HK40124060A - Application of bifidobacterium breve synergistic lactoferrin in preparation of products for regulating gut microbiota or resisting bacterial infection - Google Patents

Description

Application of Bifidobacterium breve in synergistic use with lactoferrin in the preparation of products for regulating gut microbiota or fighting bacterial infections

Technical Field

This invention belongs to the field of biomedical technology, specifically relating to the application of Bifidobacterium breve in conjunction with lactoferrin in the preparation of products that regulate intestinal flora or fight bacterial infections.

Background Technology

Regulating the gut microbiota and resisting bacterial infections are crucial for maintaining human health. Currently, various related products exist on the market; however, these products have revealed numerous shortcomings in practical applications.

Common products for regulating gut microbiota include probiotic preparations, prebiotic products, and some functional foods. Probiotic preparations are mostly available in capsule, powder, or oral liquid form, designed to supplement the gut with beneficial microorganisms. Prebiotic products promote the growth and reproduction of beneficial bacteria by providing them with "food." Some fermented functional foods, such as yogurt and fermented soy products, also claim to regulate gut microbiota. However, while prebiotic products can promote the growth of beneficial bacteria, their effects are relatively singular, and some people may experience discomfort such as bloating due to prebiotic intake. The content of beneficial components in functional foods is unstable and greatly affected by processing techniques and raw material quality, making it difficult to precisely regulate gut microbiota.

Antibacterial infection products mainly cover antibiotics and some natural extracts with antibacterial effects. Antibiotics are widely used in clinical treatment and can quickly and effectively inhibit or kill bacteria. Natural extracts, such as those containing allicin and tea polyphenols, also claim to have antibacterial effects. However, antibiotics have significant drawbacks. Long-term use can easily lead to increased bacterial resistance, disrupt the balance of the body's normal flora, and cause a series of adverse reactions, such as diarrhea and fungal infections. Diarrhea, as a common symptom of digestive system infections, has complex causes and diverse effects. Prolonged or severe diarrhea can lead to dehydration, resulting in the loss of large amounts of water and electrolytes. Mild dehydration manifests as thirst, dry skin, and decreased urine output; moderate dehydration presents as sunken eyes and lethargy; and severe dehydration can even lead to shock. Simultaneously, it can cause electrolyte imbalances, such as hypokalemia leading to muscle weakness and arrhythmia, and hyponatremia leading to headaches and drowsiness, as well as malnutrition, affecting physical growth and development and immune function. Natural extract products often have weak antibacterial effects and are difficult to deal with severe bacterial infections. Furthermore, the extraction and preservation technologies for their active ingredients are not yet perfect, resulting in inconsistent product quality.

The emergence of probiotics and glycoproteins has brought new hope to solving this problem. Patent CN119424485A encapsulates tannins and mucin in *Escherichia coli* Nissle 1917 and *Lactobacillus plantarum* NC8, resulting in a mucin-tannin encapsulated probiotic preparation. This probiotic preparation exhibits excellent resistance to the harsh environment of the gastrointestinal tract and enhances its adhesion in the intestines, thereby strengthening the colonization and growth of probiotics in the mucus layer. Simultaneously, it can effectively treat bacterial enteritis, especially enteritis caused by enterotoxigenic *Escherichia coli* (ETEC).

In terms of antibacterial activity, glycoproteins can bind to bacterial surface receptors, interfering with bacterial physiological activities and regulating the body's immune response, enhancing the phagocytic capacity of immune cells against bacteria. Regarding the regulation of gut microbiota, glycoproteins can create a more favorable environment for probiotics, promoting their growth and metabolism; the synergistic effect of the two has enormous potential. Providing the application of *Bifidobacterium breve* ( B. breve ) HH079 in synergy with lactoferrin (LF) in regulating gut microbiota or combating bacterial infections has extremely high application value and significant importance.

