caco-2 cell biofilms.

[0023] Figure 2 Flowchart of the protocol for designing in vivo experiments.

[0024] Figure 3 The graph shows the weight changes of mice in each group; * in the graph indicates significant difference compared with the CR group. p <0.05.

[0025] Figure 4 is the colon length of mice in each group; ** in the figure represents significant difference compared with the CR group. p <0.01.

[0026] Figure 5 is the bacterial load in mouse feces; * in the figure indicates significant difference compared with the CR group. p <0.05.

[0027] Figure 6 for EspB Gene quantitative detection results; * in the figure represents significant difference compared with the CR group. p <0.05.

[0028] Figure 7 The results of colon histopathological examination of mice in each group are shown in Figure 3.

[0029] Figure 8 The results of serum inflammatory factors determination of mice in each group; A in the figure is the result of TNF-α determination; B is the result of IL-6 determination; C is the result of IL-10 determination; different lowercase letters in the figure represent significant differences between the groups. p <0.05.

[0030] Figure 9Figure 2 is the change of SCFA in mouse feces; A in the figure is the result of acetic acid determination; B is the result of propionic acid determination; C is the result of butyric acid determination; different lowercase letters in the figure represent significant differences between the groups. p <0.05.

[0031] Figure 10 This is the result of α diversity analysis.

[0032] Figure 11 The results of principal coordinate analysis.

[0033] Figure 12 This is an analysis of the composition of the intestinal microbiome; A in the figure is the family level classification; B and the right figure are the genus level classification.

[0034] Figure 13 These are the results of α-diversity analysis before infection.

[0035] Figure 14 The results of principal coordinate analysis before infection.

[0036] Figure 15 This is an analysis of the intestinal microbial community 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); and D is the classification of microorganisms at the genus level (5 parallels per group). DETAILED DESCRIPTION

[0037] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0038] Example 1 In vitro experiment

[0039] 1. Effects of E. coli biofilm adhesion to epithelial cells on biofilm biomass and supernatant biomass

[0040] (1) Place 1×10 4 Caco-2 cells were seeded into DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin and incubated for 24 hours at 37° C. Before adding the bacterial culture, the medium was replaced with DMEM without antibiotics.

[0041] (2) 1×10 5 CFU / mL E. coli O157:H7 was inoculated into the culture medium, and at the same time, different composition preparations were inoculated into the culture medium and incubated at 37°C for 8 hours. The preparations were:

[0042] Control group: treated with sterile PBS;

[0043] HH-8 group: Bifidobacterium breve HH079 (1×10 8 CFU) were treated individually;

[0044] HH-10 group: Bifidobacterium breve HH079 (1×10 10 CFU) were treated individually;

[0045] LF-1 group: Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (1 mg, 12.5 μM);

[0046] LF-10 group: Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (10 mg, 125 μM);

[0047] LF-100 group: Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (100 mg, 1250 μM).

[0048] LF-1000 group: Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (1000 mg, 12500 μM).

[0049] (3) Biofilm biomass in cell adhesion: After the incubation period, the culture supernatant was removed and the biofilm was washed with PBS. Finally, the biofilm was resuspended in sterile PBS and serially diluted, then inoculated onto BHI agar plates and incubated at 37°C for 24 hours before measurement.

[0050] 2. Effects of E. coli on Caco-2 biofilms after adhesion

[0051] (1) Place 1×10 4 Caco-2 cells were seeded into DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin and incubated for 24 hours at 37° C. Before adding the bacterial culture, the medium was replaced with DMEM without antibiotics.

[0052] (2) 1×10 5 CFU / mL E. coli O157:H7 was inoculated into the culture medium and incubated for 24 h to form a biofilm;

[0053] (3) After biofilm formation, different composition preparations were inoculated into culture medium and incubated at 37°C for 8 hours. The preparations were:

[0054] Control group: treated with sterile PBS;

[0055] LF-0 group: Bifidobacterium breve HH079 (1×10 8 CFU) were treated individually;

[0056] LF-1 group: Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (1 mg, 12.5 μM);

[0057] LF-10 group: Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (10 mg, 125 μM);

[0058] LF-100 group: Bifidobacterium breve HH079 (1×10 8 CFU) combined with LF (100 mg, 1250 μM).

