A fermented microbial preparation for inhibiting c. jejuni in calves, and a preparation method and application thereof

Abstract

This invention discloses a fermented microbial preparation for inhibiting Campylobacter jejuni in calves, its preparation method, and its application. The fermented microbial preparation is a co-fermentation product of a mixture of Lygodium japonicum decoction and liquid culture medium, inoculated with Lactobacillus paracasei; the Lactobacillus paracasei is... Lacticaseibacillus paracasei KQNIU. Comparative experiments in this invention revealed that, compared to other types of traditional Chinese medicine, the inhibitory effect of fermented Lygodium japonicum on Campylobacter jejuni is significantly higher than that of other types of traditional Chinese medicine. Furthermore, the co-fermentation of the traditional Chinese medicine Lygodium japonicum and the probiotic Lactobacillus paracasei KQNIU achieves a synergistic effect between the two, overcoming the problem of limited efficacy when used alone.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a fermentation microbial preparation for inhibiting Campylobacter jejuni in calves, its preparation method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Calves' gastrointestinal tracts are not fully developed, making them susceptible to infections from bacteria, viruses, and parasites, which can cause diarrhea. Common bacterial diarrhea in calves is mainly caused by Escherichia coli (E. coli). Escherichia coli Salmonella ( Salmonella Campylobacter jejuni ( Campylobacter jejuni ) and Clostridium perfringens ( Clostridium perfringens Diarrhea caused by Campylobacter jejuni infection, also known as "winter dysentery," mainly occurs in autumn and winter. Campylobacter jejuni first invades the intestinal mucosa, then colonizes the intestinal epithelial cells, and subsequently invades other tissues, causing inflammation. Calf feces are foul-smelling, watery, brown, and loose; 50%–80% of affected calves have blood in their feces.

[0004] Traditional treatment for bacterial diarrhea in calves involves selecting antibiotics sensitive to the causative bacteria after drug sensitivity testing. However, this reliance on antibiotics can easily lead to drug resistance and drug residues. Therefore, developing an antibiotic alternative to alleviate calf diarrhea is of great significance to the livestock industry.

[0005] Traditional Chinese medicine (TCM) possesses multiple functions, including antibacterial and anti-inflammatory effects, immune enhancement, and metabolic regulation. It can effectively prevent and control common livestock and poultry diseases, reduce morbidity and mortality, and decrease reliance on antibiotics. Its natural components are easily degradable and leave no residue, ensuring the quality and safety of meat, eggs, and dairy products from the source and helping livestock products meet market access standards. Simultaneously, TCM can regulate the intestinal health of livestock and poultry, improve digestive and absorptive efficiency, and enhance production performance, achieving safer and more productive farming results.

[0006] Probiotics, as green and safe microecological preparations, have become an indispensable input for the high-quality development of modern animal husbandry, playing a key role in ensuring livestock health, improving production efficiency, and promoting the green transformation of the industry. Probiotic fermentation of traditional Chinese medicine involves the synergistic fermentation of probiotics and traditional Chinese medicine, utilizing microbial metabolism to transform, decompose, and activate the components of the medicine, significantly improving efficacy and utilization efficiency, and possessing significant application value in animal husbandry. This method solves the problems of poor absorption and slow onset of action of traditional Chinese medicine while retaining its advantages of being green and residue-free, providing an efficient way to replace antibiotics, ensure livestock health, and improve breeding efficiency, which is of great significance to promoting the development of green animal husbandry.

[0007] However, there are no publicly available traditional Chinese medicine or probiotic preparations that are effective against calf diarrhea caused by Campylobacter jejuni. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a fermented microbial preparation for inhibiting Campylobacter jejuni in calves, its preparation method, and its application. The fermented microbial preparation is a fermentation broth co-fermented with *Lactobacillus paracasei* and *Lygodium japonicum*. This fermentation broth, through the synergistic effect of *Lactobacillus paracasei* and the traditional Chinese medicine *Lygodium japonicum*, significantly improves the prevention and inhibition of diarrhea caused by *Campylobacter jejuni* infection in calves, enhances the body's immune function, and protects the integrity of the intestinal barrier, providing a new technical solution to the problems of existing technologies.

[0009] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a fermentation microbial preparation for inhibiting Campylobacter jejuni in calves, which is a co-fermentation product of a mixture of Lygodium japonicum decoction and liquid culture medium, inoculated with Lactobacillus paracasei; wherein the Lactobacillus paracasei is Lactobacillus paracasei (… Lacticaseibacillus paracasei The *Lactobacillus paracasei* KQNIU was deposited on August 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35739, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The viable count in the fermentation microbial preparation inhibiting *Campylobacter jejuni* in calves is 10-1. 9 CFU / mL ~10 10 CFU / mL.

[0010] Lygodium japonicum is the dried, mature spore of the Lygodium japonicum plant (family Lygodiumceae). It has diuretic, anti-edema, and litholytic effects, and is mainly used clinically for urinary tract diseases such as urinary tract infections, urinary stones, and hematuria, as well as biliary tract diseases such as gallstones and biliary obstruction. This invention compared the antibacterial effects of fermentation broths of 10 traditional Chinese medicines—Pulsatilla chinensis, Glycyrrhiza uralensis, Reed tsao-ko, Hippophae rhamnoides, Isatis indigotica, Morus alba leaves, Lygodium japonicum, Pyrrosia lingua, Viola yedoensis, and Lysimachia christinae—on Campylobacter jejuni. The results showed that Lygodium japonicum had a significantly higher inhibitory effect on Campylobacter jejuni than the other traditional Chinese medicines.

[0011] In a second aspect, the present invention provides a method for preparing the fermented microbial preparation for inhibiting Campylobacter jejuni as described in the first aspect, wherein Lygodium japonicum and Lactobacillus paracasei are co-fermented to obtain a fermentation product.

[0012] Furthermore, the preparation method is as follows: prepare a decoction of Lygodium japonicum, mix the decoction of Lygodium japonicum with a liquid culture medium, inoculate with Lactobacillus paracasei, and then co-ferment.

[0013] In some embodiments of the present invention, the co-fermentation conditions include: a pH range of 6.2 to 7.2, preferably 6.5; a fermentation temperature range of 30°C to 40°C, preferably 37°C; a fermentation time range of 24 h to 96 h, preferably 72 h; and the preferred fermentation method is anaerobic static fermentation. Comparative experiments of the present invention have shown that the inhibitory effect on Campylobacter jejuni is significantly stronger under these conditions than under other conditions.

