Biological agent, preparation method thereof and application of biological agent in preparation of products for inhibiting escherichia coli

Abstract

The invention belongs to the field of microorganisms and fermentation, and relates to a biological preparation, a preparation method thereof and application of the biological preparation in preparation of products for inhibiting escherichia coli. And the biological agent is a co-fermentation product of fructus forsythiae and bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The biological preparation provided by the invention can obviously improve the secretion of IFN-alpha in a mouse body, reduce the production of IL-1beta, IL-6 and TNF-alpha, increase the proportion of CD3 + CD4 + and CD3 + CD8 + T cells, and improve the expression of ZO-1 and Occludin in duodenum and jejunum. Compared with single use of fructus forsythiae and bacillus amyloliquefaciens, the biological preparation shows a stronger synergistic effect, can effectively prevent and inhibit escherichia coli infection, and is not easy to generate drug resistance. The preparation method is simple, raw materials are easy to obtain, and the feed additive is suitable for developing antibacterial 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] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Colibacillosis in calves is a common and seriously harmful disease in the cattle industry, posing a great threat to the health, growth and development of calves. Escherichia coli, as a common conditional pathogen, widely exists in the natural environment. When the immunity of calves declines or the breeding environment is poor, it is extremely easy to cause infection. Escherichia coli has a complex antigenic structure, mainly composed of three types: somatic antigen (O), surface antigen (K), and flagellar antigen (H). Currently, 171 types of O antigens, 100 types of K antigens, and 56 types of H antigens are known. Different antigen combinations form numerous serotypes. For example, serotypes such as O101 and O103 are relatively common in the occurrence of colibacillosis in calves, and the pathogenicity of these serotypes is often stronger, which is closely related to the occurrence of calf diarrhea. It has been clearly determined that Escherichia coli is one of the main pathogenic bacteria causing calf diarrhea, and there are differences in the pathogenicity of different serotypes of Escherichia coli. Serotypes such as O101 and O103 are considered common dominant serotypes. Studies have also found that low immunity of calves themselves, poor breeding environment, stress factors, etc. may all increase the risk of Escherichia coli infection. Newborn calves are unable to obtain sufficient colostrum in time and cannot obtain maternal antibodies, resulting in insufficient immunity and being easily invaded by Escherichia coli. After calves are infected with Escherichia coli, the main manifestation is diarrhea, and the feces are watery or pasty, and the color can range from light yellow to white, often accompanied by a foul smell, and sometimes there may be blood streaks. Diseased calves may also show systemic symptoms such as listlessness, loss of appetite, and elevated body temperature. In severe cases, it will lead to dehydration, acidosis, and even death. In the intensive farming mode, the incidence of colibacillosis in calves shows an upward trend. The occurrence of colibacillosis in calves will also have a potential impact on the quality and safety of beef and milk. Diseased calves may carry drug-resistant Escherichia coli, and these drug-resistant bacteria are transmitted through the food chain, which may pose a threat to human health.

[0004] Currently, the drugs used to treat colibacillosis in calves are mainly antibiotics. However, traditional antibiotics are prone to inducing drug resistance in Escherichia coli in calves, and at the same time, they will also have a certain inhibitory effect on other beneficial bacteria, and there are also problems such as antibiotic residues. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is 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, increase CD3 + CD4 + and CD3 + CD8 + The invention can reduce the proportion of T cells and improve the expression of ZO-1 and Occludin in the duodenum and jejunum. Compared with the use of Forsythia suspensa and Bacillus amyloliquefaciens alone, the biological preparation shows a stronger synergistic effect, can effectively prevent and inhibit Escherichia coli infection, and is not easy to produce drug resistance. The preparation method of the invention is simple, the raw materials are easy to obtain, and it is suitable for the development of antibacterial drugs and feed additives.

[0006] In order to achieve the above object, the technical solution of the present invention is: In the first aspect, a biological preparation is provided, wherein the biological preparation is a combination of Forsythia suspensa and Bacillus amyloliquefaciens ( Bacillus amylolyticus ) of the co-fermentation products.

[0007] In some embodiments, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens ( Bacillus amylolyticus )NM1101, deposited in China Center for Type Culture Collection (CCTCC), with the deposit number CCTCC NO: M20241003, the deposit date is May 20, 2024, and the deposit address is Wuhan University, Wuhan, China.

[0008] In some embodiments, the forsythia suspensa is a decoction of forsythia suspensa. The forsythia suspensa described in the present invention complies with the standards of the Chinese Pharmacopoeia.

[0009] In some embodiments, the co-fermentation product is a whole fermentation broth.

[0010] 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.

[0011] In in-vivo experiments, the biological agent provided by the present invention exhibited: preventive effects, including maintaining animal body weight, increasing survival rate, and improving diarrhea; safety, including no adverse reactions, no organ toxicity, and good tolerance during drug administration, etc.

[0012] In a second aspect, a preparation method of the above biological agent includes the following steps: Co-ferment Forsythia suspensa and Bacillus amyloliquefaciens to obtain a co-fermentation product.

[0013] In some embodiments, the technological parameters of co-fermentation are as follows: pH value 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; the fermentation method is preferably anaerobic static fermentation.

[0014] In some embodiments, make Forsythia suspensa into a water decoction, and then inoculate Bacillus amyloliquefaciens for co-fermentation.

[0015] Specifically, mix the water decoction of Forsythia suspensa with a liquid medium, and then inoculate Bacillus amyloliquefaciens for co-fermentation. More specifically, the liquid medium is MRS medium. More specifically, mix the water decoction of Forsythia suspensa with the liquid medium, adjust the pH value and then perform sterilization treatment, and then inoculate Bacillus amyloliquefaciens for co-fermentation. More specifically, the volume ratio of the water decoction of Forsythia suspensa to the liquid medium is 1:15 - 20.

[0016] Specifically, the inoculation amount of Bacillus amyloliquefaciens is 8 - 10% (by volume). The inoculated bacterial liquid is the logarithmic growth phase bacterial liquid activated for 24 - 36 hours.

[0017] Specifically, the method for making Forsythia suspensa into a water decoction is as follows: crush Forsythia suspensa into coarse powder, sieve it through a 30 - 50 mesh sieve, add distilled water for soaking according to the material-liquid ratio of 1:8 - 12 (g / mL), soak for 12 - 24 hours, heat to boiling after soaking, keep it boiling gently for decoction, filter after the decoction is finished, and collect the filtrate; repeat the decoction of the filter residue according to the above method, combine the filtrates; concentrate the combined filtrates under reduced pressure to obtain the traditional Chinese medicine concentrated liquid, which is the water decoction of Forsythia suspensa.

