Biological agent, preparation method thereof and use thereof in preparing product for inhibiting Escherichia coli

Abstract

The present invention belongs to the field of microorganisms and fermentation, and relates to a biological preparation and a preparation method thereof and its application in the preparation of a product for inhibiting Escherichia coli. The biological preparation is a mixture of Forsythia suspensa and Bacillus amyloliquefaciens ( Bacillus amylolyticus The biological preparation provided by the present invention can significantly increase the secretion of IFN-α in mice, reduce the production of IL-1β, IL-6, and TNF-α, and increase CD3 + CD4 + and CD3 + CD8 + The biopharmaceutical preparation demonstrated a stronger synergistic effect than the use of Forsythia suspensa and Bacillus amyloliquefaciens alone, effectively preventing and inhibiting Escherichia coli infection and preventing the development of drug resistance. The preparation method is simple, the raw materials are readily available, and the preparation is suitable for the development of antimicrobial drugs and feed additives.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms and fermentation, and relates to a biological preparation, a preparation method thereof, and an application thereof in the preparation of a product for inhibiting Escherichia coli. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Calf Escherichia coli diarrhea is a common and serious disease in the cattle industry, posing a significant threat to the health and growth of calves. Escherichia coli, a common opportunistic pathogen, is widely present in the natural environment. When calves have weakened immunity or are reared in poor conditions, they are highly susceptible to infection. Escherichia coli has a complex antigenic structure, primarily composed of somatic antigens (O), surface antigens (K), and flagellar antigens (H). Currently, there are 171 known O antigens, 100 K antigens, and 56 H antigens. These different antigen combinations form numerous serotypes. For example, serotypes such as O101 and O103 are common in cases of E. coli diarrhea in calves. These serotypes are often highly pathogenic and are closely associated with the development of calf diarrhea. It has been established that E. coli is one of the main pathogens causing calf diarrhea, and the pathogenicity of different E. coli serotypes varies. Serotypes such as O101 and O103 are considered to be the most common and dominant serotypes. Research has also found that low immunity in calves, poor rearing environments, and stress factors can increase the risk of E. coli infection. Newborn calves, lacking adequate colostrum intake, lack access to maternal antibodies, resulting in compromised immunity and increased susceptibility to E. coli infection. Calf infection with E. coli primarily manifests as diarrhea, with feces that are watery or mushy, ranging in color from pale yellow to white, often with a foul odor and sometimes streaked with blood. Affected calves may also experience systemic symptoms such as lethargy, loss of appetite, and elevated body temperature. Severe cases can lead to dehydration, acidosis, and even death. The incidence of E. coli diarrhea in calves is increasing under intensive farming practices. E. coli diarrhea in calves also has potential impacts on the quality and safety of beef and milk. Infected calves may carry drug-resistant E. coli, which can spread through the food chain and pose a threat to human health.

[0004] At present, the drugs used to treat E. coli diarrhea in calves are mainly antibiotics. However, traditional antibiotics can easily induce drug resistance in E. coli in calves, and also have a certain inhibitory effect on other beneficial bacteria. There are also problems such as antibiotic residues. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the present invention aims to provide a biological preparation and a preparation method thereof and its application in the preparation of a product for inhibiting Escherichia coli. The biological preparation provided by the present invention can significantly increase the secretion of IFN-α in mice, reduce the production of IL-1β, IL-6, and TNF-α, and increase CD3 + CD4 + and CD3 + CD8 + The biological preparation demonstrated a stronger synergistic effect compared to using Forsythia suspensa and Bacillus amyloliquefaciens alone, effectively preventing and inhibiting Escherichia coli infection and preventing the development of drug resistance. The preparation method is simple, and the raw materials are readily available, making it suitable for the development of antimicrobial drugs and feed additives.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, a biological preparation is provided, wherein the biological preparation is a combination of Forsythia suspensa and Bacillus amyloliquefaciens ( Bacillus amylolyticus ) co-fermentation products.

[0008] In some embodiments, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101, deposited in the China Center for Type Culture Collection (CCTCC), the deposit number is CCTCC NO: M20241003, the deposit date is May 20, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0009] In some embodiments, the forsythia suspensa is a decoction of forsythia suspensa. The forsythia suspensa of the present invention complies with the standards of the Chinese Pharmacopoeia.

[0010] In some embodiments, the co-fermentation product is a whole fermentation broth.

[0011] It has been verified that the biological preparation provided by the present invention has the following beneficial effects: immunomodulatory effect, including: significantly increasing the secretion of IFN-α in mice, reducing the production of IL-1β, IL-6, and TNF-α, and increasing CD3 + CD4 + and CD3 + CD8 + T cell ratio, increase the expression of ZO-1 and Occludin in the duodenum and jejunum, maintain intestinal tissue integrity, and reduce inflammatory response.

[0012] In in vivo experiments, the biological preparation provided by the present invention showed: preventive effects, including maintaining animal weight, improving survival rate, and improving diarrhea; safety, including no adverse reactions during administration, no organ toxicity, and good tolerance.

[0013] In a second aspect, a method for preparing the above-mentioned biological agent comprises the following steps:

[0014] Forsythia suspensa and Bacillus amyloliquefaciens are co-fermented to obtain a co-fermentation product.

[0015] In some embodiments, the process parameters of co-fermentation are as follows: pH range: 6.5-7.2, preferably 6.8-7.0; fermentation temperature: 30-40°C, preferably 37°C; fermentation time: 24-96 hours, preferably 72 hours; and the fermentation method is preferably anaerobic static fermentation.

[0016] In some embodiments, the Forsythia suspensa is prepared into a Forsythia suspensa decoction, which is then inoculated with Bacillus amyloliquefaciens for co-fermentation.

[0017] Specifically, a Forsythia suspensa decoction is mixed with a liquid culture medium, and then inoculated with Bacillus amyloliquefaciens for co-fermentation. More specifically, the liquid culture medium is MRS medium. More specifically, the Forsythia suspensa decoction is mixed with the liquid culture medium, the pH is adjusted, and the mixture is sterilized, and then inoculated with Bacillus amyloliquefaciens for co-fermentation. More specifically, the volume ratio of the Forsythia suspensa decoction to the liquid culture medium is 1:15-20.

[0018] Specifically, the inoculation amount of Bacillus amyloliquefaciens is 8-10% (volume), and the inoculated bacterial solution is a bacterial solution in the logarithmic growth phase that has been activated for 24-36 hours.

[0019] Specifically, the method for making Forsythia suspensa water decoction is as follows: crush Forsythia suspensa into coarse powder, sieve it, add distilled water to soak it, heat it to boiling after soaking, keep it in a slightly boiling state and decoct it, filter it after decoction, and collect the filtrate; repeatedly decoct the filter residue according to the above method, and combine the filtrate; decompress the combined filtrate to obtain a Chinese medicine concentrate, which is Forsythia suspensa water decoction.