[0014] In some embodiments of the present invention, the effects of traditional Chinese medicines such as Pulsatilla chinensis, Glycyrrhiza uralensis, Reed ferox, Hippophae rhamnoides, Isatis indigotica, Morus alba, Lygodium japonicum, Pyrrosia lingua, Viola yedoensis, and Lysimachia christinae with Lactobacillus paracasei KQNIU were compared. Unexpectedly, it was found that when Lactobacillus paracasei KQNIU was co-fermented with Lygodium japonicum decoction for 24-72 hours, the bacterial count in the co-fermentation system was significantly higher than that of other traditional Chinese medicines. At 72 hours of fermentation, the Lactobacillus paracasei KQNIU bacterial count reached 1.89 × 10⁻⁶. 8 The CFU / mL concentration is 2.3 to 5 times that of other traditional Chinese medicine ingredients. This indicates that Lygodium japonicum significantly promotes the growth and reproduction of Lactobacillus paracasei, allowing the bacterial count to reach the target level more quickly after co-fermentation. This significantly shortens the production cycle and greatly improves the production efficiency of microbial preparations based on the co-fermentation of Lygodium japonicum and Lactobacillus paracasei KQNIU, which is beneficial for the efficient production of therapeutic drugs for Campylobacter jejuni in calves.

[0015] In some embodiments of the present invention, compared with the inhibitory effect of Lactobacillus paracasei KQNIU and Lygodium japonicum alone on Campylobacter jejuni in calves, the product obtained by co-fermentation of the two has a significantly improved inhibitory effect on Campylobacter jejuni in calves. The co-fermentation technology based on Lactobacillus paracasei KQNIU and Lygodium japonicum provided by the present invention further enhances the prevention and treatment of inflammatory response and calf diarrhea caused by Campylobacter jejuni infection in calves.

[0016] In some embodiments of the present invention, the aqueous decoction of Lygodium japonicum is mixed with the liquid culture medium at a volume ratio of 1:8 to 1:12, and preferably, 10% by volume of the aqueous decoction of Lygodium japonicum is added to the liquid culture medium.

[0017] In some embodiments of the present invention, the inoculation conditions for *Lactobacillus paracasei* are as follows: the inoculation amount is 8% to 12%. Preferably, the inoculated bacterial culture is a logarithmic growth phase culture activated for 24 to 36 hours.

[0018] In some embodiments of the present invention, the preparation method of the Lygodium japonicum decoction includes: crushing Lygodium japonicum into coarse powder, sieving it, adding distilled water, soaking it, placing it in an extraction tank, heating it to boiling, maintaining a gentle boil while decocting, filtering it after decoction, and collecting the filtrate; repeating the decoction of the residue according to the above method, and combining the filtrates; concentrating the combined filtrate under reduced pressure to obtain the concentrated Chinese medicine liquid, which is the Lygodium japonicum decoction.

[0019] In some embodiments of the present invention, the preparation method of the decoction of Lygodium japonicum includes the following steps: S1. Raw material pretreatment: Crush the Lygodium japonicum into coarse powder and sieve it; add distilled water at a material-to-liquid ratio of 1:8g / mL to 1:12g / mL and soak. S2. Extraction process: Place the soaking solution in an extraction tank; heat to boiling and then maintain a gentle boil; collect the filtrate after simmering; S3. Concentration treatment: The filtrate prepared in step S2 is concentrated under reduced pressure, and the temperature is controlled at 60℃±5℃, until the relative density is 1.2±0.1. Finally, sterilize the concentrated Chinese medicine solution and cool it to room temperature for later use.

[0020] Thirdly, the present invention provides the use of the fermented microbial preparation for inhibiting Campylobacter jejuni described in the first aspect in the preparation of a drug or feed additive for inhibiting Campylobacter jejuni in calves.

[0021] Fourthly, the present invention provides the use of the fermented microbial preparation of Campylobacter jejuni described in the first aspect in the preparation of drugs or feed additives for the prevention, relief and / or treatment of calf diarrhea.

[0022] Furthermore, the aforementioned calf diarrhea is caused by Campylobacter jejuni (… Campylobacter jejuni )cause.

[0023] In some embodiments of the present invention, the fermented microbial preparation has a preventive effect, which is manifested in its activation of the level of secretory immunoglobulin A in the body's intestine, which can protect calves from Campylobacter jejuni infection, resist Campylobacter jejuni infection, enhance animal immunity, effectively prevent the decline in cellular immune function caused by Campylobacter jejuni infection, and improve production performance, etc.

[0024] In some embodiments of the present invention, the fermented microbial preparation has therapeutic effects, including inhibiting the production of TNF-α, IFN-γ, IL-1β and IL-10 induced by Campylobacter jejuni infection in calves, and increasing the levels of IL-4 and IFN-γ, which manifests as relieving infection symptoms, reducing inflammatory response, repairing intestinal damage, and improving clinical prognosis.

[0025] In some embodiments of the present invention, the form of the drug or feed additive includes liquid formulations and solid formulations, wherein the liquid formulations are selected from injection solutions or oral solutions, etc.; and the solid formulations are selected from lyophilized powders or granules, etc.

[0026] In some embodiments of the present invention, the composition of the drug or feed additive may be adjusted by adding suitable excipients depending on the type of formulation. For example, protective agents, excipients, and stabilizers may be added as needed, and the content of the active ingredient may be adjusted as required when using excipients.

[0027] In some embodiments of the present invention, the drug may be a veterinary drug, an anti-infective drug, an intestinal drug, an immunomodulator, etc.

[0028] In some embodiments of the present invention, the feed additive may be a microecological preparation, a preventive additive, a nutritional promoter, an immune enhancer, etc.

[0029] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. This invention provides a fermented microbial preparation for inhibiting Campylobacter jejuni in calves. In the treatment of Campylobacter jejuni infection in calves, this invention achieves a synergistic effect of co-fermentation of the traditional Chinese medicine Lygodium japonicum and the probiotic Lactobacillus paracasei KQNIU, overcoming the problem of limited efficacy when used alone; at the same time, it can be prepared into various dosage forms, which is convenient for administration to livestock.

[0030] 2. This invention provides a traditional Chinese medicine ingredient, Lygodium japonicum, which has a higher effect on promoting the growth and reproduction of the probiotic Lactobacillus paracasei KQNIU. Comparative experiments showed that, compared with other traditional Chinese medicine ingredients, when Lactobacillus paracasei KQNIU was co-fermented with Lygodium japonicum for 72 hours, the bacterial count was 2.3 to 5 times higher than that of other traditional Chinese medicine ingredients. This demonstrates that Lygodium japonicum's effect on promoting the growth and reproduction of Lactobacillus paracasei KQNIU is far superior to other traditional Chinese medicine ingredients. This significantly shortens the production cycle of the microbial preparation provided by this invention and greatly improves production efficiency, which is beneficial for the efficient production of therapeutic drugs for Campylobacter jejuni in calves.

[0031] 3. The fermented microbial preparation of *Lactobacillus paracasei* and *Lygodium japonicum* provided by this invention exhibits excellent immunomodulatory effects. In enhancing the body's immune function, the fermentation broth can significantly reduce the production of pro-inflammatory factors TNF-α and IL-1β; in regulating immune cells, this invention increases CD4+... + IL-4 enhances the proportion of T cells, boosting humoral immunity and maintaining CD8 levels. + IFN-γ T cell levels enhance cellular immunity, exhibiting stronger immunomodulatory capabilities than either component alone. Regarding secreted antibodies, this invention significantly increases the expression of secretory immunoglobulin A and reduces diarrhea rates; these effects are superior to those of using any single component.