[0018] The preferred preparation method of the water decoction of Forsythia suspensa includes: 1. Raw material pretreatment: Crush Forsythia suspensa into coarse powder and sieve it through a 30 - 50 mesh sieve; Add distilled water according to the material-liquid ratio of 1:8 - 12 (g / mL), and soak for 12 - 24 hours; 2. Extraction process: Place the soaking solution in an extraction tank; Heat to boiling and then keep it boiling gently; Decoct for the first time for 1.5 - 2 hours; Filter with 3 - 5 layers of gauze and collect the filtrate; The filter residue is decocted with water and repeated 1 - 2 times, and the filtrates are combined; 3. Concentration treatment: The combined filtrates are concentrated under reduced pressure, controlling the temperature at 60 - 65 °C, and concentrated to a relative density of 1.1 - 1.2; Sterilize at 120 - 122 °C for 15 - 25 minutes and cool to room temperature for standby.

[0019] In a third aspect, an application of the above - mentioned biological agent in preparing a product for inhibiting Escherichia coli.

[0020] In some embodiments, the product is a drug or a feed additive.

[0021] Specifically, the drug can be used for preventing and / or alleviating infectious diseases caused by Escherichia coli infection. The prevention refers to preventing Escherichia coli infection, enhancing animal immunity, improving intestinal health, improving production performance, etc. The treatment refers to alleviating infection symptoms, reducing inflammatory responses, repairing intestinal damage, improving clinical prognosis, etc.

[0022] Specifically, the drug can be a veterinary drug, an anti - infective drug, an intestinal drug, an immunomodulator, etc.

[0023] Specifically, the feed additive can be a probiotic preparation, a preventive additive, a nutrition promoter, an immune enhancer, etc.

[0024] 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 freeze - dried powder, a granule, etc.

[0025] In some embodiments, the product further includes excipients. The excipients can be added according to the type of the preparation. The excipients can be protective agents, excipients, stabilizers, etc. When using excipients, the content of the biological agent (co - fermentation product) as the active ingredient can be adjusted as needed.

[0026] In some embodiments, the target user of the product is cattle, especially calves.

[0027] The beneficial effects of the present invention are as follows: The Bacillus amyloliquefaciens and Forsythia suspensa co - fermentation composition of the present invention shows 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 the pro - inflammatory factors IL - 1β, IL - 6, and TNF - α, and this effect is significantly better than using Forsythia suspensa or Bacillus amyloliquefaciens alone. In terms of immune cell regulation, the present invention increases CD3 + CD4 +The proportion of T cells enhances humoral immunity and maintains CD3 + CD8 + The level of T cells enhances cellular immunity and exhibits stronger immunomodulatory ability than any single 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 tissues, reduce congestion, edema and mucosal damage, and the protective effect is better than that of using the components alone.

[0028] Compared with the prior art, the present invention realizes a synergistic effect through the co-fermentation of traditional Chinese medicine and probiotics, overcoming the problem of limited effects when used alone; compared with traditional antibiotics, the present invention is not prone to drug resistance, has no residue problem, and has little impact on the 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 both drug development and feed additive development. The above technical effects and excellent effects have been fully verified through multiple experimental indicators such as body weight change, survival rate, immune indexes, and histopathology, indicating that the present invention has significant practical value. Brief Description of the Drawings

[0029] The specification drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0030] Figure 1 It is the curve of the body weight change of mice in the embodiment of the present invention.

[0031] Figure 2 It is the result diagram of the fecal diarrhea score of mice in the embodiment of the present invention.

[0032] Figure 3 It is the survival curve of mice in the embodiment of the present invention.

[0033] Figure 4 It is the result diagram of the change of cytokines in the serum of mice after infecting with Escherichia coli in the embodiment of the present invention. A: The content of IFN-α in the serum of mice; B: The content of IL-1β in the serum of mice; C: The content of IL-6 in the serum of mice; D: TNF-α in the serum of mice; among them, **** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns (no significance) P >0.05.

[0034] Figure 5 It is the result diagram of the number of spleen T cells of mice in each test group in the embodiment of the present invention. A: CD3 in the spleen of mice+ CD4 + Number of T cells; B: CD3 + CD8 + Number of T cells, **** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns (no significance) P >0.05。

[0035] Figure 6 It is the result graph of the relative expression levels of intestinal tight junction proteins in the duodenum of mice in each experimental group in the embodiments of the present invention; A: Relative expression level of Occludin in the duodenum; B: Relative expression level of ZO-1 in the duodenum; among them, **** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns (no significance) P >0.05。

[0036] Figure 7 It is the result graph of the relative expression levels of intestinal tight junction proteins in the jejunum of mice in each experimental group in the embodiments of the present invention; A: Relative expression level of Occludin in the jejunum; B: Relative expression level of ZO-1 in the jejunum; among them, **** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns (no significance) P >0.05。

[0037] Figure 8 It is the result graph of the content of secretory sIgA in the feces of mice in each group in the embodiments of the present invention, among which, **** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns (no significance) P >0.05。

[0038] Figure 9 It is the pathological tissue section of the duodenum of mice in each group in the embodiments of the present invention; Scale bar 100 μm.

[0039] Figure 10This is the jejunum pathological tissue section diagram of each group of mice in the embodiments of the present invention; scale bar: 50 μm. Detailed implementation manners

[0040] 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 in conjunction with specific embodiments.

[0041] Example 1 Screening of compositions for inhibiting the growth of Bacillus amyloliquefaciens 1. Test materials: In this example, 10 traditional Chinese medicines such as Forsythia suspensa, Scutellaria baicalensis, Coptis chinensis, Phellodendron amurense, Lonicera japonica, Isatis indigotica, Taraxacum mongolicum, Isatis tinctoria, Houttuynia cordata, and Sophora flavescens were selected as the research objects. These traditional Chinese medicines were all purchased from local regular traditional Chinese medicine pharmacies and were identified to meet the relevant standards of the Chinese Pharmacopoeia.

[0042] Strains: Escherichia coli ( Escherichia coli ), the strain was calf Escherichia coli, and Escherichia coli was purchased from Bena Biotechnology - Henan Engineering Technology Research Center for Industrial Microbial Strains (BNCC), with the BNCC number of BNCC364150. In subsequent experiments, it was simply referred to as Escherichia coli. This strain was preserved in cooked meat medium and placed in a 4°C refrigerator. Subculture was carried out 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 h. A single colony was picked and inoculated into thioglycolate broth medium, and cultured statically at 37°C for 6-8 h to prepare a bacterial suspension, and the bacterial concentration was adjusted to 1×10 6 -1×10 7 CFU / mL for standby.