[0020] The preferred preparation method of the forsythia suspensa decoction comprises:

[0021] 1. Raw material pretreatment:

[0022] Grind the forsythia into coarse powder and pass it through a 30-50 mesh sieve;

[0023] Add distilled water at a material-liquid ratio of 1:8-12 (g / mL) and soak for 12-24 hours;

[0024] 2. Extraction process:

[0025] Place the soaking liquid in the extraction tank;

[0026] Heat to boiling and keep it at a simmer;

[0027] The first decoction is 1.5 to 2 hours;

[0028] Filter through 3 to 5 layers of gauze and collect the filtrate;

[0029] Add water to the residue and boil it repeatedly 1-2 times, and combine the filtrate;

[0030] 3. Concentration treatment:

[0031] The combined filtrate was concentrated under reduced pressure, the temperature was controlled at 60-65°C, and the relative density was 1.1-1.2;

[0032] Sterilize at 120-122℃ for 15-25 minutes, cool to room temperature and set aside.

[0033] In a third aspect, a use of the above biological preparation in the preparation of a product for inhibiting Escherichia coli.

[0034] In some embodiments, the product is a medicine or a feed additive.

[0035] Specifically, the drug can be used to prevent and / or alleviate infectious diseases caused by E. coli infection. Prevention refers to preventing E. coli infection, enhancing animal immunity, improving intestinal health, and enhancing production performance. Treatment refers to alleviating infection symptoms, reducing inflammatory responses, repairing intestinal damage, and improving clinical prognosis.

[0036] Specifically, the drug can be a veterinary drug, an anti-infective drug, an intestinal drug, an immunomodulator, etc.

[0037] Specifically, the feed additives can be probiotic preparations, preventive additives, nutritional enhancers, immune enhancers, etc.

[0038] In some embodiments, the product can be a liquid preparation or a solid preparation. The liquid preparation can be a fermentation broth, an oral solution, etc. The solid preparation can be a lyophilized powder, a granule, etc.

[0039] In some embodiments, the product further includes excipients. These excipients can be added depending on the type of formulation. These excipients can include preservatives, excipients, stabilizers, and the like. When excipients are used, the content of the biological preparation (co-fermentation product) as the active ingredient can be adjusted as needed.

[0040] In some embodiments, the product is used for cattle, especially calves.

[0041] The beneficial effects of the present invention are:

[0042] The co-fermentation composition of Bacillus amyloliquefaciens and Forsythia suspensa of the present invention exhibits significant technical effects and excellent immunomodulatory effects. In terms of enhancing the body's immune function, the present invention can significantly increase the secretion of the antiviral factor IFN-α and reduce the production of pro-inflammatory factors IL-1β, IL-6, and TNF-α. This effect is significantly better than using Forsythia suspensa or Bacillus amyloliquefaciens alone. In terms of immune cell regulation, the present invention can significantly increase the secretion of the antiviral factor IFN-α and reduce the production of pro-inflammatory factors IL-1β, IL-6, and TNF-α. + CD4 + T cell ratio enhances humoral immunity and maintains CD3 + CD8 + The T-cell level enhances cellular immunity, demonstrating stronger immunomodulatory capabilities than any component used alone. In terms of protecting the intestinal barrier, the present invention can significantly increase the expression of tight junction proteins ZO-1 and Occludin, maintain the morphological integrity of intestinal tissue, and reduce congestion, edema, and mucosal damage, with a protective effect superior to that of using any component alone.

[0043] Compared with the prior art, the present invention achieves a synergistic effect through the co-fermentation of traditional Chinese medicine and probiotics, overcoming the problem of limited effect of using them alone; compared with traditional antibiotics, the present invention is not easy to produce drug resistance, has no residue problem, and has little effect on intestinal flora. In addition, the present invention has the characteristics of simple and feasible preparation method and wide source of raw materials, and can be used for drug development as well as the development of feed additives. The above technical effects and excellent effects have been fully verified by multiple experimental indicators such as weight change, survival rate, immune indicators, and tissue pathology, indicating that the present invention has significant practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0045] Figure 1 This is the weight change curve of mice in the embodiment of the present invention.

[0046] Figure 2 This is a graph showing the diarrhea scoring results of mouse feces in an embodiment of the present invention.

[0047] Figure 3 This is the mouse survival curve in the embodiment of the present invention.

[0048] Figure 4 The graph shows the changes in cytokines in mouse serum after infection with E. coli in the examples of the present invention. A: IFN-α content in mouse serum; B: IL-1β content in mouse serum; C: IL-6 content in mouse serum; D: TNF-α content in mouse serum; where **** P <0.0001;*** P<0.001;** P <0.01;* P <0.05; ns (no significance) P >0.05.

[0049] Figure 5 The results of the experiment in the present invention are as follows: A: CD3 T cells in the spleen of mice + CD4 + The number of T cells; B: CD3 + CD8 + The number of T cells, **** P <0.0001;*** P <0.001;** P <0.01;* P <0.05; ns (no significance) P >0.05.

[0050] Figure 6 The results of the relative expression of tight junction proteins in the duodenum of mice in each group of experiments in the present invention are shown in FIG. A: relative expression of Occludin in the duodenum; B: relative expression of ZO-1 in the duodenum; where **** P <0.0001;*** P <0.001;** P <0.01;* P <0.05; ns (no significance) P >0.05.

[0051] Figure 7 The results of the relative expression of tight junction proteins in the jejunum of each group of mice in the experiment in the present invention are shown in FIG. A: relative expression of Occludin in the jejunum; B: relative expression of ZO-1 in the jejunum; where **** P <0.0001;*** P <0.001;** P <0.01;* P <0.05; ns (no significance) P >0.05.

[0052] Figure 8 The results of the secretory sIgA content in the feces of each group of mice in the embodiment of the present invention are shown in FIG. P <0.0001;*** P <0.001;** P <0.01;* P<0.05; ns (no significance) P >0.05.

[0053] Figure 9 These are pathological tissue sections of the duodenum of mice in each group in the examples of the present invention; scale bar: 100 μm.

[0054] Figure 10 Schematic diagram of the jejunum pathological tissue sections of mice in each group in the examples of the present invention; scale bar 50 μm. DETAILED DESCRIPTION

[0055] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0056] Example 1 Screening of compositions for inhibiting the growth of Bacillus amyloliquefaciens

[0057] 1. Experimental Materials: This example selected 10 Chinese medicinal herbs as research subjects, including Forsythia suspensa, Scutellaria baicalensis, Coptis chinensis, Phellodendron amurense, Honeysuckle, Radix Isatidis, Taraxacum mongolicum, Folium Isatidis, Herba Houttuyniae, and Sophora flavescens. These herbs were purchased from local reputable Chinese medicinal herb pharmacies and were found to meet the relevant standards of the Chinese Pharmacopoeia.