[0032] 4. Furthermore, this invention features a simple and feasible preparation method and a wide range of raw material sources, making it suitable for both drug development and feed additive development. The aforementioned technical effects and superior results have been fully verified through multiple experimental indicators, including calf weight change, survival rate, immune indicators, and histopathology, demonstrating the significant practical value of this invention. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 The images show the results of strain identification; where A: colony isolation results; B: Gram staining results; and C: 16S rDNA identification results.

[0035] Figure 2 This is the result of the strain phylogenetic tree.

[0036] Figure 3 The results show the growth performance of the strain; where A: growth curve of the strain; B: acid production curve of the strain.

[0037] Figure 4 The image shows the acid resistance results of the strain; among them, P <0.0001; P <0.01; P <0.05;ns(nosignificance) P >0.05.

[0038] Figure 5 This is a graph showing the bile salt tolerance results of the strain; among them, P <0.0001; P <0.001; P <0.01; P <0.05; ns (no significance) P >0.05.

[0039] Figure 6 The graph shows the weight changes of calves in each group.

[0040] Figure 7 The graph shows the diarrhea score results for each group of calves.

[0041] Figure 8 This is a comparison of IFN-γ levels in the serum of calves in different groups after infection with Campylobacter jejuni; among them, P <0.0001; P <0.001; P <0.01; ns (no significance) P >0.05.

[0042] Figure 9 This is a comparison of IL-1β levels in the serum of calves in different groups after infection with Campylobacter jejuni; among them, P <0.0001; P <0.001; P <0.01; P <0.05; ns (no significance) P >0.05.

[0043] Figure 10 This is a comparison of IL-10 levels in the serum of calves in different groups after infection with Campylobacter jejuni; among them, P <0.001; P <0.01; P <0.05; ns (no significance) P >0.05.

[0044] Figure 11 This is a comparison of TNF-α levels in the serum of calves in different groups after infection with Campylobacter jejuni; among them, P <0.0001; P <0.05; ns (no significance) P >0.05.

[0045] Figure 12 To test the CD4 levels in the peripheral blood of calves in each group + Percentage comparison chart of IL-4 T; where, P <0.001; P <0.01; P <0.05; ns (no significance) P >0.05.

[0046] Figure 13 To test the CD8 levels in the peripheral blood of calves in each group + Percentage comparison chart of IFN-γT; where, P <0.001; P <0.01; P <0.05; ns (no significance) P >0.05.

[0047] Figure 14 The graph shows the results of sIgA content in calf feces in each experimental group; among them, P <0.0001; P <0.001; P <0.01; ns (no significance) P >0.05. Detailed Implementation

[0048] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0049] The present invention will be further described below with reference to the embodiments.

[0050] Example 1: Screening of strains and traditional Chinese medicines that inhibit Campylobacter jejuni in calves. 1. Experimental Materials: Ten kinds of traditional Chinese medicines, including Pulsatilla chinensis, Glycyrrhiza uralensis, Reed ferox, Hippophae rhamnoides, Isatis indigotica, Morus alba leaves, Lygodium japonicum, Pyrrosia lingua, Viola yedoensis, and Lysimachia christinae, were used as research subjects. These Chinese medicines were all purchased from local regular Chinese medicine pharmacies and were identified as meeting the relevant standards of the Chinese Pharmacopoeia.

[0051] Strains: Campylobacter jejuni ( Campylobacter jejuni The strains were provided and activated by the laboratory strain bank of the Inner Mongolia Autonomous Region Beef Cattle Disease Prevention and Control Engineering Technology Research Center.

[0052] 2. Main reagents: MRS liquid medium, MRS agar medium, LB medium, Gram staining kit, Columbia blood agar medium, pepsin, trypsin, bovine bile salts, antibiotic sensitivity tablets and Oxford cups were all purchased from Tianjin Sikesai Biotechnology Co., Ltd.

[0053] 3. Major Instruments: HZQ-F160 full-temperature shaking 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 Shenan Medical Instrument Factory), TDL-5-A centrifuge (Shanghai Anting Scientific Instrument Factory), RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), Agilent 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, Inc., USA).

[0054] 4. Test methods 4.1 Screening of strains inhibiting Campylobacter jejuni in calves In a clean bench, 5g of fresh calf feces sample was taken and placed in a 95mL Erlenmeyer flask containing sterile physiological saline. The mixture was thoroughly shaken to prepare the initial bacterial suspension. Subsequently, a tenfold serial dilution method was used: 1mL of the bacterial suspension was placed in a centrifuge tube, and 100µL was added to 900µL of sterile physiological saline. The mixture was vortexed, and this process was repeated 10 times. -2 10 -3 10 -4 10 -5 10 -6 10 -7Serial dilutions were performed; 100 µL of each serial dilution was spread onto the surface of MRS agar medium and incubated at 37°C for 48 h. After colony formation, typical white, round colonies with smooth and plump edges were selected, and single colonies were obtained through three streak purifications. The obtained single colonies were inoculated into MRS liquid medium and incubated at 37°C for 24 h to obtain the screening strain, named KQNIU, for subsequent strain identification.

[0055] 4.1.1 Colony and Bacterial Identification Gram staining was performed on the KQNIU strain, and its morphological characteristics were observed under an oil immersion microscope. Colonies that stained purple and had morphological characteristics consistent with lactic acid bacteria were selected. Single colonies were subjected to a hydrogen peroxide test, and the results were observed. The presence of bubbles indicated a positive result, while the absence of bubbles indicated a negative result.

[0056] 4.1.2 Biochemical identification of strains Biochemical characteristics of bacterial strains were analyzed using bacterial biochemical identification tubes. The test strain was added to each biochemical identification tube using a sterile loop. Immediately after inoculation, the tubes were sealed with sealing film and incubated at 37°C for 12 hours. Color changes were observed; a color change indicated a positive result, while no color change indicated a negative result. DNA from KQNIU was extracted using a bacterial DNA extraction kit. PCR amplification was performed using universal primers 27F and 1492R for the bacterial 16S rDNA gene. The PCR amplification program was as follows: initial denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, 35 cycles; extension at 72°C for 90 s; total extension at 72°C for 10 min; storage at 4°C. The amplified products were detected by 1.5% agarose gel electrophoresis. Samples meeting the requirements were sent to Sangon Biotech Co., Ltd. for sequencing. Based on the sequencing results, a phylogenetic tree was constructed using the NJ method in MEGA software.

[0057] 4.1.3 Determination of the biological characteristics of the strain The tested bacterial strain was inoculated into MRS liquid medium at a 2% inoculum and cultured at 37℃ for 36 h. Samples were taken at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, and 28 h, and absorbance was measured at a wavelength of 600 nm. The results were repeated three times. A growth curve was plotted with culture time on the x-axis and absorbance on the y-axis. The bacterial culture was also inoculated into 10 mL of MRS liquid medium with a pH of 6.5 at a 2% inoculum and cultured at 37℃. pH was measured every 2 h using a portable pH meter. A pH change curve was plotted with culture time on the x-axis and pH on the y-axis to represent the trend of acid production rate of the strain at different fermentation stages.