[0059] (4) Biofilm biomass in cell adhesion: After the incubation period, the culture supernatant was removed and the biofilm was washed with PBS. Finally, the biofilm was resuspended in sterile PBS and serially diluted, then inoculated onto BHI agar plates and incubated at 37°C for 24 hours before measurement.

[0060] The results of the test are as follows Figure 1 The results showed that compared with the untreated 8 Treatment with CFU Bifidobacterium breve HH079 alone or with different concentrations of LF (12.5, 125, 1250 μM) combined with Bifidobacterium breve HH079 inhibited more than 95% of biofilm formation, and the higher the LF concentration, the worse the biofilm formation.

[0061] Example 2 Experimental Grouping and Dosage

[0062] Newborn C57BL / 6J mice with an average weight of approximately 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), a LF group (n=8), a LF+HH079 group (n=8), and a WPC+HH079 group (n=8).

[0063] From the 10th day to the 29th day after birth, mice in the HH079 group were given 1×10 live bacteria per day. 9 CFU Bifidobacterium breve HH079 were orally administered; mice in the LF group were orally administered with 2.5 mg lactoferrin (LF) every day; mice in the LF+HH079 group were orally administered with a synergistic combination of LF+HH079 (2.5 mg lactoferrin and 1×10 9CFU Bifidobacterium breve HH079) were orally administered; mice in the WPC+HH079 group were given a WPC+HH079 composition (2.5 mg whey protein (WPC) and 1×10 9 The mice in the Con and CR groups were orally gavaged with an equal volume of vehicle solution PBS every day.

[0064] On day 22 after birth, mice in the CR, HH079, LF, LF+HH079, and WPC+HH079 groups were orally administered with 1×10 9 CFU Citrobacter rodentium ( C. rodentium , CR); the Con group was orally gavaged with an equal volume of vehicle solution PBS. The experimental design process is shown in Figure 2 .

[0065] Example 2 Phenotype of mice

[0066] The weight changes of mice in each group were recorded on days 0, 1, 3, 5, and 7 after CR bacterial infection (i.e., days 22, 23, 25, 27, and 29 after birth). Figure 3 As shown, significant weight loss was observed in mice infected with C. rodentium, but treatment with either LF or Bifidobacterium breve HH079 alleviated this weight loss. Supplementation with B. breve HH079 and WPC (WPC+HH079 group) also tended to promote weight gain. In contrast, mice infected with B. breve HH079 and LF (LF+HH079 group) showed significant weight gain.

[0067] 24 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 to measure the length of the colon. Figure 4 CR bacterial infection significantly shortened the length of the mouse colon, while LF, Bifidobacterium breve HH079, and the LF+HH079 synergistic combination intervention promoted the reduction of the mouse colon length, indicating that Bifidobacterium breve HH079 synergistically with LF intervention has a positive effect on colon inflammation caused by CR bacterial infection.

[0068] Example 3 Bacterial load / virulence factors in mouse feces

[0069] Feces of mice in the CR, HH079, LF, LF+HH079, and WPC+HH079 groups were collected on days 0, 1, 3, 5, and 7 after CR bacterial infection (i.e., days 22, 23, 25, 27, and 29 after birth). Fresh feces (0.1 g) were resuspended in PBS (1 mL) and vortexed to disperse the fecal sediment. The fecal suspension was serially diluted to 10 -8The number of viable bacteria in stool samples was counted on LB agar plates.

[0070] Bacterial load in feces Figure 5 As shown, the feces of CR group mice had C. rodentium The counts increased significantly 5 days after CR bacterial infection. C. rodentium Reach 10 9 Seven days after CR bacterial infection, the feces of mice in the HH079 group, LF group, LF+HH079 group, and WPC+HH079 group were higher than those in the CR group. C. rodentium The load was significantly reduced, which showed that LF treatment alone, Bifidobacterium breve HH079 treatment alone, LF+HH079 synergistic composition treatment, WPC+HH079 combination treatment had a significant effect on the C. rodentium Infection resistant.