[0058] Strain: Escherichia coli ( Escherichia coli ) strain is calf Escherichia coli, and Escherichia coli was purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC), with the BNCC number BNCC364150. In subsequent experiments, it is referred to as Escherichia coli. The strain was stored in cooked meat culture medium and placed in a 4°C refrigerator. It was subcultured every 1-2 weeks to maintain its activity. Before the experiment, Bacillus amyloliquefaciens was inoculated on an anaerobic blood agar plate and cultured in an anaerobic incubator (37°C, 5% CO2, 10% H2, 85% N2) for 18-24 hours. A single colony was picked and inoculated into a thioglycollate fluid culture medium and cultured at 37°C for 6-8 hours to prepare a bacterial suspension. The bacterial concentration was adjusted to 1×10 6 -1×10 7 CFU / mL, reserve for future use.

[0059] Bacillus amyloliquefaciens ( Bacillus amylolyticus ) strain is Bacillus amyloliquefaciens ( Bacillus amylolyticus) NM1101 was kindly provided by the College of Veterinary Medicine, Jilin Agricultural University, and deposited with the China Center for Type Culture Collection (CCTCC) as CCTCC NO: M 20241003 on May 20, 2024, at Wuhan University, Wuhan, China. In subsequent experiments, it will be referred to as Bacillus amyloliquefaciens. This strain was maintained on MRS medium slants at 4°C and passaged monthly. For use, Bacillus amyloliquefaciens was inoculated into MRS liquid medium and incubated at 30°C for 24-36 hours. Then, a 2%-3% inoculum was transferred to fresh MRS liquid medium and cultured until the logarithmic growth phase for fermentation experiments.

[0060] 2. Main reagents: MRS medium, cooked meat medium, anaerobic blood agar plate, thioglycollate fluid medium, glucose, yeast extract powder, peptone, sodium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, calcium carbonate, ethanol, ethyl acetate, n-butanol, petroleum ether, hydrochloric acid, sodium hydroxide, chloroform, methanol, acetonitrile, etc., all of analytical grade reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0061] 3. Main instruments: HZQ-F160 full-temperature oscillating incubator (Harbin Donglian Electronic Technology Development Co., Ltd.), SPX-25B biochemical incubator (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory), SW-CJ-2FD double-person single-sided clean bench (Suzhou Purification Equipment Co., Ltd.), LDZX-50KBS vertical pressure steam sterilizer (Shanghai Shen'an Medical Equipment Factory), TDL-5-A centrifuge (Shanghai Anting Scientific Instrument Factory), RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), etc.

[0062] 4. Experimental methods

[0063] 1) Preparation of Chinese medicine solution

[0064] Ten Chinese medicinal herbs, including Forsythia suspensa, Scutellaria baicalensis, Coptis chinensis, Phellodendron amurense, Honeysuckle, Radix Isatidis, Herba Taraxaci, Folium Isatidis, Herba Houttuyniae, and Sophora flavescens, were pulverized into coarse powder using a grinder and passed through a 40-mesh sieve. For each coarse powder, the following steps were performed: a certain amount of coarse powder was weighed and added to distilled water at a solid-liquid ratio of 1:10 (g / mL). The powder was then soaked for 12 hours. The soaked solution was then placed in a multifunctional extraction tank, heated to boiling, and maintained at a slight boil for 1.5 hours. After decoction, the solution was filtered through four layers of gauze, and the filtrate was collected. The residue was decocted once again according to the above method, and the filtrates were combined. The combined filtrates were concentrated under reduced pressure to a relative density of 1.1 (at 60°C), approximately 1 / 5 of the original volume, to obtain a concentrated Chinese medicinal solution (referred to as the Chinese medicinal solution in subsequent experiments). Finally, the concentrated Chinese medicinal solution was sterilized at 121°C for 20 minutes, cooled to room temperature, and set aside for co-fermentation with Bacillus amyloliquefaciens and in vitro antibacterial experiments in this example.

[0065] 2) Cultivation of Bacillus amyloliquefaciens and co-fermentation with traditional Chinese medicine

[0066] Bacillus amyloliquefaciens strains stored on an MRS slant were inoculated into MRS liquid medium and incubated at 30°C for 24 hours for activation. The activated strains were then transferred to fresh MRS liquid medium at a 2% volume inoculum and cultured until the logarithmic growth phase, serving as a seed culture. The prepared traditional Chinese medicine solution was mixed evenly with the MRS liquid medium at a volume of 6% (volume) of the MRS liquid medium, and the pH was adjusted to 6.8. The seed culture was then inoculated with 10% of the volume of the Bacillus amyloliquefaciens strains and fermented anaerobically at 37°C for 72 hours. During the fermentation process, samples were collected every 24 hours to measure the bacterial count and pH to monitor the fermentation progress.

[0067] 3) In vitro antibacterial test method

[0068] The diameter of the inhibition zone of Bacillus amyloliquefaciens was determined by the Oxford cup method. First, the Escherichia coli suspension was adjusted to a concentration of 1×10 7 CFU / mL. Evenly spread 100 μL of the bacterial suspension onto an anaerobic blood agar plate. Place three Oxford cups (6 mm inner diameter, 8 mm outer diameter, 10 mm height) on each plate in an equilateral triangle arrangement, with a minimum spacing of 25 mm between cups. Add 200 μL of the test substance to each Oxford cup. Incubate the plate in an anaerobic incubator (37°C, 5% CO₂, 10% H₂, 85% N₂) for 18 hours. Observe for the formation of inhibition zones and measure their diameters using a vernier caliper.

[0069] 4) Optimization of fermentation conditions

[0070] Chinese medicinal materials were fermented with Bacillus amyloliquefaciens, and the fermentation time, fermentation temperature, and strain inoculation amount were optimized to obtain the best fermentation method. The fermentation broth obtained by fermentation was subjected to in vitro antibacterial experiments (Oxford cup method, the same method as above). At the same time, the Bacillus amyloliquefaciens fermentation group without adding Chinese medicinal liquid was used as a control.

[0071] (1) Fermentation time optimization:

[0072] Add 6% of the traditional Chinese medicine (Fructus Forsythiae) to 50 mL of MRS liquid medium, adjust the pH to 6.8, autoclave at 121°C for 25 minutes, and inoculate with 5% of the bacterial suspension. Ferment at 37°C for 24, 48, 72, and 96 hours. Determine the inhibition zone against Bacillus amyloliquefaciens after fermentation to evaluate the optimal fermentation time.

[0073] (2) Fermentation temperature optimization:

[0074] To 50 mL of MRS liquid medium, add 6% by volume of a traditional Chinese medicine solution of Forsythia suspensa, adjust the pH to 6.8, sterilize at 121°C for 25 minutes, inoculate with 5% of the bacterial solution, and adjust the pH to 6.8. Following the optimal fermentation time, incubate at 30°C, 35°C, 37°C, 40°C, and 42°C for 24 hours. The size of the inhibition zone against Bacillus amyloliquefaciens after fermentation was measured, and this evaluation index was used to select the optimal fermentation temperature.

