A strain of *Lactobacillus rhamnosus* for the prevention and treatment of mycotic diseases in livestock and poultry and its application

Abstract

The application belongs to the technical field of screening and application of probiotics, and particularly relates to a lactobacillus rhamnosus for preventing and treating mycosis of livestock and poultry and application thereof. The lactobacillus rhamnosus is isolated from the intestinal tract of healthy chickens, and has been preserved in the China Center for Type Culture Collection of Wuhan University on February 27, 2025, with a preservation number of CCTCC NO: M2025309. The strain has significant inhibitory and detoxification effects on various molds and their toxins, and can be added to animal medicines, animal feeds, feed additives and feed leavening agents, and has a broad application prospect.

Description

Technical Field

[0001] This invention belongs to the field of probiotic screening and application technology, specifically relating to a strain of Lactobacillus rhamnosus for the prevention and treatment of mycotic diseases in livestock and poultry and its application. Background Technology

[0002] Mold and its toxin contamination is very common in livestock and poultry farming and has become a significant factor affecting the healthy development of the industry. Mycotoxins have a wide range of sources, including feed, bedding, and transfer from breeding stock, making livestock and poultry susceptible to mycotoxin damage during their growth.

[0003] Due to the widespread presence of mycotoxins, the incidence of mycotic diseases in livestock and poultry is relatively high. Studies have shown that over 80% of raw materials and feed are contaminated with mold, over 80% of chicken farms are contaminated with molds and toxins, and over 80% of chicken flocks are infected with molds and toxins. Mycotoxins can damage multiple organs in livestock and poultry, including the liver, kidneys, and immune system, causing symptoms such as immunosuppression, growth retardation or stunting, failure to reach target weight, and decreased egg production. They also easily lead to secondary diseases such as E. coli infection, serositis, and respiratory diseases, seriously affecting the health and production performance of livestock and poultry, and directly increasing breeding costs and reducing the quality of livestock and poultry products. Mycotoxins may also remain in livestock and poultry products, thus threatening human health and increasing food safety risks.

[0004] Traditional methods for preventing mold contamination mainly involve physical and chemical methods. However, these methods cannot completely and effectively control toxins or can cause significant damage to the nutritional components of feed. Currently, biological methods for removing mycotoxins are gaining increasing attention due to their high efficiency and lack of toxic side effects.

[0005] Lactic acid bacteria are considered an ideal alternative to antibiotics, as their use in livestock and poultry diets has a positive impact on animal health and growth. Numerous studies have shown that lactic acid bacteria can promote animal growth, improve gastrointestinal function, inhibit the proliferation of harmful bacteria, regulate and maintain the balance of the gut microbiota, colonize the intestinal mucosal surface by tightly binding to intestinal mucosal cells through adhesive substances, repair the intestinal microbiota barrier, and cure intestinal diseases; they can also stimulate specific immune responses and enhance the body's immunity.

[0006] Lactic acid bacteria primarily reduce mycotoxins through a combination of mechanisms, including acid production, antibacterial activity, decomposition, and adsorption. However, most microorganisms cannot degrade mycotoxins; only carefully selected strains, developed through long-term research, possess this ability. Therefore, screening for lactic acid bacteria that can effectively degrade mycotoxins while also exhibiting good antibacterial properties is of great significance. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of *Lactobacillus rhamnosus* for the prevention and control of mycotic diseases in livestock and poultry, and its application. This strain, isolated from the intestines of healthy chickens, exhibits significant inhibitory and detoxifying effects against common molds and their toxins in livestock and poultry farming, and has important application value for the prevention and control of mycotic diseases in livestock and poultry.

[0008] This invention provides a strain of Lactobacillus rhamnosus, named Lactobacillus rhamnosus K18 ( Lacticaseibacillus rhamnosus K18 was deposited on February 27, 2025, at the China Center for Type Culture Collection, Wuhan University, China, with accession number CCTCC NO: M2025309.

[0009] The MALDI-TOF molecular weight profile of the Lactobacillus rhamnosus K18 strain is as follows: Figure 3 As shown; its Riboprinter fingerprint spectrum is as follows. Figure 4 As shown; its RAPD fingerprint spectrum is as follows. Figure 5 As shown; its rep-PCR fingerprint is as follows. Figure 6 As shown.

[0010] The 16S rDNA sequence of the Lactobacillus rhamnosus K18 strain is SEQ ID NO: 3.

[0011] This invention provides, in one aspect, the application of the Lactobacillus rhamnosus K18 strain in feed production.