[0043] Bacillus amyloliquefaciens ( Bacillus amylolyticus ), the strain was Bacillus amyloliquefaciens ( Bacillus amylolyticus ), NM1101, which was obtained as a gift from the College of Veterinary Medicine, Jilin Agricultural University. And this strain was deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number of CCTCC NO: M 20241003, the deposit date was May 20, 2024, and the deposit address was Wuhan University, China. In subsequent experiments, it was simply referred to as Bacillus amyloliquefaciens. This strain was preserved on an MRS medium slant and stored in a 4°C refrigerator, with subculture once a month. When in use, Bacillus amyloliquefaciens was inoculated into MRS liquid medium and cultured statically at 30°C for 24-36 h, and then transferred to fresh MRS liquid medium with an inoculation amount of 2%-3% and continued to be cultured until the logarithmic growth phase for fermentation experiments.

[0044] 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 which are analytical pure reagents and are purchased from Sinopharm Chemical Reagent Co., Ltd.

[0045] 3. Main instruments: HZQ-F160 constant temperature shaking incubator (Harbin Donglian Electronic Technology Development Co., Ltd.), SPX-25B biochemical incubator (Medical Equipment Factory of Shanghai Boxun Industry Co., Ltd.), SW-CJ-2FD double-sided single-person purification workbench (Suzhou Purification Equipment Co., Ltd.), LDZX-50KBS vertical pressure steam sterilizer (Shanghai Shen'an Medical Instrument Factory), TDL-5-A centrifuge (Shanghai Anting Scientific Instrument Factory), RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), etc.

[0046] 4. Experimental methods 1) Preparation of traditional Chinese medicine liquid Ten traditional Chinese medicines such as forsythia, scutellaria baicalensis, coptis chinensis, phellodendron amurense, honeysuckle, isatis root, dandelion, folium isatidis, houttuynia cordata, and sophora flavescens were respectively crushed into coarse powder with a pulverizer and passed through a 40-mesh sieve. The following operations were carried out on each kind of traditional Chinese medicine coarse powder: Weigh a certain amount of the traditional Chinese medicine coarse powder, add distilled water according to the solid-liquid ratio of 1:10 (g / mL), and soak for 12 h. Then place the soaking solution in a multi-functional extraction tank, heat to boiling, and keep boiling gently for 1.5 h. After the decoction, filter with four layers of gauze and collect the filtrate. The filter residue was decocted again according to the above method, and the filtrates were combined. The combined filtrate was concentrated under reduced pressure to a relative density of 1.1 (60 °C), about 1 / 5 of the original volume, to obtain the concentrated traditional Chinese medicine liquid (subsequently referred to as traditional Chinese medicine liquid in the following experiments). Finally, the concentrated traditional Chinese medicine liquid was sterilized at 121 °C for 20 min, cooled to room temperature, and reserved for co-fermentation with bacillus amyloliquefaciens and in vitro antibacterial experiments in this example.

[0047] 2) Culture of bacillus amyloliquefaciens and co-fermentation with traditional Chinese medicine Inoculate Bacillus amyloliquefaciens preserved on the MRS medium slant into the MRS liquid medium, and culture it statically at 30 °C for 24 h for activation. Transfer the activated Bacillus amyloliquefaciens to fresh MRS liquid medium at an inoculation amount of 2% (volume), and continue to culture it until the logarithmic growth phase to obtain the Bacillus amyloliquefaciens seed solution. Mix the prepared traditional Chinese medicine solution evenly with the MRS liquid medium, where the addition amount of the traditional Chinese medicine solution is 6% (volume) of the MRS liquid medium, and adjust the pH value to 6.8. Then inoculate the Bacillus amyloliquefaciens seed solution at an inoculation amount of 10% (volume) and ferment anaerobically at 37 °C for 72 h. During the fermentation process, sample every 24 h to measure the bacterial count and pH value in the fermentation broth to monitor the fermentation process.

[0048] 3) In vitro antibacterial experiment method The Oxford cup method was used to determine the diameter of the antibacterial circle against Bacillus amyloliquefaciens. First, adjust the concentration of the Escherichia coli suspension to 1×10 7 CFU / mL. Take 100 μL of the bacterial suspension and spread it evenly on the anaerobic blood agar plate. Place Oxford cups (inner diameter 6 mm, outer diameter 8 mm, height 10 mm), and place 3 Oxford cups on each plate in an equilateral triangle arrangement with a cup spacing of not less than 25 mm. Add 200 μL of the test substance to the Oxford cups. Place the plate in an anaerobic incubator (37 °C, 5% CO2, 10% H2, 85% N2) and culture for 18 h, observe the formation of the antibacterial circle, and measure the diameter of the antibacterial circle using a vernier caliper.

[0049] 4) Fermentation condition optimization Ferment traditional Chinese medicinal materials with Bacillus amyloliquefaciens, and optimize the fermentation time, fermentation temperature, and strain inoculation amount to obtain the best fermentation method. Conduct in vitro antibacterial experiments (Oxford cup method, the same method as above) on the fermented strain fermentation broth of the obtained strain, and use the Bacillus amyloliquefaciens fermentation group without adding traditional Chinese medicine solution as a control.

[0050] (1) Fermentation time optimization: Add 6% (volume) of Forsythia suspensa traditional Chinese medicine solution to 50 mL of MRS liquid medium, adjust the pH value to 6.8, sterilize at 121 °C for 25 min, and inoculate 5% of the bacterial liquid. Ferment statically at 37 °C for 24 h, 48 h, 72 h, and 96 h. Measure the size of the antibacterial circle against Bacillus amyloliquefaciens after fermentation, and screen the optimal fermentation time with the evaluation index.

[0051] (2) Fermentation temperature optimization: Add 6% (by volume) of Forsythia suspensa traditional Chinese medicine liquid to 50 mL of MRS liquid medium, adjust the pH value to 6.8, sterilize at 121 °C for 25 min, inoculate 5% of the bacterial liquid, and adjust the pH value to 6.8. Incubate statically at 30 °C, 35 °C, 37 °C, 40 °C, and 42 °C respectively according to the selected optimal fermentation time. Measure the size of the inhibition zone against Bacillus amyloliquefaciens after fermentation, and screen the optimal fermentation temperature with the evaluation index.

[0052] (3)Optimization of the inoculum size of the strain: Add 6% (by volume) of Forsythia suspensa traditional Chinese medicine liquid to 50 mL of MRS liquid medium, adjust the pH value to 6.8, sterilize at 121 °C for 25 min, and inoculate the fermentation broth with 0.1%, 1%, 10%, and 100% of the bacterial liquid respectively. According to the selected optimal fermentation time and optimal fermentation temperature. Measure the size of the inhibition zone against Bacillus amyloliquefaciens after fermentation, and screen the optimal inoculum size of the strain with the evaluation index.