[0058] 4.1.4 Determination of acid resistance of strains The pH of the MRS liquid medium was adjusted using 1 mol / L hydrochloric acid, setting up five gradients: 2.0, 3.0, 4.0, 5.0, and 6.0. A medium at pH 6.0 was retained as a blank control. The test strain was inoculated at a 1% inoculation ratio into the MRS media at the different pH values ​​and incubated at 37°C for 8 hours. The OD values ​​of each experimental group were then measured. 600 Calculate the survival rate.

[0059] Survival rate (%) = (Experimental group OD) 600 / Control group OD 600 )×100%.

[0060] 4.1.5 Determination of the strain's tolerance to bile salts Bovine bile salts were added to MRS liquid medium to prepare media with bile salt concentrations of 0%, 0.15%, 0.3%, and 0.6%, respectively. 0% bile salt was used as a blank control, and each concentration was repeated in triplicate. The test strains were inoculated into the MRS media with different bile salt concentrations at an inoculum rate of 1% and cultured at 37°C for 8 hours. The OD values ​​of each experimental group were measured. 600 Survival rates were calculated to analyze the effects of different bile salt concentrations on the growth of the strain.

[0061] 4.2 Preparation of Traditional Chinese Medicine Liquid 4.2.1 Preparation and Effects of Fermentation Agents Ten kinds of traditional Chinese medicines, including Pulsatilla chinensis, Glycyrrhiza uralensis, Reed ferox, Hippophae rhamnoides, Isatis indigotica, Morus alba, Lygodium japonicum, Pyrrosia lingua, Viola yedoensis, and Lysimachia christinae, were pulverized into coarse powder using a pulverizer and passed through a 40-mesh sieve. A certain amount of the coarse powder was weighed and added to distilled water at a material-to-liquid ratio of 1:10 g / mL, and soaked for 24 hours. The soaking liquid was then placed in a multi-functional extraction tank, heated to boiling, and simmered at a gentle boil for 2 hours. After simmering, the liquid was filtered through four layers of gauze and the filtrate was collected. The residue was simmered again using the same method, and the filtrates were combined. The combined filtrate was concentrated under reduced pressure to a relative density of 1.1 (60℃), approximately 1 / 5 of the original volume, to obtain a concentrated traditional Chinese medicine solution. Finally, the concentrated traditional Chinese medicine solution was sterilized at 121℃ for 20 minutes, cooled to room temperature, and set aside for use in the examples for co-fermentation with Lactobacillus paracasei and in vitro antibacterial tests.

[0062] 4.2.2 Culture and fermentation of traditional Chinese medicine with Lactobacillus paracasei *Lactobacillus paracasei* was inoculated into MRS medium and incubated statically at 30°C for 24 hours to activate it. The activated *Lactobacillus paracasei* was then transferred to MRS liquid medium at a 10% volume inoculation rate and cultured until the logarithmic growth phase to serve as the *Lactobacillus paracasei* seed culture. The prepared concentrated traditional Chinese medicine solution was mixed thoroughly with 10% MRS liquid medium at a volume ratio, and the pH was adjusted to 6.5. Then, the *Lactobacillus paracasei* seed culture was inoculated at a 10% volume inoculation rate and anaerobic fermented at 37°C for 72 hours. During fermentation, samples were taken every 20 hours to determine the number of *Lactobacillus paracasei* bacteria and the pH value in the fermentation broth to monitor the fermentation progress.

[0063] 4.2.3 Determination of the antibacterial properties of fermented medicinal liquid The diameter of the inhibition zone against Campylobacter jejuni of fermented traditional Chinese medicine was determined using the Oxford cup method. First, the Campylobacter jejuni bacterial suspension was adjusted to a concentration of 1×10⁻⁶. 7 CFU / mL. Spread 100 μL of bacterial suspension evenly onto an anaerobic blood agar plate. Place Oxford cups (6 mm inner diameter, 8 mm outer diameter, 10 mm height) on each plate, arranging three Oxford cups in an equilateral triangle with a minimum distance of 25 mm between cups. Add 200 μL of fermented traditional Chinese medicine concentrate to each Oxford cup, while using unfermented traditional Chinese medicine concentrate and MRS liquid medium as controls. Incubate the plates in an anaerobic incubator (37℃, 5% CO2, 10% H2, 85% N2) for 18 h, observe the formation of inhibition zones, and measure the diameter of the inhibition zones using calipers.

[0064] 4.3 Optimization of Fermentation Conditions Traditional Chinese medicine was fermented with Lactobacillus paracasei, and the fermentation time, fermentation temperature and strain inoculation amount were optimized to obtain the best fermentation method. The fermentation broth obtained by the strain was then used to test the inhibition of Campylobacter jejuni in calves.

[0065] 1) Fermentation time optimization: Add 10% volume of Lygodium japonicum solution to 50 mL of MRS liquid medium, adjust the pH to 6.5, autoclave at 121℃ for 25 min, and inoculate with 10% bacterial solution. Incubate at 37℃ for 24 h, 48 h, 72 h, and 96 h. Measure the size of the inhibition zone against Campylobacter jejuni after fermentation, and use the evaluation indicators to screen for the optimal fermentation time.

[0066] 2) Fermentation temperature optimization: Add 10% volume of Lygodium japonicum solution to 50 mL of MRS liquid medium, adjust the pH to 6.5, autoclave at 121℃ for 25 min, inoculate with 10% bacterial suspension, and adjust the pH to 6.5. Incubate at 30℃, 35℃, 37℃, 40℃, and 42℃ according to the selected optimal fermentation time. Measure the size of the inhibition zone against Campylobacter jejuni after fermentation, and use these indicators to screen for the optimal fermentation temperature.

[0067] 3) Optimization of inoculum size: Add 10% volume of Lygodium japonicum solution to 50 mL of MRS liquid medium, adjust the pH to 6.5, and sterilize at 121℃ for 25 min. Inoculate the fermentation broth with 5%, 10%, 15%, and 20% bacterial solutions, respectively. Based on the selected optimal fermentation time and temperature, measure the size of the inhibition zone against Campylobacter jejuni after fermentation, and use these as evaluation indicators to screen for the optimal inoculum size.

[0068] 5. Results 5.1 Isolation and Identification of Strains This experiment isolated and purified four rod-shaped, Gram-positive, and hydrogen peroxide-negative single colonies. One strain of *Lactobacillus* was ultimately selected. Its colony morphology, Gram staining, and genomic DNA electrophoresis results are as follows: Figure 1 As shown.

[0069] Depend on Figure 1 The image of the colony isolation results for bacteria A shows white dot-like colonies with regular and complete edges and raised tips.

[0070] Depend on Figure 1 The Gram staining results of the medium B strain show short purple rod-shaped bacteria, some of which can be linked together in chains.

[0071] Depend on Figure 1 The C16S rDNA identification results show that the band size is around 1400bp.