Summary of the Invention

To address the aforementioned shortcomings, this invention provides the application of *Bifidobacterium breve* synergistically with lactoferrin in the preparation of products for regulating intestinal flora or combating bacterial infections. This invention provides the application of * Bifidobacterium breve * HH079 (accession number GDMCC No: 64216) synergistically with lactoferrin in the preparation of products for regulating intestinal flora or combating bacterial infections. This invention combines *Bifidobacterium breve* HH079 with lactoferrin (LF) for synergistic action against bacterial infections. It can reverse bacterial infection-induced weight loss, colonic shortening, increased bacterial load, and colonic epithelial damage. It can significantly inhibit pro-inflammatory cytokine levels and promote anti-inflammatory cytokine expression. It can significantly increase the concentrations of acetic acid and propionic acid in the feces of infected mice and positively regulate intestinal flora, demonstrating promising application prospects in regulating intestinal flora and combating bacterial infections.

The technical solution of this invention is as follows:

On the one hand, this invention provides the application of Bifidobacterium breve in synergistic use with lactoferrin in the preparation of products that regulate intestinal flora or antibacterial infection products. The Bifidobacterium breve is Bifidobacterium breve HH079, with accession number GDMCC No: 64216, deposited at Guangdong Provincial Center for Microbial Culture Collection on December 29, 2023, and has been disclosed in patent CN118853502B.

Specifically, in the product for regulating intestinal flora or antibacterial infection, the ratio of Bifidobacterium breve HH079 to lactoferrin is ^10⁸ - ^10¹⁰ CFU: 1-1000 mg.

Preferably, in the intestinal flora regulating product or antibacterial infection product, the ratio of added Bifidobacterium breve HH079 to lactoferrin is as follows: ^10⁸ - ^10¹⁰ CFU: 1-10 mg, ^10⁸ - ^10¹⁰ CFU: 10-20 mg, ^10⁸ - ^10¹⁰ CFU: 20-30 mg, ^10⁸ - ^10¹⁰ CFU: 30-40 mg, ^10⁸ - ^10¹⁰ CFU: 40-50 mg, ^10⁸ - ^10¹⁰ CFU: 50-60 mg, ^10⁸ - ^10¹⁰ CFU: 60-70 mg, ^10⁸ - ^10¹⁰ CFU: 70-80 mg, ^10⁸ - ^10¹⁰ CFU: 80-90 mg, ^10⁸ - ^10¹⁰ CFU: 90-100 mg, ^10⁸ -10¹⁰ CFU: ^... 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 ¹⁰ CFU: 900-1000mg.

More preferably, in ^{the intestinal flora regulating product or antibacterial infection product, the ratio of added Bifidobacterium breve HH079 to lactoferrin is 10⁸-10¹⁰ CFU :} 1 mg, ^2.5 mg, ^{10 mg} , or ^{100 mg} .

More preferably, in the intestinal flora regulating product or antibacterial infection product, the ratio of added Bifidobacterium breve HH079 to lactoferrin is ^10⁸ CFU: 1 mg, ^10⁸ CFU: 10 mg, ^10⁸ CFU: 100 mg, or ^10⁹ CFU: 2.5 mg.

Specifically, the aforementioned Bifidobacterium breve HH079 includes live bacteria, inactivated bacteria, broken-cell bacteria, secretions, or metabolites of Bifidobacterium breve HH079.

Preferably, the Bifidobacterium breve HH079 is a live Bifidobacterium breve HH079.

Specifically, the products for regulating gut microbiota include food or health supplements.

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

Preferably, the food products include, but are not limited to: candy, soy milk, yogurt, canned food, biscuits, chocolate, pastries, cream, cheese, dairy products, milk powder, formula milk powder, ice cream, jam, fruit puree, candied fruit, preserved fruit, dried fruit, bread, egg rolls, protein drinks, solid beverages, lactic acid bacteria beverages, plant protein beverages, carbonated beverages, coffee, or puffed foods.

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

Preferably, the food or health product also includes nutritionally acceptable nutritional additives.

More preferably, the nutritional additives include, but are not limited to, one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

Specifically, the antibacterial infection products include food additives or pharmaceuticals.

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

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

Preferably, the medicine further includes one or more physiologically acceptable excipients.

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

The beneficial effects of this invention are as follows:

This invention combines Bifidobacterium breve HH079 with lactoferrin for synergistic action against bacterial infection. It can reverse the weight loss, colon shortening, increased bacterial load, and colonic epithelial damage caused by bacterial infection. It can significantly inhibit the level of pro-inflammatory factors and promote the expression of anti-inflammatory factors. It can significantly increase the concentration of acetic acid and propionic acid in the feces of infected mice and positively regulate the intestinal flora. It shows good application prospects in regulating intestinal flora and fighting bacterial infection.