[0053] 5. Results 1) Pre-fermentation test of Bacillus amyloliquefaciens and traditional Chinese medicine Table 1 Monitoring the change in the number of bacteria during the co-fermentation of Bacillus amyloliquefaciens and traditional Chinese medicine (10 7 CFU / ml)

[0054] Table 2 Monitoring the pH change during the co-fermentation of Bacillus amyloliquefaciens and traditional Chinese medicine

[0055] 2) In vitro inhibitory effect of traditional Chinese medicine liquid on Bacillus amyloliquefaciens Using the traditional Chinese medicine liquid as the test substance for in vitro antibacterial experiments, by measuring the size of each inhibition zone, comparing the antibacterial effects of various traditional Chinese medicine liquids, the results show that Forsythia suspensa has the best inhibitory effect, and the diameter of its inhibition zone is 23.5 ± 0.1 mm. The results are shown in Table 3. Therefore, Forsythia suspensa and Bacillus amyloliquefaciens are co-fermented in the follow-up and optimization experiments are carried out.

[0056] Table 3 Antibacterial effects of various traditional Chinese medicine liquids on Bacillus amyloliquefaciens

[0057] 3) Optimization of the fermentation of traditional Chinese medicine by Bacillus amyloliquefaciens (1)Optimization of the fermentation time Using the fermentation broth of Bacillus amyloliquefaciens and the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa as the test substances, in vitro antibacterial experiments were carried out, and the antibacterial zones of Bacillus amyloliquefaciens at different fermentation times were measured respectively. The results are shown in Table 4. The measurement results show that with the extension of the fermentation time, the diameter of the antibacterial zone shows a trend of first increasing and then stabilizing. At 48 h of fermentation, the diameter of the antibacterial zone was (24.9 ± 1.3) mm, reaching the maximum; at 72 h of fermentation, it was (23.3 ± 0.8) mm; when fermented continuously to 96 h, the diameter of the antibacterial zone decreased to (21.1 ± 0.7) mm. This indicates that the fermentation time has a significant effect on the antibacterial effect of the co-fermentation broth, and the inhibitory effect on Bacillus amyloliquefaciens is the strongest at about 48 h.

[0058] Table 4 Antibacterial zone diameter of Bacillus amyloliquefaciens at different fermentation times (mm)

[0059] (2)Optimization of fermentation temperature Using the fermentation broth of Bacillus amyloliquefaciens and the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa as the test substances, in vitro antibacterial experiments were carried out, and the antibacterial zones of Bacillus amyloliquefaciens at different fermentation temperatures were measured respectively. The results are shown in Table 5. The results show that with the increase of the fermentation temperature, the diameter of the antibacterial zone shows a trend of first increasing and then decreasing. At 37 °C of fermentation, the diameter of the antibacterial zone was (23.4 ± 0.8) mm, reaching the maximum. When the fermentation temperature was increased to 42 °C, the diameter of the antibacterial zone decreased to (16.5 ± 0.9) mm. This indicates that the fermentation temperature has a significant effect on the antibacterial effect of the co-fermentation broth, and the inhibitory effect on Bacillus amyloliquefaciens is the strongest at about 37 °C.

[0060] Table 5 Antibacterial zone diameter of Bacillus amyloliquefaciens at different fermentation temperatures (mm)

[0061] (3)Screening of inoculum size of bacterial liquid Using the fermentation broth of Bacillus amyloliquefaciens and the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa as the test substances, in vitro antibacterial experiments were carried out, and the antibacterial zone diameters of Bacillus amyloliquefaciens at different inoculum sizes of Bacillus amyloliquefaciens bacterial liquid were measured respectively. The results are shown in Table 6. The results show that with the increase of the inoculum size of the bacterial liquid, the diameter of the antibacterial zone shows a trend of first increasing and then decreasing. When the inoculum size of the bacterial liquid was 10%, the diameter of the antibacterial zone was (25.3 ± 0.5) mm, reaching the maximum. When the inoculum size of the bacterial liquid was 100%, the diameter of the antibacterial zone was (18.4 ± 1.1) mm. This indicates that the inoculum size of the bacterial liquid has a significant effect on the antibacterial effect of the co-fermentation broth, and the inhibitory effect on Bacillus amyloliquefaciens is the strongest at about 10%.

[0062] Table 6 Inhibitory zone diameter (mm) of Bacillus amyloliquefaciens at different inoculum amounts of bacterial solutions

[0063] Example 2 Investigation on the protective effect of the co-fermentation of Bacillus amyloliquefaciens and Forsythia suspensa on mice infected with Bacillus amyloliquefaciens In this example, Bacillus amyloliquefaciens and Forsythia suspensa were co-fermented (co-fermentation group), and mice were fed and challenged for protection experiments with the healthy group, Bacillus amyloliquefaciens group, and Forsythia suspensa group respectively. By detection methods such as ELISA, Flow Cytometry, and H&E staining, the changes in mouse body weight, the contents of IFN-α, TNF-α, IL-1β, and IL-6 in serum, the activation of the body's immune organs and immune cells, and the pathological changes of the small intestine of mice were detected and analyzed to study the protective effect of the co-fermentation product of Forsythia suspensa and Bacillus amyloliquefaciens on mice infected with Bacillus amyloliquefaciens.

[0064] 1. Experimental strains: Escherichia coli ( Escherichia coli ) The strain is calf Escherichia coli. Escherichia coli was purchased from Bena Biotechnology - Henan Engineering Technology Research Center for Industrial Microbial Strains (BNCC), with the strain number BNCC364150, and is simply referred to as Escherichia coli in subsequent experiments.

[0065] Bacillus amyloliquefaciens ( Bacillus amylolyticus ) The strain is Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101, which was kindly donated by the College of Veterinary Medicine, Jilin Agricultural University. And this strain is preserved in the China Center for Type Culture Collection (CCTCC), with the strain number CCTCC NO: M 20241003, the preservation date is May 20, 2024, and the preservation address is Wuhan University, China. It is simply referred to as Bacillus amyloliquefaciens in subsequent experiments.

[0066] 2. Experimental animals Fifty healthy 30-day-old mice were selected and randomly divided into 5 groups, with 10 mice in each group. They were raised in isolation and fed and watered at 8:00, 14:00, and 20:00 every day.