[0075] (3) Optimization of bacterial inoculation amount:

[0076] Add 6% volume of Forsythia suspensa (Chinese herbal medicine) to 50 mL of MRS liquid medium, adjust the pH to 6.8, and sterilize at 121°C for 25 minutes. Then, inoculate the fermentation broth with 0.1%, 1%, 10%, and 100% of the bacterial suspension, respectively. Optimal fermentation time and temperature were determined. The inhibition zone size against Bacillus amyloliquefaciens was measured after fermentation, and the inoculum size of the optimal strain was selected.

[0077] 5. Results

[0078] 1) Pre-fermentation test of Bacillus amyloliquefaciens and traditional Chinese medicine

[0079] Table 1 Monitoring of bacterial count changes in co-fermentation of Bacillus amyloliquefaciens and traditional Chinese medicine (10 7 CFU / ml)

[0080]

[0081] Table 2 Monitoring pH changes during co-fermentation of Bacillus amyloliquefaciens and traditional Chinese medicine

[0082]

[0083] 2) In vitro inhibitory effect of Chinese herbal medicine on Bacillus amyloliquefaciens

[0084] In vitro antibacterial experiments were conducted using traditional Chinese medicine solutions as test substances. By measuring the size of each inhibition zone and comparing the antibacterial effects of various traditional Chinese medicine solutions, the results showed that Forsythia suspensa had the best inhibitory effect, with an inhibition zone diameter of 23.5 ± 0.1 mm. The results are shown in Table 3. Therefore, Forsythia suspensa was subsequently co-fermented with Bacillus amyloliquefaciens and optimized.

[0085] Table 3 Antibacterial effect of various Chinese medicinal solutions on Bacillus amyloliquefaciens

[0086]

[0087] 3) Optimization of Traditional Chinese Medicine Fermentation by Bacillus amyloliquefaciens

[0088] (1) Optimization of fermentation time

[0089] In vitro antibacterial experiments were conducted using fermentation broths of Bacillus amyloliquefaciens and co-fermentation broths of Bacillus amyloliquefaciens and Forsythia suspensa. The inhibition zones of B. amyloliquefaciens were determined at different fermentation times. The results are shown in Table 4. The inhibition zone diameter initially increased and then stabilized with increasing fermentation time. At 48 h of fermentation, the inhibition zone diameter reached a maximum of (24.9 ± 1.3) mm. After 72 h of fermentation, the zone diameter reached (23.3 ± 0.8) mm. After 96 h of fermentation, the inhibition zone diameter decreased to (21.1 ± 0.7) mm. This indicates that fermentation time significantly influences the antibacterial effect of the co-fermentation broth, with the strongest inhibitory effect against B. amyloliquefaciens occurring around 48 h.

[0090] Table 4 Inhibition zone diameters of Bacillus amyloliquefaciens at different fermentation times (mm)

[0091]

[0092] (2) Optimization of fermentation temperature

[0093] In vitro antibacterial experiments were conducted using fermentation broth of Bacillus amyloliquefaciens and co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa. The inhibition zones of B. amyloliquefaciens were determined at different fermentation temperatures. The results are shown in Table 5. The results show that the diameter of the inhibition zone initially increased and then decreased with increasing fermentation temperature. At 37°C, the maximum inhibition zone diameter was (23.4 ± 0.8) mm. When the fermentation temperature was increased to 42°C, the inhibition zone diameter decreased to (16.5 ± 0.9) mm. This indicates that fermentation temperature significantly affects the antibacterial effect of the co-fermentation broth, with the strongest inhibitory effect on B. amyloliquefaciens occurring at around 37°C.

[0094] Table 5 Diameters of inhibition zones against Bacillus amyloliquefaciens at different fermentation temperatures (mm)

[0095]

[0096] (3) Screening of bacterial inoculum volume

[0097] In vitro antibacterial experiments were conducted using fermentation broth of Bacillus amyloliquefaciens and co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa. The inhibition zone diameters of B. amyloliquefaciens were determined at different inoculum concentrations. The results are shown in Table 6. The results show that the inhibition zone diameter initially increased and then decreased with increasing inoculum concentration. At a 10% inoculum concentration, the inhibition zone diameter reached a maximum of (25.3 ± 0.5) mm. At a 100% inoculum concentration, the inhibition zone diameter reached (18.4 ± 1.1) mm. This indicates that the inoculum concentration significantly affects the antibacterial effect of the co-fermentation broth, with the strongest inhibitory effect against B. amyloliquefaciens occurring at approximately 10%.

[0098] Table 6 Inhibition zone diameter of Bacillus amyloliquefaciens at different bacterial inoculation amounts (mm)

[0099]

[0100] Example 2 Investigation of the protective effect of co-fermentation of Bacillus amyloliquefaciens and Forsythia suspensa on mice infected with Bacillus amyloliquefaciens

[0101] In this example, Bacillus amyloliquefaciens co-fermented with Forsythia suspensa (co-fermentation group) was fed to mice in a challenge protection test, along with a healthy control group, a Bacillus amyloliquefaciens group, and a Forsythia suspensa group. ELISA, flow cytometry, and H&E staining were used to analyze indicators such as mouse weight change, serum levels of IFN-α, TNF-α, IL-1β, and IL-6, activation of immune organs and cells, and pathological changes in the small intestine. This study examined the protective effect of the Forsythia suspensa and Bacillus amyloliquefaciens co-fermentation product against Bacillus amyloliquefaciens infection in mice.

[0102] 1. Experimental strains:

[0103] Escherichia coli ( Escherichia coli ) strain is calf Escherichia coli, and Escherichia coli was purchased from Beina Bio-Henan Industrial Microbial Strain Engineering Technology Research Center (BNCC), with the strain number BNCC364150, and is referred to as Escherichia coli in subsequent experiments.

[0104] Bacillus amyloliquefaciens ( Bacillus amylolyticus ) strain is Bacillus amyloliquefaciens ( Bacillus amylolyticus) NM1101 was kindly obtained from the College of Veterinary Medicine of Jilin Agricultural University and deposited in the China Center for Type Culture Collection (CCTCC) with the number CCTCC NO: M 20241003. The deposit date is May 20, 2024, and the deposit address is Wuhan University, Wuhan, China. In subsequent experiments, it is referred to as Bacillus amyloliquefaciens.

[0105] 2. Experimental Animals

[0106] Fifty healthy 30-day-old mice were selected and randomly divided into 5 groups, with 10 mice in each group. They were housed in isolation and fed with food and water at 8:00, 14:00, and 20:00 every day.