Attached Figure Description

Figure 1 shows the in vitro experimental verification of the destructive effect of the composition formulation on the biofilm; A in the figure shows the effect of the composition formulation on the biofilm biomass during the E. coli biofilm adhesion to epithelial cells; B shows the effect of the composition formulation on the supernatant biomass during the E. coli biofilm adhesion to epithelial cells; C shows the destructive effect of the composition formulation on the biofilm after E. coli adheres to the Caco-2 cell biofilm.

Figure 2 is a flowchart of the in vivo experimental design scheme.

Figure 3 shows the changes in body weight of mice in each group; * in the figure represents a significant difference from the CR group, p < 0.05.

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

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

Figure 6 shows the quantitative detection results of the EspB gene; * in the figure represents a significant difference from the CR group, p < 0.05.

Figure 7 shows the results of histopathological examination of the colon tissue of mice in each group.

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

Figure 9 shows the changes in SCFA in mouse feces; A in the figure represents the results of acetic acid measurement; B represents the results of propionic acid measurement; C represents the results of butyric acid measurement; different lowercase letters in the figure represent significant differences between groups, p < 0.05.

Figure 10 shows the results of the α diversity analysis.

Figure 11 shows the results of the principal coordinate analysis.

Figure 12 shows the composition analysis of the gut microbiota; A in the figure represents the family-level classification; B and the right figure represent the genus-level classification.

Figure 13 shows the results of the pre-infection α diversity analysis.

Figure 14 shows the results of the principal coordinate analysis before infection.

Figure 15 shows the composition of the gut microbiota before infection; A in the figure represents the classification of microorganisms at the phylum level; B represents the classification of microorganisms at the family level; C represents the classification of microorganisms at the genus level (within-group average); D represents the classification of microorganisms at the genus level (5 replicates per group).

Detailed Implementation

The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

Example 1 In vitro experiment

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

(1) Inoculate 1× ^10⁴ Caco-2 cells per well into DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin, and incubate at 37°C for 24 hours. Replace the medium with antibiotic-free DMEM before adding bacterial culture.

(2) 1× ^10⁵ CFU/mL E. coli O157:H7 was inoculated into the culture medium, and at the same time, different formulations were inoculated into the culture medium and incubated at 37°C for 8 hours. The formulations were:

Control group: treated with sterile PBS;

HH-8 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) was used as a single treatment;

HH-10 group: Bifidobacterium breve HH079 (1× ^10¹⁰ CFU) was used as a single treatment;

LF-1 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) combined with LF (1 mg, 12.5 μM);

LF-10 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) combined with LF (10 mg, 125 μM);

LF-100 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) combined with LF (100 mg, 1250 μM).

LF-1000 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) combined with LF (1000 mg, 12500 μM).

(3) Biofilm biomass in cell adhesion: After incubation, 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 incubated at 37°C for 24 hours before measurement.

2. The effect of E. coli adhering to Caco-2 cell biomembranes on biomembranes.

(1) Inoculate 1× ^10⁴ Caco-2 cells per well into DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin, and incubate at 37°C for 24 hours. Replace the medium with antibiotic-free DMEM before adding bacterial culture.

(2) Inoculate 1× ^10⁵ CFU/mL E. coli O157:H7 into the culture medium and incubate for 24 h to form a biofilm;

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

Control group: treated with sterile PBS;

LF-0 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) was used as a single treatment;

LF-1 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) combined with LF (1 mg, 12.5 μM);

LF-10 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) combined with LF (10 mg, 125 μM);

LF-100 group: Bifidobacterium breve HH079 (1× ^10⁸ CFU) combined with LF (100 mg, 1250 μM).

(4) Biofilm biomass in cell adhesion: After incubation, 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 incubated at 37°C for 24 hours before measurement.

The results are shown in Figure 1. The results show that, compared with the untreated, treatment with 1× ^10⁸ CFU of Bifidobacterium breve HH079 alone, and combined treatment with different concentrations of LF (12.5, 125, 1250 μM) and Bifidobacterium breve HH079, all prevented more than 95% of biofilm formation. Moreover, the higher the LF concentration, the worse the biofilm formation.