[0067] 3. Reagents 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 Enzyme Immunoassay (Jiangsu) Industrial Co., Ltd. Mouse CD3-PE-Cy7 antibody, CD4-PE antibody, and CD8-FITC antibody (BD); 1640 cell culture medium (Hyclone); Blocking mouse serum was stored in the laboratory; Antibody diluent: 1% BSA; Red blood cell lysate, Fluor 488-labeled goat anti-rabbit IgG(H + L) (Beyotime Biotechnology Co., Ltd.); FACS solution (1000 mL PBS for culturing cells, 10 mL FBS, 0.9 g sodium azide); AXYGEN PCRSTRIP TUBES (Corning, USA); Mini BEST Viral RNA / DNA Extraction Kit Ver5.0 (CodeNo.9766 Takara); Blocking goat serum; DAPI staining solution; Anti-quenching mounting medium; 4% paraformaldehyde solution; Sodium citrate antigen retrieval solution.

[0068] 4. Main experimental equipment: ABI Prism7500QT-qPCR instrument (USA) ABI; Heparin sodium anticoagulant tubes and clot-promoting tubes were purchased from Kangjian Medical Supplies Co., Ltd. (Jiangsu); Tissue embedding machine, paraffin slicer, and DMi8 fluorescence inverted microscope were purchased from Leica Instruments Co., Ltd. (Germany); The tissue homogenizer is domestic.

[0069] 5. Experimental methods: 1) Experimental protocol Grouping of experimental animals: Healthy group (n = 10), Bacillus amyloliquefaciens group (n = 10), Forsythia suspensa group (n = 10), Co-fermentation group (n = 10), Control group (n = 10). Male and female were randomly assigned. The grouping is shown in Table 7 below.

[0070] Table 7 Grouping of animal experiments

[0071] Immunization and challenge procedures: Each group of mice was immunized on days 1, 3, and 5. Except for the healthy group and the control group which were gavaged with 0.5 mL PBS, the other groups were given corresponding drug gavage. Each mouse in the Bacillus amyloliquefaciens group was gavaged with 1×10 9 cfu / mL, 0.5 mL, each mouse in the Forsythia suspensa group was gavaged with 0.5 mL of Forsythia suspensa Chinese medicine solution, and each mouse in the co-fermentation group was gavaged with 0.5 mL of the co-fermentation solution of Bacillus amyloliquefaciens and Forsythia suspensa.

[0072] Among them, the preparation method of Bacillus amyloliquefaciens fermentation broth: Inoculate 10% of Bacillus amyloliquefaciens (5 mL) in 50 mL of MRS liquid medium and ferment at 37 °C for 72 hours. After fermentation, take the whole fermentation broth after shaking well, ensuring that the viable count remains at 1×10 9 cfu / mL, and shake well before use.

[0073] The preparation method of Forsythia suspensa Chinese medicine liquid: Take Forsythia suspensa and crush it into coarse powder with a pulverizer, pass through a 40-mesh sieve, weigh the coarse powder and add distilled water according to the solid-liquid ratio of 1:10 (g / mL) (for example: add 250 mL of distilled water to 25 g of Forsythia suspensa coarse powder), and soak for 12 h. Then place the soaking solution in a multi-functional extraction tank, heat to boiling, and keep boiling gently for 1.5 h. After boiling, filter with 4 layers of gauze and collect the filtrate. The filter residue is decocted again according to the above method once, and the filtrates are combined. The combined filtrate is concentrated under reduced pressure to a relative density of 1.1 (60 °C), about 1 / 5 of the original volume, to obtain 50 mL of Chinese medicine concentrated liquid. Finally, sterilize the Chinese medicine concentrated liquid at 121 °C for 20 min, cool to room temperature, and that's it. Shake well before use.

[0074] The preparation method of the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa: Add 6% of the aforementioned Forsythia suspensa Chinese medicine liquid (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 fermentation, take the whole fermentation broth after shaking well, ensuring that the viable count remains at 1×10 9 cfu / mL, and shake well before use.

[0075] After immunization, measure the body weight of the mice once a day, record the data and calculate the average daily weight gain. On the 5th day, except for the healthy group which was intraperitoneally injected with 0.5 mL of PBS, the other groups were intraperitoneally injected with an Escherichia coli suspension (concentration of 1×10 9 CFU / mL, injection volume of 0.5 mL).

[0076] 2) Detection of growth performance indicators Body weight: Weigh the mice once a day, record the body weight change data and draw a body weight change curve. Record the fecal conditions of the mice every day and score them. The fecal scoring method: Collect and evaluate the rectal feces of each mouse, and a professional veterinarian uses these photos to score the feces for consistency. The fecal consistency score ranges from -2 to 2, where 0 = normal; 1 = soft feces; 2 = hard feces; -1 = mild diarrhea; -2 = severe diarrhea.

[0077] 3) Detection methods for immune indicators IFN-α, TNF-α, IL-1β, IL-6 After infection with Escherichia coli, blood was collected from the eyes of mice in each group. 1 mL of blood was collected, centrifuged in a centrifuge tube, and the serum was separated and stored at -80 °C. The contents of IFN-α, TNF-α, IL-1β, and IL-6 in the serum were detected by ELISA method.

[0078] 4) Preparation of single-cell suspension Preparation of spleen single-cell suspension: Take a part of the spleen and obtain a single-cell suspension by gentle grinding. Pass it through a 200-mesh filter into a corresponding 15 mL centrifuge tube, balance, centrifuge at 1650 rpm, 4 °C for 5 min. Discard the supernatant completely, resuspend the cells with 1 mL of red blood cell lysate, lyse on ice for 10 min, take it out at 5 min in the middle, and shake for 30 s. Add 10 mL of PBS buffer to terminate, balance, centrifuge at 1650 rpm, 4 °C for 5 min. Discard the supernatant, resuspend the cells with 1 mL of FACS buffer, and count.

[0079] Preparation of mesenteric lymph node single-cell suspension: Carefully and gently grind all the lymph nodes into a single-cell suspension, pass it through a 200-mesh filter into a corresponding 15 mL centrifuge tube, balance, centrifuge at 1650 rpm, 4 °C for 5 min. Discard the supernatant, resuspend the cells with 0.2 mL of FACS buffer, and count.

[0080] 5) Flow cytometry detection Flow antibody staining (1) Divide the above single-cell suspensions into tubes to ensure that each tube contains 1×10 6 cells (5×10 5 cells for peripheral blood), and the total volume is 100 μL; (2) According to the antibody titer, add the corresponding volume of flow antibody for cell surface markers.

[0081] (3) After shaking and mixing evenly, place it in the dark at 4 °C for 30 min (4) After labeling, add 3.5 mL of ice-cold FACS buffer to the tube, centrifuge at 1650 rpm, 4 °C for 5 min, discard the supernatant, and resuspend the cells with a small amount of FACS buffer.