[0107] 3. Reagents

[0108] Mouse interleukin-1β (IL-1β), mouse interferon-α (IFN-α), mouse tumor necrosis factor-α (TNF-α), and mouse interleukin-6 (IL-6) ELISA kits were purchased from ELISA (Jiangsu) Industrial Co., Ltd. Mouse CD3-PE-Cy7 antibody, CD4-PE antibody, and CD8-FITC antibody were all purchased from BD Biosciences; 1640 cell culture medium was purchased from Hyclone; blocked mouse serum was stored in the laboratory; antibody diluent: 1% BSA; red blood cell lysis buffer, Fluor 488-labeled goat anti-rabbit IgG (H + L) (Biyuntian Biotechnology Co., Ltd.); FACS solution (1000 mL PBS for cell culture, 10 mL FBS, 0.9 g sodium azide); AXYGEN PCRSTRIP TUBES (Corning Incorporated, USA); Mini BEST Viral RNA / DNA Extraction Kit Ver5.0 (Code No. 9766 Takara); goat serum for blocking; DAPI staining solution; antifade mounting medium; 4% paraformaldehyde solution; sodium citrate antigen retrieval solution.

[0109] 4. Main experimental equipment:

[0110] ABI Prism7500QT-qPCR instrument (ABI, USA); heparin sodium anticoagulant tubes and procoagulant tubes were purchased from (Jiangsu) Kangjian Medical Supplies Co., Ltd.; tissue embedding machine, paraffin slicer, and DMi8 fluorescence inverted microscope were purchased from (Germany) Leica Instrument Co., Ltd.; tissue homogenizer was domestically produced.

[0111] 5. Experimental methods:

[0112] 1) Experimental plan

[0113] The experimental animals were divided into the following groups: healthy group (n=10), Bacillus amyloliquefaciens group (n=10), Forsythia suspensa group (n=10), co-fermentation group (n=10), and control group (n=10). Male and female animals were randomly assigned. The groups are shown in Table 7 below.

[0114] Table 7 Animal test groups

[0115]

[0116] Immunization and virus attack procedures:

[0117] Each group of mice was immunized on day 1, day 3, and day 5. Except for the healthy group and the control group, which were gavaged with 0.5 mL PBS, the other groups were gavaged with the corresponding drugs. Each mouse in the Bacillus amyloliquefaciens group was gavaged with 1×10 9 cfu / mL, 0.5 mL. Each mouse in the Forsythia group was orally administered with 0.5 mL of Forsythia solution, and each mouse in the co-fermentation group was orally administered with 0.5 mL of the co-fermentation solution of Bacillus amyloliquefaciens and Forsythia.

[0118] The preparation method of Bacillus amyloliquefaciens fermentation broth is as follows: 10% Bacillus amyloliquefaciens (5 mL) was inoculated into 50 mL of MRS liquid medium and fermented at 37 °C for 72 hours. After the fermentation was completed, the whole fermentation broth was shaken thoroughly and the viable cell count was maintained at 1 × 10 9 cfu / mL, shake well before use.

[0119] Preparation of Forsythia suspensa Traditional Chinese Medicine Liquid: Grind Forsythia suspensa into coarse powder using a grinder, pass through a 40-mesh sieve, weigh the coarse powder, and add it to distilled water at a material-liquid ratio of 1:10 (g / mL) (for example, 25g of Forsythia suspensa coarse powder to 250mL of distilled water). Soak for 12 hours. The soaked liquid is then placed in a multifunctional extraction tank, heated to boiling, and maintained at a slight boil for 1.5 hours. After decoction, filter through four layers of gauze, and collect the filtrate. Repeat the decoction once with the residue as described above, and combine the filtrates. The combined filtrates are concentrated under reduced pressure to a relative density of 1.1 (at 60°C), approximately 1 / 5 of the original volume, to obtain 50mL of the traditional Chinese medicine concentrate. Finally, sterilize the traditional Chinese medicine concentrate at 121°C for 20 minutes, cool to room temperature, and shake thoroughly before use.

[0120] Preparation method of co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa: add 6% of the aforementioned Forsythia suspensa Chinese medicine solution (i.e., 3 mL) to 50 mL of MRS liquid medium, adjust the pH to 6.8, sterilize at 121°C for 20 min, inoculate 10% of Bacillus amyloliquefaciens (5 mL) and ferment at 37°C for 72 hours. After the fermentation is completed, shake thoroughly and take the entire fermentation broth to ensure that the number of viable bacteria remains at 1×10 9 cfu / mL, shake well before use.

[0121] After immunization, the mice were weighed once a day and the data were recorded to calculate the average daily weight gain. On day 5, except for the healthy group, which was injected with 0.5 mL PBS intraperitoneally, the other groups were injected with Escherichia coli suspension (concentration of 1×10 9 CFU / mL, injection volume was 0.5 mL).

[0122] 2) Growth performance index testing

[0123] Body weight: Mice were weighed daily, and weight changes were recorded and plotted. Feces were recorded and scored daily. Fecal scoring: Rectal feces were collected and evaluated for each mouse, and a veterinarian used these images to score the consistency of the feces. Fecal consistency scores ranged from -2 to 2, where 0 = normal; 1 = loose stools; 2 = hard stools; -1 = mild diarrhea; and -2 = severe diarrhea.

[0124] 3) Detection methods for immune indicators IFN-α, TNF-α, IL-1β, and IL-6

[0125] After infection with E. coli, blood was collected from the eyeballs of each group of mice. One mL of blood was collected and centrifuged in a centrifuge tube. Serum was separated and stored at -80°C. Serum levels of IFN-α, TNF-α, IL-1β, and IL-6 were measured using ELISA.

[0126] 4) Preparation of single-cell suspension

[0127] Preparation of spleen single-cell suspension: Gently triturate spleen sections to obtain a single-cell suspension. Pass the suspension through a 200-mesh strainer into a corresponding 15 mL centrifuge tube, balance, and centrifuge at 1650 rpm at 4°C for 5 min. Discard the supernatant and resuspend the cells in 1 mL of red blood cell lysis buffer. Lyse on ice for 10 min. Remove the tube 5 minutes in between and shake for 30 seconds. Add 10 mL of PBS buffer to terminate the suspension. Balance the tube and centrifuge at 1650 rpm at 4°C for 5 min. Discard the supernatant and resuspend the cells in 1 mL of FACS buffer. Count the cells.

[0128] Preparation of mesenteric lymph node single-cell suspension: Carefully and gently grind all lymph nodes into a single-cell suspension. Pour the suspension through a 200-mesh strainer into a corresponding 15 mL centrifuge tube. Centrifuge at 1650 rpm, 4°C, for 5 min. Discard the supernatant and resuspend the cells in 0.2 mL of FACS buffer and count.

[0129] 5) Flow cytometry

[0130] Flow cytometry antibody staining

[0131] (1) Divide the above single cell suspension into tubes, ensuring that each tube contains 1×10 6 cells (peripheral blood ensures 5×10 5cells), with a total volume of 100 μL;

[0132] (2) Add the corresponding volume of flow cytometry antibody against cell surface markers according to the antibody titer.