[0012] The present invention also provides the use of the Lactobacillus rhamnosus K18 strain in the preparation of medicines for the prevention or treatment of mycosis in livestock and poultry.

[0013] The aforementioned fungal disease is a disease caused by fungal infection, which mainly harms livestock and poultry through mycotoxins, the metabolic products of fungi.

[0014] The mold mentioned is one or more of Penicillium spp., Aspergillus flavus, Fusarium graminearum, and Fusarium moniliforme;

[0015] The mycotoxins mentioned are one or more of aflatoxin, chrysoraxin, zearalenone, vomitoxin, and T-2 toxin.

[0016] This invention also provides the use of Lactobacillus rhamnosus strain K18 in the preparation of medicines for the prevention or treatment of mixed fungal and bacterial infections, secondary bacterial infections of fungal diseases, or bacterial infections in livestock and poultry;

[0017] The bacteria mentioned are any one or more of Escherichia coli, Salmonella, Staphylococcus aureus, or Clostridium perfringens.

[0018] The present invention also provides a probiotic preparation comprising at least one of the following: live bacteria, inactivated bacterial cells, extracellular metabolites, or intracellular extracts of the Lactobacillus rhamnosus K18 strain.

[0019] The present invention also provides Lactobacillus rhamnosus K18, which can significantly inhibit the growth of common molds in livestock and poultry farming. The fermentation broth containing live Lactobacillus rhamnosus K18 has an inhibition rate of more than 80% against Penicillium spp., Aspergillus flavus, Fusarium graminearum and Fusarium moniliforme.

[0020] The Lactobacillus rhamnosus K18 provided by this invention also has a strong inhibitory effect on the growth of common bacteria in livestock and poultry farming, such as Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens, with an inhibition zone diameter as high as 22.74 mm.

[0021] The Lactobacillus rhamnosus K18 provided by this invention has strong antioxidant and cholesterol-degrading capabilities. Its DPPH free radical scavenging rate is 52.57%, and the scavenging rates of its cells and supernatant on hydroxyl free radicals are 41.36% and 53.35%, respectively. Its lipid peroxidation inhibition rates are 10.74% and 32.55%, respectively, and its cholesterol degradation rate reaches 32.15%.

[0022] The Lactobacillus rhamnosus K18 provided by this invention has strong tolerance to simulated porcine gastrointestinal fluid and can successfully reach the intestine to exert its probiotic effect.

[0023] The adhesion index of Lactobacillus rhamnosus K18 to Caco-2 cells provided by this invention is 11.25, indicating that this strain has strong adhesion to intestinal epithelial cells, providing favorable conditions for its adhesion and colonization in the intestine.

[0024] The *Lactobacillus rhamnosus* K18 provided by this invention can significantly reduce the incidence and mortality of fungal diseases in broiler chickens and improve their growth performance. Compared with Group III broiler chickens fed only with slightly moldy diets, Group II broiler chickens fed with *Lactobacillus rhamnosus* K18 simultaneously had an average daily weight gain and average daily feed intake that increased by 27.25% and 13.26%, respectively, and a feed conversion ratio that decreased by 11.33%.

[0025] The Lactobacillus rhamnosus K18 provided by this invention has no toxic effects on the body, is sensitive to common antibiotics, and has good biosafety. It can be added to animal medicines, animal feed, feed additives, and feed fermentation agents, and has broad application prospects. Attached Figure Description

[0026] Figure 1 These are colony images and Gram staining images of strain K18; where A is the colony image and B is the Gram staining image.

[0027] Figure 2The image shows the API test results for strain K18.

[0028] Figure 3 MALDI-TOF ribosomal protein fingerprint of strain K18;

[0029] Figure 4 Riboprinter fingerprint of strain K18;

[0030] Figure 5 RAPD fingerprint of strain K18;

[0031] Figure 6 The rep-PCR fingerprint of strain K18;

[0032] Figure 7 This is a diagram showing the cytotoxicity of strain K18 on Caco-2 cells. Detailed Implementation

[0033] This invention screened a strain of *Lactobacillus rhamnosus* that has a significant inhibitory and detoxifying effect on *Penicillium spp.*, *Aflatoxin*, and other pathogenic fungi and mycotoxins, which has very important application value for the prevention and control of fungal diseases in livestock and poultry.

[0034] The screening process for Lactobacillus rhamnosus K18 provided by this invention is carried out in accordance with regulatory requirements at every step. Polyphasic taxonomic identification has confirmed that Lactobacillus rhamnosus K18 is a novel strain of Lactobacillus rhamnosus.