[0082] (5) Repeat step (4) once to fully remove the unbound antibody and reduce non-specific staining on the cell surface. The BD flow cytometer is used to detect the samples, and the FlowJo_v10.6.2 software is used for data analysis and graph processing.

[0083] 6) Fluorescent quantitative PCR detection of intestinal tight junction proteins Extracting RNA from sample tissues or cells includes the following steps: First, add the sample to a lysis buffer and break it to release RNA. Then, separate the RNA from the supernatant using a phenol / chloroform mixture and precipitate the RNA with isopropanol or ethanol. Subsequently, wash the RNA precipitate to remove impurities and finally dissolve the RNA in RNase-free water or buffer. The extracted RNA can be detected for its concentration and purity by colorimetry or a fluorescence analyzer and stored in a -80 °C refrigerator to prevent degradation. These steps may be adjusted according to the specific experimental purpose and sample type. The relative quantitative q-PCR method is used to detect the transcriptional levels of two barrier proteins, ZO-1 and Occludin, in the duodenum and colon of mice.

[0084] 7) Detection method for secreted antibodies in feces After infecting with Escherichia coli, collect the feces of each group of mice. Dissolve with sterile water, aspirate the supernatant, and detect the content of sIgA in the feces by ELISA method.

[0085] 8) H&E staining to detect intestinal pathological changes To further observe the pathological changes in the small intestine segment of mice, obtain the middle duodenum and middle jejunum of mice. Take the same part of each intestinal segment, place it in 4% paraformaldehyde solution for fixation for more than two days, then cut the fixed tissue neatly, embed, section, and perform H&E staining. The steps are as follows: (1) Dehydration: Sequentially place the sample into 70% alcohol, 80% alcohol, 85% alcohol for 2 h each, 90% alcohol for overnight dehydration, 95% I alcohol and 95% II alcohol for 1 h each, and 100% I alcohol and 100% II alcohol for 1 h each. (2) Clearing: Place the sample into xylene I solution and xylene II solution in sequence, with the clearing time being 5 min each until the reddish skin-like appearance can be seen with the naked eye, indicating the end of clearing.

[0086] (3) Infiltration with wax: Sequentially place the tissue blocks in wax I, wax II, and wax III, and infiltrate with wax for 1 h each at 56 °C, then place the tissue blocks in an embedding cassette for embedding.

[0087] (4) Sectioning: Cut tissue sections with a thickness of 3.5 μm from each embedding block, spread the sections on a clean glass slide in a 41 °C water bath. After placing the glass slide in an 80 °C drying oven for 1 h to bake the slide, perform H&E staining.

[0088] (5)The H&E staining procedure is as follows: The sample is placed in xylene I and xylene II for 8 minutes in sequence, then in 100% alcohol I, 100% alcohol II, 95% alcohol, 80% alcohol, and 70% alcohol for 1 minute, and rinsed in ultrapure water to remove the excess alcohol. Then it is stained with hematoxylin, rinsed with ultrapure water, differentiated in 0.5% hydrochloric acid in an alcohol bath for 5 seconds, then rinsed, then placed in light ammonia water for 2 minutes, then rinsed with water, placed in 0.5% eosin aqueous solution for 5 minutes for staining, rinsed with water again, rinsed in an 80% alcohol bath, observe the eosin staining situation, extract the sample repeatedly 5 times in the alcohol baths of 95% alcohol, 100% alcohol I, and 100% alcohol II in sequence, then place it in xylene I and xylene II for 2 minutes in sequence, and seal the slide with neutral gum.

[0089] 6. Experimental results: 1) Detection results of growth performance indicators During the experiment, the body weight changes were recorded. The results showed that the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa could protect mice from Escherichia coli infection and reduce the decline in body weight.

[0090] Specifically, during the immunization period from day 0 to day 5, the body weights of mice in all groups showed a stable growth trend, and the growth conditions of each group were similar. After the challenge treatment (intraperitoneal infection with Escherichia coli) on day 5, the growth of the healthy group was basically unaffected, the body weight of the control group decreased significantly, reflecting the negative impact of Escherichia coli infection. The Forsythia suspensa group and the Bacillus amyloliquefaciens group could relieve the sudden drop in body weight to a certain extent, and there were significant differences in the body weight changes of the mice in the co-fermentation group. The above changes in body weight indicated that the mice fed with the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa could resist Escherichia coli infection to a certain extent and play a preventive and protective role. The results are shown in Figure 1 .

[0091] When scoring the diarrhea conditions of each group of mice, the results showed that the score of the healthy group occasionally increased but basically remained around 2 points, indicating that the fecal state was normal; the score of the control group gradually reached -2 points, indicating obvious diarrhea symptoms and reaching a more severe level on day 5; the score of the Forsythia suspensa group gradually dropped below 0, with mild diarrhea; the score of the Bacillus amyloliquefaciens group basically remained around 1 point, with occasional mild diarrhea; this indicated that the Forsythia suspensa group and the Bacillus amyloliquefaciens group could relieve the diarrhea degree to a certain extent compared with the control group. However, the score of the co-fermentation group always remained between 1 and 2 points, indicating that the fecal state was normal. This showed that the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa could relieve the diarrhea of mice caused by Escherichia coli infection. The results are shown in Figure 2 .

[0092] Figure 3The survival of each group is shown. During the 0-5-day immunization period, the survival rate of each group was 100%, indicating that there were no deaths during the immunization period and the drug administration safety was good. After challenging with bacteria (intraperitoneal infection with Escherichia coli) on the 5th day, the healthy group and the co-fermentation group maintained a 100% survival rate, indicating that the mice fed with the co-fermented liquid of Bacillus amyloliquefaciens and Forsythia suspensa showed excellent preventive and protective effects against Escherichia 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 that of the control group. The survival rate of the control group decreased significantly after challenging and dropped to 0 on the 8th day. The results are shown in Figure 3 .

[0093] 2) Detection results of immune indexes IFN-α, TNF-α, IL-1β, and IL-6 The collected serum was used to detect the secretion of cytokines IFN-α, TNF-α, IL-1β, and IL-6 by EILSA. The results are shown in Figure 4 .