[0133] (3) After shaking and mixing, place in a dark place at 4℃ for 30 minutes

[0134] (4) After labeling, add 3.5 ml of ice-cold FACS buffer to the tube, centrifuge at 1650 rpm and 4°C for 5 min, discard the supernatant, and resuspend the cells with a small amount of FACS buffer.

[0135] (5) Repeat step (4) once to fully remove the unbound antibodies and reduce nonspecific staining on the cell surface. Samples were detected using a BD flow cytometer, and data analysis and graphics processing were performed using FlowJo_v10.6.2 software.

[0136] 6) Detection of intestinal tight junction proteins by fluorescence quantitative PCR

[0137] Extracting RNA from sample tissues or cells involves the following steps: First, the sample is added to a lysis buffer and disrupted to release RNA. Next, RNA is separated from the supernatant using a phenol / chloroform mixture and precipitated with isopropanol or ethanol. The RNA precipitate is then washed to remove impurities and finally dissolved in RNase-free water or buffer. The extracted RNA can be assayed for concentration and purity using a colorimetric or fluorescence analyzer and stored at -80°C to prevent degradation. These steps may be adjusted depending on the specific experimental objectives and sample type. Relative quantitative q-PCR was used to detect the transcript levels of two barrier proteins, ZO-1 and occludin, in the duodenum and colon of mice.

[0138] 7) Detection of secretory antibodies in feces

[0139] After infection with E. coli, feces of each group of mice were collected, dissolved in sterile water, and the supernatant was aspirated. The sIgA content in the feces was measured by ELISA.

[0140] 8) H&E staining to detect intestinal pathological changes

[0141] To further observe the pathological changes in the mouse small intestine, the mid-duodenum and mid-jejunum were obtained from the mice. The same part of the intestine was cut from each group and fixed in 4% paraformaldehyde solution for more than two days. The fixed tissue was then neatly cut, embedded, sectioned, and stained with H&E as follows:

[0142] (1) Dehydration: Place the sample in 70% alcohol, 80% alcohol, and 85% alcohol in sequence for 2 hours each, dehydrate in 90% alcohol overnight, dehydrate in 95% alcohol I and 95% alcohol II for 1 hour each, and dehydrate in 100% alcohol I and 100% alcohol II for 1 hour each.

[0143] (2) Transparency: Place the sample in xylene I and xylene II in sequence for 5 minutes each, until the reddish skin sample can be seen with the naked eye.

[0144] (3) Wax immersion: Place the tissue blocks in wax I, wax II, and wax III in sequence. After immersing in wax at 56°C for 1 hour, place the tissue blocks in an embedding box for embedding.

[0145] (4) Sectioning: Cut 3.5 μm thick tissue slices from each embedded block and spread them on clean slides in a 41°C water bath. Place the slides in an 80°C drying oven for 1 hour and then perform H&E staining.

[0146] (5) The H&E staining procedure is as follows: Place the sample in xylene I and xylene II for 8 minutes, then place it in 100% alcohol I, 100% alcohol II, 95% alcohol, 80% alcohol, and 70% alcohol for 1 minute, and rinse it in ultrapure water to remove the part exceeding the alcohol. Then stain it with hematoxylin, rinse it with ultrapure water, differentiate it with 0.5% hydrochloric acid in an alcohol bath for 5 seconds, then rinse it, then place it in light ammonia water for 2 minutes, then rinse it with water, place it in 0.5% eosin aqueous solution for 5 minutes, rinse it with water, rinse it in 80% alcohol bath, observe the eosin staining, and extract it repeatedly in 95% alcohol, 100% alcohol I, and 100% alcohol II alcohol baths 5 times in sequence, then place it in xylene I and xylene II for 2 minutes in sequence, and seal it with neutral gum.

[0147] 6. Experimental results:

[0148] 1) Growth performance index test results

[0149] Body weight changes were recorded during the experiment, and the results showed that the co-fermentation liquid of Bacillus amyloliquefaciens and Forsythia suspensa could protect mice from Escherichia coli infection and reduce the weight loss.

[0150] Specifically, during the immunization period from day 0 to day 5, the weight of mice in all groups showed a steady growth trend, and the growth conditions of each group were similar. After the 5th day of toxicity treatment (intraperitoneal infection with E. coli), the growth of the healthy group was basically unaffected, while the weight of the control group dropped significantly, reflecting the negative impact of E. coli infection. The Forsythia group and the Bacillus amyloliquefaciens group could alleviate the sudden drop in weight to a certain extent, and the weight changes of mice in the co-fermentation group were significantly different. The above weight changes suggest that mice fed with the co-fermentation liquid of Bacillus amyloliquefaciens and Forsythia can resist E. coli infection to a certain extent, playing a preventive and protective role. The results are shown in Figure 1 .

[0151] When scoring the diarrhea of ​​each group of mice, the results showed that the healthy group occasionally increased, but basically maintained at around 2 points, indicating that the feces were normal; the control group's score gradually dropped to -2 points, indicating obvious diarrhea symptoms, which reached a more serious level on the 5th day; the Forsythia group's score gradually dropped below 0, indicating mild diarrhea; the Bacillus amyloliquefaciens group's score basically remained at around 1 point, with occasional mild diarrhea; this indicates that the Forsythia group and the Bacillus amyloliquefaciens group can alleviate the degree of diarrhea to a certain extent compared with the control group, but the co-fermentation group's score has been maintained at 1-2 points, indicating that the feces are normal, which shows that the co-fermentation liquid of Bacillus amyloliquefaciens and Forsythia can alleviate the diarrhea of ​​mice caused by Escherichia coli infection. Figure 2 .

[0152] Figure 3 The survival status of each group is shown. During the 0-5 day immunization period, the survival rate of each group was 100%, indicating that there was no death during the immunization period and the drug administration was safe. After the 5th day of infection (intraperitoneal infection with E. coli), the healthy group and the co-fermentation group maintained a 100% survival rate, indicating that the mice fed with the co-fermentation liquid of Bacillus amyloliquefaciens and Forsythia suspensa showed an excellent preventive and protective effect against E. coli infection; the survival rate of the Forsythia suspensa group and the Bacillus amyloliquefaciens group began to decline from the 6th day, and dropped to 60% on the 6th day, but was still better than the control group. The survival rate of the control group dropped significantly after the infection and dropped to 0 on the 8th day. Figure 3 .

[0153] 2) Immune indicators IFN-α, TNF-α, IL-1β, and IL-6 test results

[0154] The collected serum was used to detect the secretion of cytokines IFN-α, TNF-α, IL-1β, and IL-6 using EILSA. The results are shown in Figure 4 .