[0035] On February 27, 2025, the applicant deposited Lactobacillus rhamnosus K18 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025309.

[0036] The screening method described in this invention is not limited to the embodiments. Any known method capable of achieving the screening purpose can be used. The screening descriptions in the embodiments are merely illustrative of this invention and are not intended to limit the scope of protection of this invention. Any modifications or substitutions made to the methods, steps, or conditions of this invention without departing from the spirit and substance of this invention are within the scope of this invention.

[0037] The present invention will now be described in detail with reference to specific embodiments.

[0038] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0039] Among them, Aspergillus flavus ( Aspergillus flavus The accession number for *Penicillium chlorophyll* is BNCC 142801. Penicillium viridicatum The accession number for *Fusarium graminearum* is BNCC 337558. Fusarium graminearum The accession number for *Fusarium moniliforme* is BNCC 113713. Fusarium verticillioides The accession number of the sample is BNCC 340687, and all samples were purchased from BeiNa Biotechnology.

[0040] Escherichia coli ( Escherichia coli The accession number for the bacteria was CMCC 44102, purchased from the China Medical Bacterial Culture Collection Center; Salmonella enteritidis (… Salmonella enteritidis The accession number for Staphylococcus aureus is ATCC 14028. Staphylococcus aureus The accession number of the sample is ATCC 29213, purchased from the China Industrial Microbial Culture Collection Center; Escherichia coli EC 7.1, EC 6.1, EC 8.1, EC 1.1, Salmonella SE 1.1, SE 6.1, SE 5.1, SE 3.1, Staphylococcus aureus SA 4.1, SA 5.1, SA 7.1, SA 8.1, and Clostridium perfringens CP 2.1 are all clinically isolated pathogenic bacteria, preserved by our company.

[0041] Example 1: Isolation and screening of Lactobacillus rhamnosus K18

[0042] 1.1 Initial screening

[0043] Prepare MRS agar medium, adjust pH to 6.2-6.5, and autoclave at 121℃ for 15 min.

[0044] Sample source: 16 cecal contents of healthy white-feathered broiler chickens from Laixi Farm, Qingdao City, Shandong Province, collected on January 11, 2024.

[0045] Take 1g of chicken intestinal contents, dilute with sterile physiological saline, place in a sterile sample bag, and homogenize using a homogenizer. Take 100μL of the mixture, serially dilute it, spread it on MRS agar medium, and incubate anaerobically at 37℃ for 48h. After single colonies grow on the plates, examine them under a microscope. Based on the colony morphology and microscopic examination results, the applicant screened out a total of 63 potential lactic acid bacteria strains, named K01, K02, ..., K17, K18, ..., K63.

[0046] 1.2 Secondary screening

[0047] 1.2.1 Screening of Lactic Acid Bacteria with High Efficiency in Detoxifying Herbadoxime (OTA)

[0048] The 63 lactic acid bacteria strains obtained from the initial screening were cultured in MRS liquid medium to achieve a bacterial cell concentration of 2.0 × 10⁻⁶. 10CFU / mL, take 1 mL of fresh bacterial culture, centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the bacterial pellet twice with PBS, and resuspend it in PBS solution containing OTA. Set up two replicates for each strain, with an uninoculated PBS solution containing OTA as a blank control. Incubate at 37℃ for 2 h, then centrifuge at 8000 rpm for 5 min, collect the supernatant for detection. Determine the OTA concentration in the supernatant according to the method provided by the ELISA kit, and calculate the OTA detoxification rate of the strain.

[0049] 1.2.2 Screening of lactic acid bacteria that inhibit Penicillium spp.

[0050] The inhibition of *Penicillium chlorophyll* by 20 lactic acid bacteria strains with high OTA detoxification rates was determined using a double-layer plate method. First, MRS solid medium was poured into petri dishes as the lower layer. After solidification, an inoculation loop was used to streak two parallel lines (2-3 cm) of activated lactic acid bacteria solution in the logarithmic growth phase onto the plate. The plates were incubated at 37°C for 48 hours until mycelial growth occurred. Then, a plate containing *Penicillium chlorophyll* spores at a concentration of 10... 6 Pour PDA medium at a concentration of 1000 / mL into a plate and cover it on the lower medium containing lactic acid bacteria. Incubate at 30°C for 3-5 days, measure the size of the inhibition zone around the lactic acid bacteria growth band, and calculate the inhibition rate.