[0094] After each group of immunized mice was infected with Escherichia coli, compared with the healthy group, the content of IFN-α in the control group decreased significantly (** P <0.01), indicating that after infection with Escherichia coli, the secretion of IFN-α in mice would be inhibited; there was no significant difference between the Forsythia suspensa group and the control group (ns), indicating that the Forsythia suspensa group did not significantly improve the decrease in IFN-α caused by Escherichia coli infection; the difference in IFN-α between the co-fermentation group and the control group was extremely significant (**** P <0.0001), indicating that the co-fermented liquid could significantly increase the secretion of IFN-α, and the secretion amount was also increased compared with the healthy group. This indicates that the co-fermented liquid can significantly improve the immunity of mice and resist Escherichia coli infection. The results are as shown in Figure 4 A in

[0095] After each group of immunized mice was infected with Escherichia coli, compared with the healthy group, the content of IL-1β in the control group increased extremely significantly (**** P <0.0001), indicating that after infection with Escherichia coli, a large amount of pro-inflammatory factor IL-1β would be induced in mice; compared with the control group, the level of IL-1β in the Forsythia suspensa group and the Bacillus amyloliquefaciens group decreased significantly (**** P <0.0001), but was still significantly higher than that of the healthy group, indicating that both of these treatments could 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-fermented liquid could significantly inhibit the production of IL-1β. This shows that the co-fermented liquid has the strongest anti-inflammatory effect and can effectively inhibit the inflammatory response caused by Escherichia coli infection. The results are as shown in Figure 4 B in

[0096] After immunization, each group of mice was infected with Escherichia coli. Compared with the healthy group, the content of IL-6 in the control group increased significantly (**** P <0.0001), indicating that after infection with Escherichia coli, it would promote the secretion of the pro-inflammatory factor IL-6 in mice; compared with the control group, the level of IL-6 in the Forsythia suspensa group and the Bacillus amyloliquefaciens group was significantly different (*** P <0.001), indicating that these two treatments had obvious effects on maintaining the level of IL-6; the difference in IL-6 between the co-fermentation group and the control group was significant (**** P <0.0001), and the level of IL-6 was significantly higher than that of the control group and close to that of the healthy group, indicating that the co-fermentation broth could inhibit the secretion of a higher level of IL-6, which showed that the co-fermentation broth could effectively maintain the pro-inflammatory ability of the body and help fight against the inflammatory response caused by Escherichia coli infection. The results were as Figure 4 shown in C below.

[0097] After immunization, each group of mice was infected with Escherichia coli. Compared with the healthy group, the content of TNF-α in the control group increased extremely significantly (**** P <0.0001), indicating that after infection with Escherichia coli, it would induce a large amount of the pro-inflammatory factor TNF-α in mice; compared with the control group, the level of TNF-α in the Forsythia suspensa group decreased significantly (**** P <0.0001), and the Bacillus amyloliquefaciens group showed a more significant decrease (**** P <0.0001), indicating that these two treatments both had the effect of inhibiting inflammation. The difference in TNF-α between the co-fermentation group and the control group was extremely significant (**** P <0.0001), and its TNF-α level was the lowest, close to the level of the healthy group, indicating that the co-fermentation broth had the strongest anti-inflammatory effect and could significantly inhibit the production of TNF-α induced by Escherichia coli infection, further confirming its excellent anti-inflammatory effect. The results were as Figure 4 shown in D below.

[0098] 3) Effects of the co-fermentation product of Bacillus amyloliquefaciens and Forsythia suspensa on TH2 response TH2-type cellular immunity participates in humoral immunity by secreting IL-4. Therefore, the activation level of TH2 cells in the spleen was detected. The results showed that after infection with Escherichia coli, Bacillus amyloliquefaciens, the Chinese medicine solution of Forsythia suspensa, and the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa could all activate CD4 + T cells to secrete IL-4. Importantly, the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa could stimulate a higher level of IL-4 and had the best immune protection effect in the co-fermentation. The results were as Figure 5 shown in A below.

[0099] According to Figure 5It can be seen that compared with the healthy group, the proportion of CD4 + IL-4 T cells in the spleen of the control group decreased extremely significantly (*** P <0.001), indicating that after infection with Escherichia coli, the Th2-type immune response in mice was significantly inhibited; compared with the control group, the proportion of CD4 + IL-4 T cells in the Forsythia suspensa group and the Bacillus amyloliquefaciens group increased significantly (* P <0.1), indicating that both of these treatments could maintain the Th2-type immune response to a certain extent; the difference between the co-fermentation group and the control group was extremely significant (*** P <0.001), and the proportion of its CD4 + IL-4 T cells was close to the level of the healthy group, indicating that the co-fermentation broth could significantly improve the Th2-type immune response, which showed that the co-fermentation broth could effectively maintain the immune balance of the body and enhance the defense ability against Bacillus amyloliquefaciens infection.

[0100] 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, the traditional Chinese medicine liquid of Forsythia suspensa, and the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa could activate CD3 + CD8 + T cells to secrete higher levels of IFN-γ, improving the cellular immune response, and the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa had a more significant effect, as shown in Figure 5 Figure B.

[0101] As Figure 5 shown in Figure B, compared with the healthy group, the proportion of CD8 + IFN-γ T cells in the control group was not significant (ns), and the proportion of CD8 + IFN-γ T cells in the Forsythia suspensa group and the Bacillus amyloliquefaciens group was not significant (ns) compared with the control group, indicating that the effect of using these two treatments alone on maintaining cellular immunity was not good; while the level of CD8 + IFN-γ T cells in the co-fermentation group was significantly different from that in the control group (*** P <0.001), indicating that the co-fermentation broth could effectively maintain the level of CD8 + IFN-γ T cells, 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.

[0102] 4) Quantitative analysis results of tight junction proteins in the small intestine of mice Quantify the tight junction proteins ZO-1 (Zonula occludens-1) and Occludin in the duodenum and jejunum of each group of mice. Among them, ZO-1 is a scaffold protein located inside the cell, mainly responsible for connecting other proteins to the cytoskeleton. Occludin is a transmembrane protein that spans the cell membrane and interacts with Occludin of adjacent cells. These two proteins together constitute the intestinal barrier. They work together to maintain tight junctions between cells, control the selectivity of substances passing through the intercellular space, and thus protect the intestine from harmful substances. When the expression of these proteins decreases, it usually means that the intestinal barrier function is impaired. Therefore, by detecting the expression levels of ZO-1 and Occludin, the integrity and functional status of the intestinal barrier can be evaluated.

[0103] After immunizing mice with Bacillus amyloliquefaciens, Forsythia suspensa traditional Chinese medicine solution, and the co-fermentation broth of Bacillus amyloliquefaciens and Forsythia suspensa, compared with the control group, the relative expression levels of ZO-1 and Occludin were significantly increased. The results showed that Escherichia coli could damage the duodenum and reduce the expression of tight junction proteins in the duodenum. The results are shown in Figure 6 .