Example 2 Experimental grouping and administration method

This invention uses newborn C57BL/6J mice as experimental animals, with an average weight of approximately 12.5g. 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).

From day 10 to day 29 after birth, mice in the HH079 group were orally administered 1× ^10⁹ CFU of live bacteria *Bifidobacterium breve* HH079 daily; mice in the LF group were orally administered 2.5 mg of lactoferrin (LF) daily; mice in the LF+HH079 group were orally administered the LF+HH079 synergistic combination (2.5 mg lactoferrin and 1× ^10⁹ CFU of live bacteria *Bifidobacterium breve* HH079) daily; mice in the WPC+HH079 group were orally administered the WPC+HH079 combination (2.5 mg whey protein (WPC) and 1× ^10⁹ CFU of live bacteria *Bifidobacterium breve* HH079) daily; and mice in the Con and CR groups were orally administered an equal volume of carrier solution PBS daily.

On day 22 after birth, mice in the CR, HH079, LF, LF+HH079, and WPC+HH079 groups were administered 1× ^10⁹ CFU of Citrobacter rodentium ( C. rodentium , CR) by gavage; mice in the Con group were administered an equal volume of PBS orally. The experimental design is shown in Figure 2.

Example 2: Mouse physical signs and phenotypes

Body weight changes in mice were recorded at 0, 1, 3, 5, and 7 days after CR bacterial infection (i.e., days 22, 23, 25, 27, and 29 after birth), as shown in Figure 3. Significant weight loss was observed in mice infected with *Citrobacter brevis*, while the weight loss was alleviated after treatment with LF or *Bifidobacterium brevis* HH079. *Bifidobacterium brevis* HH079 combined with WPC supplementation (WPC+HH079 group) also showed a trend towards promoting weight gain. In contrast, infected mice treated with a mixture of *Bifidobacterium brevis* HH079 and LF (LF+HH079 group) showed a significant increase in body weight.

Twenty-four hours after the last gavage, mice were euthanized by _CO2 asphyxiation, and colonic tissue was immediately collected from each group of mice for colonic length measurement. The results are shown in Figure 4. CR bacterial infection significantly shortened the colonic length in mice, while intervention with LF, Bifidobacterium breve HH079, and the synergistic combination of LF and HH079 further promoted the reduction in colonic length, indicating that the synergistic intervention of Bifidobacterium breve HH079 and LF has a positive effect on colonic inflammation induced by CR bacterial infection.

Example 3: Bacterial load/virulence factor in mouse feces

Fecal samples from mice in the CR, HH079, LF, LF+HH079, and WPC+HH079 groups were collected at 0, 1, 3, 5, and 7 days after CR bacterial infection (i.e., days 22, 23, 25, 27, and 29 after birth). Fresh fecal samples (0.1 g) were resuspended in PBS (1 mL) and vortexed to disperse the precipitate. The fecal suspensions were serially diluted to a concentration of ^10⁻⁸ , and the number of viable bacteria in the fecal samples was counted on LB agar plates.

The bacterial load in feces is shown in Figure 5. Throughout the infection period, the C. rodentium count in the feces of mice in the CR group increased significantly, reaching ^10⁹ CFU on day 5 post-CR infection. On day 7 post-CR infection, compared to the CR group, the C. rodentium load in the feces of mice in the HH079, LF, LF+HH079, and WPC+HH079 groups was significantly reduced. This indicates that LF treatment alone, Bifidobacterium breve HH079 treatment alone, LF+HH079 synergistic treatment, and WPC+HH079 combination treatment provided resistance to C. rodentium infection.

For the quantitative detection of the EspB gene in feces, fecal DNA extraction and purity determination are described in Case Study 7. The target gene was quantified using a SYBR kit on a PCR detection system (CFX384, BioRad, USA) according to the following procedure: 95°C, 30 seconds; 40 cycles, 95°C, 10 seconds and 60°C, 30 seconds. Primers designed for the EspB gene (as shown in Table 1) were used to quantify the C. rodentium load, and the cycle threshold (Ct) was normalized to the total bacterial count.