[0072] 5.2 Biochemical identification of strains The biochemical identification results are shown in Table 1. This strain was negative for hydrogen peroxide and could decompose sucrose, glucose, maltose, mannitol, raffinose, fructose, xylose, lactose, esculin, galactose, sorbitol, cellobiose, melibiose, salicin, and rhamnose, but could not ferment arabinose. The 16S rDNA sequencing result (SEQ ID NO.1) showed 99.74% homology with the *Lactobacillus paracasei* sequence in the database, confirming this strain as *Lactobacillus paracasei*. A phylogenetic tree was constructed using the NJ method in MEGA 11 software; the results are shown in Table 1. Figure 2 This strain is related to the genus *Lactobacillus paracasei*. Lacticaseibacillus paracasei ATCC25302, Lacticaseibacillus paracaseiNBRC 15889 belongs to the same branch. Based on physiological and biochemical identification, this strain is *Lactobacillus paracasei*, and is named *Lactobacillus paracasei* KQNIU. Lacticaseibacillus paracasei KQNIU.

[0073] Table 1 Biochemical identification results of the strains

[0074] 5.3 Determination of biological characteristics of the strain 5.3.1 Growth curves and acid production performance of the strain like Figure 3 As shown in Figure A, the growth curve of *Lactobacillus paracasei* KQNIU reveals its growth dynamics at different culture stages: growth is slow before 6 hours, enters the logarithmic growth phase after 6 hours with accelerated growth, and enters the stationary growth phase after 18 hours, where growth tends to stabilize. The acid-producing capacity of *Lactobacillus paracasei* KQNIU is shown in Figure A. Figure 3 As shown in Figure B, during the period from 0h to 6h of culture, Lactobacillus paracasei KQNIU has a weak acid production capacity and relatively slow metabolism, and is in the adaptation period; during the period from 6h to 18h, the acid production capacity is strong and the metabolism is accelerated; after 18h, the pH stabilizes at 3.5 to 3.7.

[0075] 5.3.2 Acid resistance of the strain The tolerance of a bacterial strain to gastric acid is one of the important indicators for assessing its probiotic potential. The acid tolerance test results for *Lactobacillus paracasei* KQNIU are as follows: Figure 4 With pH 6.0 as the control group, the viable count of *Lactobacillus paracasei* KQNIU decreased significantly with decreasing pH. At pH 5.0, the activity was high, with a survival rate of 92.43%; at pH 4.0, the survival rate significantly decreased to 78.49%; at pH 3.0, the survival rate significantly decreased to 63.84%; and at pH 2.0, the growth of *Lactobacillus paracasei* KQNIU was severely affected, with the survival rate decreasing to 44.82%. These results indicate that *Lactobacillus paracasei* KQNIU has strong acid resistance, suggesting stronger tolerance and survival ability in the intestine.

[0076] 5.3.3 Bile salt tolerance of the strain Figure 5 The study presented results on the bile salt tolerance of *Lactobacillus paracasei* KQNIU. Data showed that the survival rate of *Lactobacillus paracasei* KQNIU decreased with increasing bile salt concentration. The survival rate reached 89.16% at a bile salt concentration of 0.15%, and remained above 72.43% at 0.3%. While the survival rate decreased significantly at 0.6%, it remained at 56.91%, indicating a certain degree of bile salt tolerance.

[0077] 5.4 Experiment on the Pre-fermentation of Lactobacillus paracasei KQNIU and Traditional Chinese Medicine The results of the pre-fermentation experiments of *Lactobacillus paracasei* KQNIU with different traditional Chinese medicines are shown in Table 2, and the pH changes are shown in Table 3. As can be seen from Tables 2 and 3, the growth and reproduction of *Lactobacillus paracasei* KQNIU were significantly better than those of other traditional Chinese medicines during co-fermentation with *Lygodium japonicum*. Furthermore, the pH value of the co-fermentation system significantly decreased after co-fermentation of *Lactobacillus paracasei* KQNIU and *Lygodium japonicum*.

[0078] Table 2. Monitoring of changes in bacterial counts of *Lactobacillus paracasei* KQNIU during fermentation of traditional Chinese medicine (10) 8 CFU / mL

[0079] Table 3. Monitoring of pH changes during the fermentation of traditional Chinese medicine by *Lactobacillus paracasei* KQNIU.

[0080] 5.4.1 In vitro inhibitory effect of traditional Chinese medicine on Campylobacter jejuni in calves By measuring the size of each inhibition zone, the antibacterial effects of various traditional Chinese medicines were compared. The results showed that Lygodium japonicum exhibited the best inhibitory effect, with an inhibition zone diameter of 23.5 ± 0.5 mm. The results are shown in Table 4. Therefore, Lygodium japonicum was used for fermentation in subsequent fermentation of traditional Chinese medicines with Lactobacillus paracasei KQNIU.

[0081] Table 4. Antibacterial effects of various fermented herbal decoctions on Campylobacter jejuni in calves.

[0082] 5.4.2 Optimization of Traditional Chinese Medicine Fermentation by Lactobacillus paracasei KQNIU Optimization of fermentation time The results of the inhibition zone diameter determination of *Campylobacter jejuni* in fermented herbal broths with different fermentation times (see Table 5) show that the inhibition zone diameter initially increased and then stabilized with increasing fermentation time. At 48 h of fermentation, the inhibition zone diameter was (25.4 ± 0.6) mm; at 72 h, the diameter reached its maximum of (30.7 ± 0.7) mm; and after 96 h of fermentation, the diameter decreased to (27.6 ± 0.9) mm. This indicates that fermentation time significantly affects the antibacterial effect of the fermented herbal broth, with the strongest inhibitory effect on *Campylobacter jejuni* observed around 72 h.

[0083] Table 5. Diameter of the inhibition zone (mm) of traditional Chinese medicine fermentation broth against Campylobacter jejuni at different fermentation times.

[0084] Optimization of fermentation temperature The results of the inhibition zone diameter determination of *Campylobacter jejuni* by fermentation broths of traditional Chinese medicine at different fermentation temperatures (see Table 6) show that the inhibition zone diameter first increases and then decreases with increasing fermentation temperature. At 37℃, the inhibition zone diameter is (30.3±0.5) mm, reaching its maximum. When the fermentation temperature is increased to 42℃, the inhibition zone diameter is (19.1±1.7) mm. This indicates that fermentation temperature has a significant impact on the antibacterial effect of the fermentation broth of traditional Chinese medicine, with the strongest inhibitory effect on *Campylobacter jejuni* observed at around 37℃.

[0085] Table 6. Diameter of inhibition zones (mm) of traditional Chinese medicine fermentation broth against Campylobacter jejuni at different fermentation temperatures.

[0086] Screening of bacterial inoculum size The results of the inhibition zone diameter determination of traditional Chinese medicine fermentation broth with different inoculum amounts of *Lactobacillus paracasei* KQNIU on *Campylobacter jejuni* were shown in Table 7. The inhibition zone diameter initially increased and then decreased with increasing inoculum amount. The largest inhibition zone diameter was (31.6 ± 1.3) mm when the inoculum amount was 10%. The largest inhibition zone diameter was (20.3 ± 0.1) mm when the inoculum amount was 20%. This indicates that the inoculum amount significantly affects the antibacterial effect of the fermentation broth, with the strongest inhibitory effect on *Campylobacter jejuni* observed at around 10%.