[0104] Specifically, as shown in Figure 6 A, compared with the healthy group, the relative mRNA expression level of Occludin in the jejunum of the control group decreased extremely significantly (*** P <0.001), indicating that after infection with Escherichia coli, the expression of the tight junction protein Occludin in the small intestine was significantly damaged; compared with the control group, the expression levels of Occludin in the Forsythia suspensa group and the Bacillus amyloliquefaciens group were slightly (* P <0.05), indicating that these two treatments had a certain effect on maintaining the integrity of the intestinal barrier; the difference between the co-fermentation group and the control group was extremely significant (** P <0.01), and its Occludin expression level was closest to that of the healthy group, indicating that the co-fermentation broth could effectively maintain the expression of the tight junction protein Occludin in the small intestine, suggesting that the co-fermentation broth had the strongest intestinal barrier protection effect.

[0105] As shown in Figure 6 B, compared with the healthy group, the relative mRNA expression level of ZO-1 in the duodenum of the control group decreased extremely significantly (**** P <0.0001), indicating that after infection with Escherichia coli, the expression of the tight junction protein ZO-1 in the duodenum was significantly damaged; the difference between the Bacillus amyloliquefaciens group and the control group was significant (** P <0.01), indicating that the effect of using these two treatments alone on maintaining the integrity of the intestinal barrier was limited; the difference between the co-fermentation group and the control group was extremely significant (**** P(<0.0001), and its ZO-1 expression level is closest to that of the healthy group, indicating that the co-fermented broth can effectively maintain the expression of tight junction protein ZO-1 in the duodenum, suggesting that the co-fermented broth has the strongest intestinal barrier protection effect.

[0106] After immunizing mice with Bacillus amyloliquefaciens, Forsythia suspensa traditional Chinese medicine solution, and the co-fermented broth of Bacillus amyloliquefaciens and Forsythia suspensa, compared with the control group, the relative expression levels of ZO-1 and Occludin were significantly increased. The results showed that Escherichia coli could damage the jejunum and reduce the expression of tight junction proteins in the jejunum. The results are shown in Figure 7 .

[0107] Specifically, as Figure 7 shown in A, compared with the healthy group, the relative mRNA expression level of Occludin in the jejunum of the control group decreased extremely significantly (**** P <0.0001), indicating that after infection with Escherichia coli, the expression of tight junction protein Occludin in the jejunum would be significantly damaged; compared with the control group, the expression level of Occludin in the Forsythia suspensa group and the Bacillus amyloliquefaciens group was not significant (ns), indicating that these two treatments had no effect on maintaining the integrity of the intestinal barrier; the difference between the co-fermented 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-fermented broth could effectively maintain the expression of tight junction protein Occludin in the small intestine, suggesting that the co-fermented broth has the strongest intestinal barrier protection effect.

[0108] As Figure 7 shown in B, compared with the healthy group, the relative mRNA expression level of ZO-1 in the jejunum of the control group decreased extremely significantly (*** P <0.001), indicating that after infection with Escherichia coli, the expression of tight junction protein ZO-1 in the jejunum would be significantly damaged; the difference between the Bacillus amyloliquefaciens group and the control group was significant (* P <0.1), indicating that the effect of using these two treatments alone on maintaining the integrity of the intestinal barrier was limited; the difference between the co-fermented 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-fermented broth could effectively maintain the expression of tight junction protein ZO-1 in the duodenum, suggesting that the co-fermented broth has the strongest intestinal barrier protection effect.

[0109] 5) Detection of sIgA in mouse feces The sIgA content in the feces of mice in each group was detected by ELISA method. The results showed that compared with the control group, the healthy group increased significantly (** P<0.01), the infection of Escherichia coli reduced the secretion of sIgA in the intestine. The content of sIgA in the Forsythia suspensa group increased significantly (** P <0.01), the content of sIgA in the Bacillus amyloliquefaciens group increased extremely significantly (*** P <0.001), the content of sIgA in the co-fermentation group increased extremely significantly (**** P <0.0001), indicating that the co-fermented traditional Chinese medicine liquid can effectively stimulate the intestine to secrete sIgA to resist the infection of Escherichia coli. The results are shown in Figure 8 .

[0110] 6) Results of pathological sections of duodenum and jejunum Observation of intestinal lesions found that the healthy group showed normal intestinal tissue structure, neat arrangement of villi, complete mucosal layer, and no obvious pathological changes. The intestines of the control group mice showed obvious lesions such as congestion, edema, and mucosal necrosis, the intestinal wall became thinner, and the intestine was filled with gas and bloody contents. To a certain extent, lesions appeared in the intestinal tissues of the Forsythia suspensa group and the Bacillus amyloliquefaciens group, but the degree was lighter than that of the control group, showing mild tissue damage and structural changes. However, the degree of intestinal lesions in the co-fermentation group mice was relatively light, the intestinal mucosa was relatively intact, congestion and edema were not obvious, and there were fewer gas and bloody contents in the intestine. These pathological results indicate that the infection of Escherichia coli can cause severe damage to the intestinal tissues of mice, while the co-fermented liquid of Bacillus amyloliquefaciens and Forsythia suspensa can significantly reduce this damage and has an obvious protective effect on intestinal tissues, and its effect is better than that of using Forsythia suspensa or Bacillus amyloliquefaciens alone. The results are shown in Figure 9 and Figure 10 , Figure 9 is the pathological section of the duodenum of mice in each group, Figure 10 is the pathological section of the jejunum of mice in each group.

[0111] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A biological preparation, characterized in that, The biological preparation is a co-fermentation product of Forsythia suspensa and Bacillus amyloliquefaciens.

2. The biological preparation according to claim 1, characterized in that, The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens ( Bacillus amylolyticus ) NM1101, which is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20241003.

3. The biological preparation according to claim 1, characterized in that, The forsythia suspensa is a decoction of the forsythia suspensa; and the co-fermentation product is a whole fermentation liquid.

4. A preparation method of the biological preparation according to claim 1, characterized in that, The steps include: The forsythia suspensa and Bacillus amyloliquefaciens are co-fermented to obtain a co-fermentation product.

5. The preparation method according to claim 4, characterized in that, in total The fermentation process parameters are: pH value range is 6.5~7.2, fermentation temperature is 30~40℃, and fermentation time is 24~96 hours.

6. The preparation method according to claim 4, characterized in that, The Forsythia suspensa was made into a Forsythia suspensa decoction, and then inoculated with Bacillus amyloliquefaciens for co-fermentation.

7. The preparation method according to claim 4, characterized in that, The decoction of Forsythia suspensa was mixed with liquid culture medium, and then inoculated with Bacillus amyloliquefaciens for co-fermentation.

8. An application of the biological preparation according to any one of claims 1 to 3 in the preparation of a product for inhibiting Escherichia coli.

9. The application according to claim 8, characterized in that, The product is a medicine or a feed additive.

10. The application according to claim 8, characterized in that, The product is used for cattle or calves.

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