Table 1

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

Figure 6 shows the quantitative detection results of the EspB gene. 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 virulence factor expression, while the LF group and the LF+HH079 group showed a significant ability to reduce EspB gene expression. This indicates 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.

Example 4: Mouse Colon Pathology

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

H&E staining revealed extensive inflammatory cell infiltration in the colon of mice infected with Citrobacter rodentus, accompanied by epithelial cell shedding and crypt structure destruction, indicating inflammatory lesions in the mouse colon.

Supplementation with LF and Bifidobacterium breve HH079 alone reduced colonic inflammation to some extent. In the LF-treated group, epithelial cells were more tightly packed, while in the Bifidobacterium breve HH079 intervention group, inflammatory cell infiltration was reduced. The WPC+HH079 group showed a partial reduction in colonic epithelial inflammatory infiltration.

The LF+HH079 group exhibited a more intact intestinal epithelial structure, reduced inflammatory granulocyte infiltration, and an increased number of goblet cells. This indicates that the synergistic intervention of Bifidobacterium breve HH079 and LF significantly enhances the resistance to Citrobacterium cirrhosa infection in mice.

Example 5: Mouse serum inflammatory factors

Serum samples were collected from mice immediately 24 hours after the last gavage. TNF-α, IL-6, and IL-10 levels in the mouse serum samples were measured using an ELISA kit. The procedure was performed according to the instructions provided with the ELISA kit. The results are shown in Figure 8.

Seven days after infection with C. rodentium , inflammatory cytokines in mouse serum were found to significantly increase the production of serum pro-inflammatory factors (IL-6, TNF-α) and decrease the expression of serum anti-inflammatory factor (IL-10) compared with the blank control group.

Supplementation with LF alone, Bifidobacterium breve HH079 alone, and the synergistic combination of LF and HH079 all significantly inhibited the expression of pro-inflammatory cytokines and promoted the expression of anti-inflammatory factors in mouse serum. Among these, the LF+HH079 group showed a more pronounced decrease in TNF-α, indicating that the synergistic effect of LF and Bifidobacterium breve HH079 is more effective in regulating the inflammatory imbalance induced by Citrobacterium rodentii infection in mice. Although the WPC+HH079 group also showed a trend of decreasing pro-inflammatory cytokines and increasing anti-inflammatory factors, the overall effect of the LF+HH079 group was the best.

Example 6 Changes in SCFA in mouse feces

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

The results are shown in Figure 9. After infection with Citrobacter rodentia, the concentration of short-chain fatty acids, including acetic acid and propionic acid, in mouse feces decreased significantly.

The HH079 group, LF group, LF+HH079 group, and WPC+HH079 group all significantly increased the acetic acid content in mouse feces. LF supplementation alone showed a certain increasing trend in propionic and butyric acid. HH079 supplementation alone showed an increasing trend in propionic acid and significantly increased butyric acid concentration. Co-supplementation with WPC and HH079 promoted the production of acetic, propionic, and butyric acids. 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 short-chain fatty acid concentrations in mouse feces.

Example 7 Changes in the gut microbiota of mice

Changes in intestinal flora in the feces of mice were measured on day 7 after infection.

Total DNA was extracted from mouse fecal samples using the QIAGEN DNA Mini-Kit. DNA was quantified using Nanodrop, and the 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 reverse primer SEQ ID NO.7 (5′-GGACTACHVGGGTWTCTAAT-3′), and the amplification products were purified and recovered using magnetic beads. Sequencing libraries were prepared using the Illumina TruSeq Nano DNA LT Library Prep Kit. Sequencing was performed using a MiSeq sequencer. The raw high-throughput sequencing data were screened, supplemented, and divided into libraries and samples. Chimeras and barcode sequences were removed, followed by noise reduction and ASV clustering. The composition of each sample at different species taxonomic levels was explored in conjunction with existing databases. Based on different OUT distributions, the alpha diversity level of the samples and the beta diversity differences between different groups are assessed, and the differences in community structure between different groups are further measured.

Alpha diversity (Chao1, Pielou, Shannon, and Simpson indices) showed that the richness and evenness of the fecal microbial community in the CR group were significantly reduced compared to the Con group. All interventions reversed the alpha diversity index, indicating that LF/Bifidobacterium breve HH079 intervention alone or in combination affected the gut microbial diversity of the pups (Figure 10).