[0087] Table 7. Diameter of inhibition zone (mm) of traditional Chinese medicine fermentation broth with different inoculum amounts against Campylobacter jejuni.

[0088] Example 2: Feeding trial to evaluate the preventive and therapeutic effects of biological agent fermentation broth on diarrhea in calves. In this embodiment, *Lactobacillus paracasei* KQNIU and *Lygodium japonicum* were co-fermented (co-fermentation group), and calves were fed with a control group, a *Lactobacillus paracasei* KQNIU group, a *Lygodium japonicum* group, and a healthy group to conduct an infection experiment with *Campylobacter jejuni*. Using ELISA and flow cytometry, the changes in calf body weight, serum levels of IFN-γ, TNF-α, IL-1β, and IL-10, and the activation of immune organs and immune cells were analyzed to study the protective effect of the co-fermentation product of *Lactobacillus paracasei* KQNIU and *Lygodium japonicum* against *Campylobacter jejuni* infection in calves.

[0089] 1. Test strain: Lactobacillus paracasei KQNIU was deposited on August 25, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35739. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0090] 2. Experimental animals: Twenty-five beef calves (weighing 46±5 kg) that gave birth naturally and were in similar physical condition were selected as experimental animals.

[0091] 3. Test location: Inner Mongolia Keerqin Beef Cattle Breeding Co., Ltd.

[0092] 4. Reagents and equipment Bovine interleukin-1β (IL-1β), bovine interferon-gamma (IFN-γ), bovine tumor necrosis factor-α (TNF-α), and bovine interleukin-10 (IL-10) ELISA kits were purchased from Enzyme Immunosorbent Assay (Jiangsu) Co., Ltd. Bovine CD3-PE-Cy7 antibody, CD4-PE antibody, and CD8-FITC antibody (BD Biosciences); 1640 cell culture medium (Hyclone); blocking serum was stored in the laboratory; antibody dilution buffer: 1% BSA; red blood cell lysis buffer; FACS solution (1000 mL PBS for cell culture, 10 mL FBS, 0.9 g sodium azide); AXYGEN PCR STRIP TUBES (Corning Electron, Inc.); Mini BEST Viral RNA / DNA Extraction Kit Ver5.0 (Code No. 9766 Takara).

[0093] The ABI Prism 7500 QT-qPCR instrument (USA) was purchased from ABI Corporation; heparin sodium anticoagulant tubes and coagulation-promoting tubes were purchased from Kangjian Medical Supplies Co., Ltd. (Jiangsu); the ELISA reader was purchased from Thermo Fisher Scientific Co., Ltd. (USA); and the tissue homogenizer was domestically produced.

[0094] 5. Experimental Design: The experiment employed a single-factor randomized experimental design, selecting 25 7-day-old calves and dividing them into 5 groups: a control group, a *Lactobacillus paracasei* (KQNIU) group, a *Lygodium japonicum* (Hydrocotyle vulgaris) group, a co-fermentation group (KQNIU and *Lygodium japonicum*), and a healthy group. Each group contained 5 calves, and the experiment lasted 14 days. Calves were administered the bacteria via gavage for 7 consecutive days. Fecal condition was observed over the 7 days following infection with *Campylobacter jejuni*. Calf weight was measured daily, and the average daily weight gain was calculated.

[0095] The preparation method of Lactobacillus paracasei KQNIU fermentation broth is as follows: 10% Lactobacillus paracasei KQNIU (5 mL) is inoculated into 50 mL of MRS liquid medium and fermented at 37℃ for 72 h. After fermentation, the broth is thoroughly shaken and the total fermentation broth is collected, ensuring that the viable cell count is maintained at 1×10⁻⁶. 9 CFU / mL, shake well before use.

[0096] Preparation method of Lygodium japonicum decoction: Grind Lygodium japonicum into coarse powder using a pulverizer, pass through a 40-mesh sieve, weigh the coarse powder and add it to distilled water at a material-to-liquid ratio of 1:10 g / mL (e.g., 25g of Lygodium japonicum coarse powder to 250mL of distilled water), and soak for 12 hours. Then, place the soaking solution in a multi-functional extraction tank, heat to boiling, and maintain a gentle boil for 1.5 hours. After decoction, filter through four layers of gauze and collect the filtrate. Repeat the above method to decoct the residue once more, and combine the filtrates. Concentrate the combined filtrate under reduced pressure to a relative density of 1.1 (60℃), approximately 1 / 5 of the original volume, to obtain 50mL of concentrated traditional Chinese medicine solution. Finally, sterilize the concentrated traditional Chinese medicine solution at 121℃ for 20 minutes, cool to room temperature, and shake well before use.

[0097] Preparation method of co-fermentation broth of *Lactobacillus paracasei* KQNIU and *Lygodium japonicum*: Add 10% of the aforementioned *Lygodium japonicum* herbal solution (i.e., 5 mL) to 50 mL of MRS liquid medium, adjust the pH to 6.8, sterilize at 121℃ for 20 min, inoculate with 10% *Lactobacillus paracasei* KQNIU (5 mL) and ferment at 37℃ for 72 hours. After fermentation, shake thoroughly and take the whole fermentation broth, ensuring that the viable count is maintained at 1×10⁻⁶. 9 CFU / mL, shake well before use.

[0098] 5.1 Growth performance index testing Weight: Calves were weighed once a day, and weight change data were recorded and a weight change curve was plotted. The condition of calf feces was recorded daily and scored. The feces scoring method is shown in Table 8, with a score range of 1 to 4, where 1 = normal; 2 = mild diarrhea; 3 = moderate diarrhea; and 4 = severe watery diarrhea.

[0099] Table 8 Evaluation Criteria for Calf Feces Morphology

[0100] 5.2 Detection methods for immune markers IFN-γ, TNF-α, IL-1β, and IL-10 After infection with Campylobacter jejuni in calves, blood was collected from the anterior vena cava of each group of calves. 1 mL of blood was collected, centrifuged in centrifuge tubes, and the serum was separated and stored at -80°C. The levels of IFN-γ, TNF-α, IL-1β, and IL-10 in the serum were detected using ELISA.

[0101] 5.3 Detection of immune cells in peripheral blood of calves 5.3.1 Preparation of single-cell suspension Single-cell suspension preparation: Take 5 mL of anticoagulated blood from each group of calves, separate the peripheral blood cells using peripheral blood lymphocyte separation medium, aspirate the cells from the middle layer, resuspend the cells in 1 mL of erythrocyte lysis buffer, lyse on ice for 10 min, remove after 5 min and shake for 30 s. Add 10 mL of PBS buffer to stop the reaction, balance the liquid, centrifuge at 1650 rpm, 4℃, for 5 min. Discard the supernatant, resuspend the cells in 1 mL of FACS buffer, and count the cells.