[0155] After immunization, mice in each group were infected with E. coli. The IFN-α content in the control group decreased significantly compared with the healthy group (** P<0.01), which indicates that infection with E. coli inhibits the secretion of IFN-α in mice; the difference between the Forsythia group and the control group was not significant (ns), indicating that the Forsythia group did not significantly improve the reduction of IFN-α caused by E. coli infection; the difference in IFN-α between the co-fermentation group and the control group was particularly significant (**** P <0.0001), indicating that the co-fermentation liquid can significantly increase the secretion of IFN-α, and the secretion amount is also increased compared with the healthy group, which shows that the co-fermentation liquid can significantly improve the immunity of mice and resist the infection of Escherichia coli. Figure 4 As shown in A.

[0156] After immunization, mice in each group were infected with E. coli. Compared with the healthy group, the IL-1β content in the control group increased significantly (**** P <0.0001), indicating that infection with E. coli induces the production of a large amount of pro-inflammatory factor IL-1β in mice; compared with the control group, the IL-1β level in the Forsythia suspensa group and the Bacillus amyloliquefaciens group was significantly reduced (**** P <0.0001), but still significantly higher than the healthy group, indicating that both treatments can inhibit the inflammatory response to a certain extent; the difference in IL-1β between the co-fermentation group and the control group was extremely significant (**** P <0.0001), and its IL-1β level was closest to that of the healthy group, indicating that the co-fermentation liquid could significantly inhibit the production of IL-1β. This indicates that the co-fermentation liquid has the strongest anti-inflammatory effect and can effectively inhibit the inflammatory response caused by E. coli infection. Figure 4 As shown in B.

[0157] After immunization, mice in each group were infected with E. coli. The IL-6 content in the control group was significantly increased compared with the healthy group (**** P <0.0001), indicating that infection with E. coli promotes the secretion of the pro-inflammatory factor IL-6 in mice; the IL-6 levels in the Forsythia suspensa group and the Bacillus amyloliquefaciens group were significantly different from those in the control group (*** P <0.001), indicating that the two treatments had a significant effect on maintaining IL-6 levels; the IL-6 levels of the co-fermentation group and the control group were significantly different (**** P <0.0001), the IL-6 level was significantly higher than that of the control group and close to that of the healthy group, indicating that the co-fermentation liquid can inhibit the secretion of higher levels of IL-6, which shows that the co-fermentation liquid can effectively maintain the body's pro-inflammatory ability and help to fight the inflammatory response caused by E. coli infection. Figure 4 As shown in C.

[0158] After immunization, mice in each group were infected with E. coli. Compared with the healthy group, the TNF-α content in the control group increased significantly (**** P<0.0001), indicating that infection with E. coli induces the production of a large amount of pro-inflammatory factor TNF-α in mice; the TNF-α level in the Forsythia group was significantly lower than that in the control group (**** P <0.0001, while the Bacillus amyloliquefaciens group showed a more significant decrease (**** P <0.0001), indicating that both treatments have the effect of inhibiting inflammation, and the difference in TNF-α between the co-fermentation group and the control group is extremely significant (**** P <0.0001), its TNF-α level was the lowest, close to the level of the healthy group, indicating that the co-fermentation liquid had the strongest anti-inflammatory effect and could significantly inhibit the production of TNF-α induced by E. coli infection, further confirming its excellent anti-inflammatory effect. Figure 4 As shown in D.

[0159] 3) Effects of Bacillus amyloliquefaciens and Forsythia suspensa co-fermentation products on TH2 response

[0160] TH2 cell immunity participates in humoral immunity by secreting IL-4, so the activation level of TH2 cells in the spleen was detected. The results showed that after infection with Escherichia coli, Bacillus amyloliquefaciens, Forsythia suspensa Chinese herbal medicine liquid and Bacillus amyloliquefaciens and Forsythia suspensa co-fermentation liquid could activate CD4 + T cells secrete IL-4. It is important that the co-fermentation liquid of Bacillus amyloliquefaciens and Forsythia suspensa can stimulate higher levels of IL-4 and the synergistic fermentation has the best immune protection effect. Figure 5 As shown in A.

[0161] according to Figure 5 It can be seen that the control group has a higher number of CD4 + The proportion of IL-4 T cells decreased significantly (*** P <0.001), which indicates that infection with E. coli significantly inhibits the Th2 immune response in mice; compared with the control group, the CD4 + The proportion of IL-4 T cells increased significantly (* P <0.1, indicating that both treatments can maintain Th2 immune response to a certain extent; the difference between the co-fermentation group and the control group was extremely significant (*** P <0.001), and their CD4 + The proportion of IL-4 T cells was close to that of the healthy group, indicating that the co-fermentation liquid could significantly enhance the Th2 immune response, which means that the co-fermentation liquid could effectively maintain the body's immune balance and enhance the defense against Bacillus amyloliquefaciens infection.

[0162] Cytotoxic T cells produce TH1 immune responses by secreting IFN-γ, so the secretion level of IFN-γ in the spleen was detected. The results showed that after infection with Bacillus amyloliquefaciens, Bacillus amyloliquefaciens, Forsythia suspensa traditional Chinese medicine liquid and Bacillus amyloliquefaciens and Forsythia suspensa co-fermentation liquid could activate CD3 + CD8 + T cells secrete higher levels of IFN-γ, improving cellular immune response, and the co-fermentation of acidobacillus and forsythia suspensa is more effective. Figure 5 As shown in B.

[0163] like Figure 5 As shown in B, the control group had a higher CD8 + The proportion of IFN-γ T cells was not significant (ns). The CD8 + The proportion of IFN-γ T cells was not significant (ns), indicating that the two treatments alone were not effective in maintaining cellular immunity; while the CD8 + The level of IFN-γ T cells was significantly different from that in the control group (*** P <0.001), indicating that the co-fermentation solution can effectively maintain CD8 + The levels of IFN-γ T cells were significantly increased, which showed that the co-fermentation broth had the strongest immune protection effect and could effectively prevent the decline of cellular immune function caused by Escherichia coli infection.

[0164] 4) Quantitative analysis of tight junction proteins in mouse small intestine

[0165] The tight junction proteins ZO-1 (Zonula occludens-1) and occludin were quantified in the duodenum and jejunum of each group of mice. ZO-1 is a scaffolding protein located on the intracellular surface, primarily responsible for connecting other proteins to the cytoskeleton. Occludin is a transmembrane protein that crosses the cell membrane and interacts with occludin in adjacent cells. Together, these two proteins form the intestinal barrier, working together to maintain tight junctions between cells and control the selectivity of substances passing through the intercellular space, thereby protecting the intestine from harmful substances. Reduced expression of these proteins generally indicates impaired intestinal barrier function. Therefore, by measuring the expression levels of ZO-1 and occludin, the integrity and functional status of the intestinal barrier can be assessed.

[0166] After mice were immunized with Bacillus amyloliquefaciens, Forsythia suspensa traditional Chinese medicine liquid and Bacillus amyloliquefaciens and Forsythia suspensa co-fermentation liquid, the relative expression of ZO-1 and Occludin were significantly increased compared with the control group. The results showed that Escherichia coli can damage the duodenum and reduce the expression of duodenal tight junction proteins. Figure 6 .