[0071] In feces EspB For gene quantification, fecal DNA extraction and purity determination, see Example 7. Target genes were quantified using the SYBR kit on a PCR detection system (CFX384, BioRad, USA) according to the following program: 95°C, 30 seconds; 40 cycles of 95°C, 10 seconds and 60°C, 30 seconds. EspB The designed primers (as shown in Table 1) were used for quantification C. rodentium Load, cycle threshold (Ct) values ​​were normalized to the total bacterial count.

[0072] Table 1

[0073]

[0074] Note: In the table, “F” stands for forward primer; “R” stands for reverse primer.

[0075] EspB Gene quantitative detection results such as Figure 6 As 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 trend of decreasing the expression of virulence factors, while the LF group and the LF+HH079 group showed a significant decrease. EspB This indicates that Bifidobacterium breve HH079 synergistically with LF intervention has a positive effect on the expression of bacterial virulence factors after CR infection.

[0076] Example 4 Mouse Colon Pathology

[0077] The colon tissues of mice in each group were fixed in 4% buffered paraformaldehyde solution for 48 hours, then embedded in paraffin, dewaxed into water, and stained with H&E. The sections were observed under an optical microscope and representative photos were taken. Figure 7 .

[0078] H&E staining revealed that after mice were infected with Citrobacter rodentium, large areas of inflammatory cell infiltration appeared in the colon, accompanied by epithelial cell shedding and destruction of crypt structure, indicating that inflammatory lesions occurred in the mouse colon.

[0079] Supplementation with either LF or B. breve HH079 reduced colonic inflammatory lesions to some extent. In the LF-treated group, epithelial cells were tightly packed, while inflammatory cell infiltration was reduced in the B. breve HH079-treated group. In the WPC+HH079 group, colonic epithelial inflammatory infiltration was partially reduced.

[0080] The LF+HH079 group showed a more complete intestinal epithelial structure, reduced inflammatory granulocyte infiltration, and increased goblet cells. This indicates that Bifidobacterium breve HH079 combined with LF intervention is effective in resisting Citrobacter rodentium infection in mice.

[0081] Example 5 Mouse serum inflammatory factors

[0082] 24 hours after the last gavage, serum samples were collected from mice. TNF-α, IL-6, and IL-10 indicators in mouse serum samples were measured using ELISA kits. The operation was performed according to the instructions provided in the ELISA kit. The results are shown in the figure below. Figure 8 shown.

[0083] Seven days after CR bacterial infection, inflammatory cytokines were found in the serum of mice, 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 factors (IL-10).

[0084] Supplementation with LF alone, Bifidobacterium breve HH079 alone, and the synergistic combination of LF and HH079 all significantly inhibited the expression of proinflammatory cytokines and promoted the expression of anti-inflammatory factors in mouse serum. The LF+HH079 group showed a more pronounced downward trend in TNF-α, suggesting that LF and Bifidobacterium breve HH079 synergistically play a more positive role in regulating the inflammatory imbalance caused by Citrobacter rodentium infection in mice. Although the WPC+HH079 group also showed a trend of reducing proinflammatory cytokines and increasing anti-inflammatory factors, the overall effect of the LF+HH079 group was the best.

[0085] Example 6 Changes in SCFA in Mouse Feces

[0086] SCFA content in feces of mice was measured on day 7 post-infection using a gas chromatograph. An internal standard mixture was prepared with 4-methylvaleric acid. 30 μL of this mixture was added to 120 μL of the fecal supernatant, mixed thoroughly, and injected into the gas chromatograph. Short-chain fatty acids in the sample were separated using a capillary column (Zebron, ZB-FFAP, 30 m × 0.25 mm × 0.25 μm) and a hydrogen ion flame detector in nitrogen at a flow rate of 1 mL / min, and their concentrations were determined. The oven temperature was initially maintained at 60°C, then increased at a rate of 20°C / min to 220°C and held at 220°C for 3 minutes. The injector and detector temperatures were set to 250°C and 280°C, respectively. After the program was completed, the peak areas were recorded and the target short-chain fatty acids in the sample were quantified by correcting the peak areas with those of the internal standard.

[0087] The results of the test are as follows Figure 9 As shown in Figure 3, after infection with Citrobacter rodentium, the concentrations of short-chain fatty acids, including acetate and propionate, in the feces of mice decreased significantly.