[0102] 5.3.2 Flow cytometry detection Flow cytometry antibody staining (1) Divide the prepared single-cell suspension into tubes, ensuring that each tube contains 1×10⁻⁶ cells. 6 5 × 10 cells (peripheral blood) 5 The total volume is 100 μL; (2) Add the appropriate volume of flow cytometry antibody containing cell surface markers according to the antibody titer; (3) After shaking and mixing, place at 4°C for 30 minutes in the dark; (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 in a small amount of FACS buffer. (5) Repeat step (4) once to thoroughly remove unbound antibodies and reduce non-specific staining on the cell surface. Samples were analyzed using a BD flow cytometer, and data analysis and graphics processing were performed using FlowJo_v10.6.2 software.

[0103] 5.4 Detection of secretory immunoglobulin A (sIgA) in calves Collect 5g of feces from each group of calves, dissolve it in PMSF (1:100) and centrifuge at 3000 rpm, 4℃ for 15 min. Collect the supernatant and perform ELISA to detect secretory immunoglobulin A (sIgA) in the feces of each group of calves.

[0104] 6. Test Results: 1) Results of growth performance index testing Weight changes were recorded during the experiment, and the results showed that the co-fermentation broth of Lactobacillus paracasei KQNIU and Lygodium japonicum could protect calves from Campylobacter jejuni infection and reduce the rate of weight loss.

[0105] Specifically, during the immunization period from day 0 to day 7, the weight of calves in all groups showed a stable growth trend, and the growth status of each group was similar. After the challenge treatment on day 7 (oral administration of Campylobacter jejuni infection), the growth of the healthy group was basically unaffected, while the control group experienced a larger weight loss, reflecting the negative impact of Campylobacter jejuni infection. The Lygodium japonicum group and the Lactobacillus paracasei KQNIU group could alleviate the sharp weight loss to some extent, and the weight changes of calves in the co-fermentation group were significantly different from those in the co-fermentation group. The above weight changes suggest that calves fed with the co-fermentation liquid of Lactobacillus paracasei KQNIU and Lygodium japonicum can resist Campylobacter jejuni infection to a certain extent, playing a preventive and protective role. The results are shown in the table below. Figure 6 .

[0106] When scoring the diarrhea status of calves in each group, the results showed that the healthy group occasionally showed an increase, but generally maintained a score of around 1, indicating normal fecal condition; the control group's score gradually increased to 3-4, indicating obvious diarrhea symptoms, reaching a more severe level on days 4-6; the score of the Lygodium japonicum group remained basically at 1, with mild diarrhea; the score of the Lactobacillus paracasei KQNIU group remained basically at around 1-2.5, with occasional mild diarrhea. This indicates that the Lygodium japonicum group and the Lactobacillus paracasei KQNIU group can alleviate the diarrhea severity to some extent compared to the control group. The co-fermentation group's score remained at 1-2, indicating normal fecal condition. This suggests that the co-fermentation broth of the Lygodium japonicum group and Lactobacillus paracasei KQNIU can alleviate diarrhea in calves caused by Campylobacter jejuni infection. Figure 7 .

[0107] 2) Detection results of immune markers IFN-γ, TNF-α, IL-1β, and IL-10 The collected serum was analyzed using EILSA to detect the secretion of cytokines IFN-γ, TNF-α, IL-1β, and IL-10. The results are shown in the figure. Figures 8-11 .

[0108] After immunization, calves in each group were infected with Campylobacter jejuni. Compared with the healthy group, the KQNIU group of Lactobacillus paracasei, the Lygodium japonicum group, and the fermentation broth group, the IFN-γ content in the control group was significantly increased, indicating that infection with Campylobacter jejuni increases IFN-γ secretion in calves. However, compared with the healthy group, the KQNIU group, the Lygodium japonicum group, and the fermentation broth group showed no significant difference (ns) in IFN-γ content, indicating that Campylobacter jejuni infection did not affect the changes in IFN-γ content in the KQNIU group, the Lygodium japonicum group, and the fermentation broth group. The results are as follows. Figure 8 As shown.

[0109] After immunization, calves in all groups were infected with Campylobacter jejuni. Compared with the healthy group, the control group showed a significantly higher level of IL-1β. P <0.0001), indicating that infection with Campylobacter jejuni induces the production of large amounts of the pro-inflammatory factor IL-1β in calves; compared with the control group, the IL-1β levels in the Lactobacillus paracasei KQNIU group and Lygodium japonicum group were significantly lower, but still significantly higher than those in the healthy group, indicating that both treatments can inhibit the inflammatory response to some extent; the difference in IL-1β between the co-fermentation group and the control group was extremely significant ( P The IL-1β level was <0.0001, and its level was closest to that of the healthy group, indicating that the co-fermentation broth could significantly inhibit the production of IL-1β. This suggests that the co-fermentation broth has the strongest anti-inflammatory effect and can effectively inhibit the inflammatory response caused by Campylobacter jejuni infection in calves. The results are as follows. Figure 9 As shown.

[0110] After immunization, calves in all groups were infected with Campylobacter jejuni. Compared with the healthy group, the control group had a significantly higher IL-10 level. P <0.001), indicating that infection with Campylobacter jejuni in calves increases the secretion of the anti-inflammatory factor IL-10; the IL-10 level in the Lygodium japonicum group was not significantly different from that in the control group (ns), indicating that Lygodium japonicum has a limited effect on maintaining IL-10 levels; the IL-10 levels in the Lactobacillus paracasei KQNIU group and the co-fermentation group were significantly different from those in the control group ( P The IL-10 level was <0.01%, significantly lower than the control group and close to that of the PBS group, indicating that the *Lactobacillus paracasei* KQNIU group and the co-fermentation broth can reduce serum IL-10. This suggests that the co-fermentation broth can effectively maintain the body's anti-inflammatory capacity and help combat the inflammatory response caused by *Campylobacter jejuni* infection in calves. Figure 10 As shown.

[0111] After immunization, calves in all groups were infected with Campylobacter jejuni. Compared with the healthy group, the control group showed a significantly higher level of TNF-α. P <0.0001), indicating that infection with Campylobacter jejuni induces the production of large amounts of the pro-inflammatory factor TNF-α in calves; compared with the control group, the levels of TNF-α in the Lactobacillus paracasei KQNIU group, Lygodium japonicum group, and fermentation broth group were significantly reduced ( P The TNF-α level was <0.0001, indicating that all three treatments had an anti-inflammatory effect. The fermentation broth group had the lowest TNF-α level, close to that of the healthy group, indicating that the co-fermentation broth had the strongest anti-inflammatory effect and could significantly inhibit TNF-α production induced by Campylobacter jejuni infection in calves, further confirming its excellent anti-inflammatory effect. The results are as follows... Figure 11 As shown.

[0112] 3) Effects of co-fermentation products of *Lactobacillus paracasei* KQNIU and *Lygodium japonicum* on Th2 response Th2-type cellular immunity participates in humoral immunity through the secretion of IL-4. Therefore, the activation level of Th2 cells in the peripheral blood of calves was detected. The results showed that infection with Campylobacter jejuni, as well as co-fermentation broths of Lactobacillus paracasei KQNIU and Lygodium japonicum, could activate CD4+. + T cells secrete IL-4, and importantly, the co-fermentation broth of *Lactobacillus paracasei* KQNIU and *Lygodium japonicum* stimulates higher levels of IL-4, with the synergistic fermentation showing the best immune protective effect. The results are as follows: Figure 12 As shown.