Principal coordinate analysis (PCoA) further revealed distinct clustering of the microbiota in different experimental groups, and the LF+HH079 group clustered more closely to the control group than the CR group, indicating that synergistic supplementation with LF and Bifidobacterium breve HH079 is more beneficial for restoring the diversity of the gut microbiota after CR infection (Figure 11).

Next, the gut microbiota composition of each group was assessed at the taxonomic level. Following infection with *Citrobacter rodentii*, the abundance of pathogenic bacteria in the mouse gut increased, including a relative increase in *Staphylococcus *, *Citrobacter A* , and *Enterococcusus H*. Different interventions reduced the abundance of gut pathogenic bacteria. LF supplementation reduced *Lactobacillus* abundance and significantly promoted * Alistipes A * abundance (reducing inflammation and producing small amounts of acetic acid and propionic acid). Supplementation with *Bifidobacterium breve* HH079 enriched *Bifidobacterium * and *Muribaculum * (potential probiotics with cross-feeding relationships with probiotics such as *Bifidobacterium* and *Lactobacillus*). Synergistic supplementation with LF and *Bifidobacterium breve* HH079 reduced gut pathogenic bacteria and promoted the enrichment of more beneficial bacteria (Figure 12).

Example 8 Changes in the gut microbiota of mice before infection

Feces were collected from mice in the Con, HH079, LF, and LF+HH079 groups 21 days after birth. Alpha diversity analysis and principal coordinate analysis were performed according to the method described in Example 7, and the gut microbiota composition of each group was assessed at the taxonomic level.

The results of α-diversity analysis (Figure 13) and principal coordinate analysis (Figure 14) showed that supplementation with LF alone or in combination with Bifidobacterium breve HH079 in early life had little effect on the α-diversity composition of the mouse gut microbiota; PCOA analysis confirmed that supplementation with LF alone or in combination with Bifidobacterium breve HH079 made the composition of the mouse gut microbiota more uniform.

Taxonomic assessment of gut microbiota composition in each group showed that LF supplementation alone promoted Alistipes A abundance (reduced inflammation, and produced small amounts of acetic acid and propionic acid), LF supplementation alone enriched Bifidobacterium breve HH079 and UBA3282 (Lachnospiraceae) , and LF and Bifidobacterium breve HH079 synergistically promoted the enrichment of more beneficial bacteria (Figure 15).

The above detailed description is a specific illustration of one feasible embodiment 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 modifications 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 should be determined by the appended claims.

Claims (13)

1. The application of Bifidobacterium breve in conjunction with lactoferrin in the preparation of products for regulating intestinal flora or antibacterial infection, characterized in that the Bifidobacterium breve is Bifidobacterium breve HH079, with accession number GDMCC No: 64216. 2. The application according to claim 1, characterized in that, in the intestinal flora regulating product or antibacterial infection product, the ratio of added Bifidobacterium breve HH079 to lactoferrin is ^10⁸ - ^10¹⁰ CFU: 1-1000 mg. 3. The application according to claim 1, wherein the Bifidobacterium breve HH079 comprises live, inactivated, cell-wall-broken, secretions, or metabolites of Bifidobacterium breve HH079. 4. The application according to claim 1, wherein the product for regulating intestinal flora includes food or health products. 5. The application according to claim 4, wherein the dosage form of the food includes a liquid dosage form, a solid dosage form, or a semi-solid dosage form. 6. The application according to claim 4, wherein 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, characterized in that the food or health product further includes nutritionally acceptable nutritional additives. 8. The application according to claim 7, wherein the nutritional additive comprises one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins. 9. The application according to claim 1, wherein the antibacterial infection product comprises food additives or pharmaceuticals. 10. The application according to claim 9, wherein the food additive is added to human food or animal food. 11. The application according to claim 9, wherein the medicine is used for the prevention, treatment or adjunctive treatment of diarrhea. 12. The application according to claim 9, wherein the dosage form of the medicine includes tablets, pills, powders, suspensions, gels, emulsions, creams, granules, capsules, suppositories, injections, sprays, or injections. 13. The application according to claim 9, wherein the pharmaceutical product further comprises one or more physiologically acceptable excipients.