[0088] The HH079, LF, LF+HH079, and WPC+HH079 groups all significantly increased the acetic acid content in the mouse feces. Supplementation with LF alone showed a certain growth trend for propionic acid and butyric acid. Supplementation with HH079 alone showed an increasing trend for propionic acid and significantly increased the concentration of butyric acid. Co-supplementation with WPC and HH079 all promoted acetic acid, propionic acid, and butyric acid. Synergistic supplementation with LF and Bifidobacterium breve HH079 significantly increased the propionic acid concentration in the feces of infected mice, indicating that synergistic supplementation has a positive effect on restoring the concentration of short-chain fatty acids in mouse feces.

[0089] Example 7 Changes in the intestinal flora of mice

[0090] The changes in intestinal flora in the feces of mice were measured on the 7th day after infection.

[0091] Total DNA was extracted from mouse fecal samples using the QIAGEN DNA Mini-Kit. DNA was quantified using Nanodrop, and extraction quality was assessed 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′). The amplified product was purified using magnetic beads. Sequencing libraries were prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit. Sequencing was performed on a MiSeq sequencer. The resulting raw, off-line high-throughput sequencing data were screened, re-sampled, and partitioned into libraries and samples. Chimeras and barcode sequences were removed, followed by denoising and ASV clustering. The taxonomic composition of each sample at different taxonomic levels was investigated using existing databases. Based on different OUT distributions, the alpha diversity level of the samples and the beta diversity differences between different groups were evaluated to further measure the differences in community structure between different groups.

[0092] The α diversity (Chao1, Pielou, Shannon, and Simpson indices) showed that the fecal microbial community richness and uniformity of the CR group were significantly reduced compared with the Con group. Different interventions reversed the α diversity index, indicating that LF / Bifidobacterium breve HH079 alone or in combination affected the intestinal microbial diversity of the pups ( Figure 10 ).

[0093] Principal coordinate analysis (PCoA) further showed distinct grouping of microbiota in different experimental groups, and the LF+HH079 group clustered closer to the control group than the CR group, indicating that synergistic supplementation of LF and Bifidobacterium breve HH079 was more beneficial for restoring the diversity of intestinal microbiota after CR infection ( Figure 11 ).

[0094] 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 (reducing inflammation, producing small amounts of acetic and propionic acid), supplemented with Bifidobacterium breve HH079 enrichment Bifidobacterium and Muribaculum(Potential probiotics, with 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 ).

[0095] Example 8 Changes in intestinal flora of mice before infection

[0096] 21 days after birth, feces were collected from the Con, HH079, LF, and LF+HH079 groups. α-diversity analysis and principal coordinate analysis were performed according to the methods described in Example 7, and the intestinal microbiota composition of each group was evaluated at the taxonomic level.

[0097] α diversity analysis ( Figure 13 ) and principal coordinate analysis ( Figure 14 ) The results showed that the individual / 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 the individual / synergistic supplementation of LF and Bifidobacterium breve HH079 made the mouse intestinal flora composition tend to be consistent.

[0098] 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 a small amount of acetic acid and propionic acid), and supplemented with Bifidobacterium breve HH079 alone. Bifidobacterium and UBA3282 (Lachnospiraceae) , synergistically supplementing LF and Bifidobacterium breve HH079 to promote the enrichment of more beneficial bacteria ( Figure 15 ).

[0099] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. Bifidobacterium breve ( Bifidobacterium breve ) Use of synergistic lactoferrin in the preparation of anti-bacterial infection products, characterized in that, The Bifidobacterium breve is Bifidobacterium breve HH079, with a deposit number of GDMCC No: 64216; the bacterium is Citrobacter rodentium; In the antibacterial infection product, the ratio of the added amount of Bifidobacterium breve HH079 and lactoferrin is 10 8 -10 10 CFU: 1-1000mg.

2. The use according to claim 1, characterized in that The anti-bacterial infection products include medicines.

3. The use according to claim 2, characterized in that The dosage forms of the medicine include tablets, pills, powders, suspensions, gels, creams, granules, capsules, suppositories, injections or sprays.

4. The use according to claim 3, characterized in that The medicine also includes one or more physiologically acceptable excipients.

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