[0167] Specifically, if Figure 6 As shown in A, the relative expression of Occludin mRNA in the jejunum of the control group decreased significantly compared with the healthy group (*** P <0.001), indicating that infection with E. coli significantly disrupts the expression of the intestinal tight junction protein Occludin; compared with the control group, the expression of Occludin in the Forsythia suspensa group and the Bacillus amyloliquefaciens group was slightly (* P <0.05), indicating that these two treatments have a certain effect on maintaining the integrity of the intestinal barrier; the difference between the co-fermentation group and the control group is extremely significant (** P <0.01), and its occludin expression level was closest to that of the healthy group, indicating that the co-fermentation liquid can effectively maintain the expression of the small intestinal tight junction protein occludin, which shows that the co-fermentation liquid has the strongest intestinal barrier protection effect.

[0168] like Figure 6 As shown in B, the relative expression of ZO-1 mRNA in the duodenum of the control group decreased significantly compared with the healthy group (**** P <0.0001), indicating that infection with E. coli significantly disrupted the expression of duodenal tight junction protein ZO-1; the difference between the Bacillus amyloliquefaciens group and the control group was significant (** P <0.01, indicating that the effects of these two treatments alone on maintaining intestinal barrier integrity were limited; the difference between the co-fermentation group and the control group was extremely significant (**** P <0.0001), and its ZO-1 expression level was closest to that of the healthy group, indicating that the co-fermentation liquid can effectively maintain the expression of duodenal tight junction protein ZO-1, which indicates that the co-fermentation liquid has the strongest intestinal barrier protection effect.

[0169] After mice were immunized with Bacillus amyloliquefaciens, Forsythia suspensa traditional Chinese medicine liquid and Bacillus amyloliquefaciens and Forsythia suspensa co-fermentation liquid, the relative expression levels of ZO-1 and Occludin were significantly increased compared with the control group. The results showed that Escherichia coli can damage the jejunum and reduce the expression of tight junction proteins in the jejunum. Figure 7 .

[0170] Specifically, if Figure 7 As shown in A, the relative expression of Occludin mRNA in the jejunum of the control group decreased significantly compared with the healthy group (**** P<0.0001), indicating that infection with E. coli significantly disrupts the expression of the tight junction protein Occludin in the jejunum; the expression of Occludin in the Forsythia suspensa and Bacillus amyloliquefaciens groups was not significant compared with the control group (ns), indicating that these two treatments had no effect on maintaining the integrity of the intestinal barrier; the difference between the co-fermentation group and the control group was extremely significant (**** P <0.0001), and its occludin expression level was closest to that of the healthy group, indicating that the co-fermentation liquid can effectively maintain the expression of the small intestinal tight junction protein occludin, which shows that the co-fermentation liquid has the strongest intestinal barrier protection effect.

[0171] like Figure 7 As shown in B, the relative expression of ZO-1 mRNA in the jejunum of the control group decreased significantly compared with the healthy group (*** P <0.001), indicating that infection with E. coli significantly disrupted the expression of tight junction protein ZO-1 in the jejunum; the difference between the Bacillus amyloliquefaciens group and the control group was significant (* P <0.1, indicating that the effects of these two treatments alone on maintaining intestinal barrier integrity were limited; the difference between the co-fermentation group and the control group was significant (** P <0.01), and its ZO-1 expression level was closest to that of the healthy group, indicating that the co-fermentation liquid could effectively maintain the expression of the jejunal tight junction protein ZO-1, which indicates that the co-fermentation liquid has the strongest intestinal barrier protection effect.

[0172] 5) Detection of sIgA in mouse feces

[0173] The sIgA content in the feces of mice in each group was detected by ELISA. The results showed that the healthy group was significantly higher than the control group (** P <0.01), E. coli infection reduced the secretion of sIgA in the intestine. The sIgA content in the Forsythia group was significantly increased (** P <0.01), the sIgA content in the Bacillus amyloliquefaciens group was significantly increased (*** P <0.001), the sIgA content in the co-fermentation group was extremely significantly increased (**** P <0.0001), indicating that the co-fermented Chinese medicine liquid can effectively stimulate the intestinal secretion of sIgA to resist Escherichia coli infection. Figure 8 .

[0174] 6) Pathological section results of duodenum and jejunum

[0175] Observation of intestinal lesions revealed that the healthy group showed normal intestinal tissue structure, with neatly arranged villi and intact mucosal layer, and no obvious pathological changes were observed. The mice in the control group showed obvious lesions such as congestion, edema, and mucosal necrosis in the intestine, with thinning of the intestinal wall and the intestine filled with gas and bloody contents. The intestinal tissues of the Forsythia group and the Bacillus amyloliquefaciens group showed a certain degree of lesions, but the degree was milder than that of the control group, with mild tissue damage and structural changes. The intestinal lesions of the mice in the co-fermentation group were milder, the intestinal mucosa was relatively intact, congestion and edema were not obvious, and there was less gas and bloody contents in the intestine. These pathological results show that Escherichia coli infection can cause serious damage to the intestinal tissue of mice, and the co-fermentation liquid of Bacillus amyloliquefaciens and Forsythia can significantly reduce this damage, and has a significant protective effect on intestinal tissue. Its effect is better than that of using Forsythia or Bacillus amyloliquefaciens alone. The results are shown in Table 2. Figure 9 and Figure 10 , Figure 9 Pathological sections of the duodenum of mice in each group. Figure 10 Pathological sections of the jejunum of mice in each group.

[0176] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that they may modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A biological agent, characterized in that: The biological preparation is a co-fermentation product of Forsythia suspensa and Bacillus amyloliquefaciens; the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101, deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241003; the forsythia suspensa is a water decoction of forsythia suspensa.

2. The biological preparation according to claim 1, characterized in that: The co-fermentation product is whole fermentation broth.

3. A method for preparing the biological agent according to claim 1, characterized in that: The steps include: Forsythia suspensa and Bacillus amyloliquefaciens are co-fermented to obtain a co-fermentation product.

4. The preparation method according to claim 3, wherein The fermentation process parameters are: pH value range of 6.5~7.2, fermentation temperature of 30~40℃, and fermentation time of 24~96 hours.

5. The preparation method according to claim 3, wherein: The Forsythia suspensa was made into a Forsythia suspensa decoction, and then inoculated with Bacillus amyloliquefaciens for co-fermentation.

6. The preparation method according to claim 3, wherein: The forsythia suspensa decoction was mixed with liquid culture medium, and then inoculated with Bacillus amyloliquefaciens for co-fermentation.

7. Use of the biological agent according to any one of claims 1 to 2 in the preparation of a product for inhibiting Escherichia coli, characterized in that: The product is a medicine or a feed additive.

8. The use according to claim 7, characterized in that: The product is intended for use with cattle or calves.

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