[0113] according to Figure 12 It can be seen that, compared with the healthy group, the control group had higher levels of CD4+ in peripheral blood. + The proportion of IL-4 T cells decreased extremely significantly. P <0.001), indicating that infection with Campylobacter jejuni significantly suppresses the Th2 immune response in calves; compared with the control group, the CD4 count in the Lactobacillus paracasei KQNIU group was significantly lower. + The proportion of IL-4 T cells increased significantly ( P <0.01, indicating that this treatment can maintain the Th2 immune response to some extent; the difference between the co-fermentation group and the control group was significant ( P <0.001), its CD4 + The proportion of IL-4 T cells was close to that of the healthy group, indicating that the co-fermentation broth could significantly improve the Th2 immune response. This suggests that the co-fermentation broth can effectively maintain the body's immune balance and enhance its defense against Campylobacter jejuni infection in calves.

[0114] 4) Effects of co-fermentation products of *Lactobacillus paracasei* KQNIU and *Lygodium japonicum* on Th1 response Killer T cells generate a Th1 immune response by secreting IFN-γ, therefore, the secretion level of IFN-γ in peripheral blood was detected. The results showed that after infection with Campylobacter jejuni, the co-fermentation broth of Lactobacillus paracasei KQNIU, Lactobacillus paracasei KQNIU, and Lygodium japonicum activated CD8. + T cells secrete higher levels of IFN-γ, enhancing cellular immune responses. The effects are even more pronounced with the co-fermentation of *Lactobacillus paracasei* KQNIU and *Lygodium japonicum*. Results are as follows... Figure 13 As shown.

[0115] like Figure 13 As shown, compared to the healthy group, the control group had CD8... + The proportion of IFN-γ T cells was extremely significantly reduced. P <0.001), indicating that Campylobacter jejuni infection in calves significantly suppressed cellular immune responses; compared with the healthy group, the CD8 counts of Lactobacillus paracasei KQNIU group and Lygodium japonicum group were significantly lower. + The decreased proportion of IFN-γ T cells indicates that the combined use of these two treatments has limited effect on maintaining cellular immunity; while the co-fermentation group showed a decrease in CD8... + The level of IFN-γ T cells was not significantly different from that in the healthy group (ns), but was significantly higher than that in the control group (ns). P <0.001), indicating that the co-fermentation broth can effectively maintain CD8. + The level of IFN-γ T cells indicates that the co-fermentation broth has the strongest immunoprotective effect and can effectively prevent the decline in cellular immune function caused by Campylobacter jejuni infection in calves.

[0116] 5) Effects of co-fermentation products of Lactobacillus paracasei KQNIU and Lygodium japonicum on secretory sIgA in calf feces Secretory immunoglobulin A (sIgA) in bovine feces is a core biomarker of intestinal mucosal immunity. This example tested the sIgA content in the feces of calves in each group. Figure 14 As shown, the results indicated that the healthy group maintained a higher level of sIgA content compared to the control group, with a significant difference ( P <0.0001, Campylobacter jejuni infection in calves has a certain impact on sIgA, and the sIgA of the Lactobacillus paracasei KQNIU group is significantly higher than that of the control group ( P<0.001). Furthermore, the difference between the co-fermentation group and the control group was extremely significant (…). P The value <0.0001 indicates that the co-fermentation broth can activate calf intestinal immune cells to produce high levels of sIgA. Furthermore, the difference between the co-fermentation group and the healthy group was significant. P The value <0.01 indicates that the sIgA level in the gut activated by the co-fermentation broth has exceeded that of the healthy group, and that its sIgA can protect calves from infection by Campylobacter jejuni.

[0117] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fermentation microbial preparation for inhibiting Campylobacter jejuni in calves, characterized in that, This is a co-fermentation product of a mixture of Lygodium japonicum decoction and liquid culture medium, inoculated with Lactobacillus paracasei; the Lactobacillus paracasei is Lactobacillus paracasei (… Lacticaseibacillus paracasei Lactobacillus paracasei KQNIU was deposited on August 25, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35739, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The fermenting microbial preparation for inhibiting C. jejuni in calves contains 10 9 CFU / mL ~ 10 10 CFU / mL.

2. The method for preparing the fermentation microbial agent for inhibiting Campylobacter jejuni of calves according to claim 1, characterized in that, To prepare the aqueous decoction of Lygodium japonicum, the aqueous decoction of Lygodium japonicum was mixed with liquid culture medium, inoculated with Lactobacillus paracasei, and then co-fermented. The co-fermentation conditions were: pH value range of 6.2-7.2, fermentation temperature range of 35℃-40℃, and fermentation time range of 24h-96h.

3. The method for preparing the fermentation microbial agent for inhibiting Campylobacter jejuni according to claim 2, characterized in that, The decoction of Lygodium japonicum and the liquid culture medium were mixed at a volume ratio of 1:8 to 1:

12.

4. The method for preparing the fermentation microbial agent for inhibiting Campylobacter jejuni according to claim 2, characterized in that, Inoculation conditions for Lactobacillus paracasei: Inoculation amount is 8%~12%.

5. The method for preparing the fermentation microbial agent for inhibiting Campylobacter jejuni in calves according to claim 2, characterized in that, The preparation method of the Lygodium japonicum decoction includes: crushing Lygodium japonicum into coarse powder, sieving it, adding distilled water, soaking it, placing it in an extraction tank, heating it to boiling, maintaining a gentle boil while decocting, filtering it after decoction, and collecting the filtrate; repeating the decoction of the residue according to the above method, and combining the filtrates; concentrating the combined filtrate under reduced pressure to obtain the concentrated Chinese medicine liquid, which is the Lygodium japonicum decoction.

6. The use of the fermented microbial preparation for inhibiting Campylobacter jejuni of calves as described in claim 1 in the preparation of drugs or feed additives for inhibiting Campylobacter jejuni of calves.

7. The use of the fermented microbial preparation for inhibiting Campylobacter jejuni of calves as described in claim 1 in the preparation of drugs or feed additives for the prevention, relief and / or treatment of calf diarrhea.

8. The application of the fermented microbial preparation for inhibiting Campylobacter jejuni according to claim 7 in the preparation of drugs or feed additives for the prevention, relief, and / or treatment of calf diarrhea, characterized in that, The calf diarrhea was caused by Campylobacter jejuni ( Campylobacter jejuni )cause.

9. The application of the fermented microbial preparation for inhibiting Campylobacter jejuni according to claim 7 in the preparation of drugs or feed additives for the prevention, relief, and / or treatment of calf diarrhea, characterized in that, The drug or feed additive is available in liquid or solid form, wherein the liquid form is selected from injections or oral solutions, and the solid form is selected from lyophilized powders or granules.

10. The application of the fermented microbial preparation for inhibiting Campylobacter jejuni according to claim 7 in the preparation of drugs or feed additives for the prevention, relief, and / or treatment of calf diarrhea, characterized in that, The drugs or feed additives also include excipients.

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