[0051] Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0052] The results showed that among the 20 Lactobacillus strains with high detoxification rates of harboratoin, strain K18 had the highest inhibition rate against Penicillium chloroform, reaching 91.21%, indicating that strain K18 had the strongest inhibitory effect on Penicillium chloroform.

[0053] Example 2: Strain Identification

[0054] 2.1 Identification of colony and cell morphology

[0055] After inoculating strain K18 onto MRS agar medium and anaerobically incubating at 37°C for 24 hours, single K18 colonies were observed to be white, moist, smooth, round, and opaque, with a diameter of approximately 0.5–2 mm. Under a microscope, the bacteria appeared as short rods, occurring singly, in pairs, or in chains. They did not form spores, were Gram-positive, non-motile, and lacked flagella. Figure 1 shows a single K18 colony and its culture state under an optical microscope.

[0056] 2.2 Carbon source metabolism experiment

[0057] The carbon source metabolism performance of strain K18 was verified using API 50CHL reagent. API 50CHL reagent can be used to identify differences in strains at the genus or species level. For experimental methods and result analysis, please refer to the API 50CHL kit instructions. Analysis showed that strain K18 had an ID value of 99.8% with *Lactobacillus rhamnosus*, indicating identical carbohydrate metabolic activity. The API test results are shown below. Figure 2 .

[0058] 2.3 Molecular biological identification

[0059] 2.3.1 16S rDNA gene sequence analysis

[0060] 1) Genomic DNA extraction

[0061] Follow the instructions in the Tiangen Bacterial Genomic DNA Extraction Kit (catalog number: DP302).

[0062] 2) 16S rDNA gene amplification

[0063] Primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2:

[0064] 27F: 5′-AGAGTTTGATCCTGGCTCA-3′ (as shown in SEQ ID NO:1);

[0065] 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (as shown in SEQ ID NO:2).

[0066] The 16S rDNA sequence of strain K18 was obtained by sequencing and is SEQ ID NO:3, as follows:

[0067]

[0068] The 16S rDNA sequence of SEQ ID NO:3 was compared with the NCBI database, and strain K18 was identified as Lactobacillus rhamnosus.

[0069] 2.3.2 MALDI-TOF-MS detection of ribosomal protein expression in strains

[0070] Fresh bacterial culture was inoculated into MRS liquid medium at an inoculum rate of 0.1%. After incubation at 37°C and 150 rpm for 48 hours, the bacterial cells were collected, washed four times with sterile water, and dried. A small amount of fresh bacterial cells was then uniformly coated onto a target plate in the form of a thin film. 1 μL of lysis buffer was added to cover the sample, and after drying, 1 μL of matrix solution was added to cover the sample again. After drying, the sample target was placed in a mass spectrometer for identification. The co-crystallized film formed by the sample and matrix was irradiated with a laser to ionize the proteins in the sample. The ions were accelerated through the flight tube under an electric field of 10–20 kV, and the molecular weight of the proteins was determined based on their flight time to the detector. Protein fingerprints were obtained using Autofms 1000 software (Autof Analyzer v1.0). The main ion peaks of strain K18 were: m / z The identification results for numbers such as 2945.129, 4447.585, 5349.271, 5887.628, and 7573.299 are as follows: Figure 3 As shown.

[0071] 2.3.3 Riboprinter fingerprint spectrum

[0072] A single purified colony was picked up from an agar plate using a sampling stick and placed into a sample tube containing buffer. The colony was then stirred with a hand stirrer to suspend it in the buffer. After inactivation in a heater, the sample was placed into the Riboprinter system. Following DNA preparation, transfer, imaging, and data processing, the Riboprinter fingerprint of strain K18 was obtained. Figure 4 ).

[0073] 2.3.4 Identification using RAPD and rep-PCR fingerprinting

[0074] 2.3.4.1 RAPD fingerprint identification

[0075] 1) Primer sequence:

[0076] 5′- GAGGGTGGCGGTTCT-3′ (as shown in SEQ ID NO:4).

[0077] 2) The RAPD reaction system is shown in Table 1.

[0078] Table 1 RAPD Reaction System

[0079] Reactive components volume Taq DNA polymerase (5 U / μL) 0.2 μL 10× Buffer (containing Mg2+) 2 μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 0.8 μL DNA template 2 μL sterile double-distilled water 14 μL Total volume 20 μL

[0080] 3) Electrophoresis

[0081] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a control. Electrophoresis was performed at a constant voltage of 100V for 80 min, and the electrophoresis pattern was finally detected using a gel imaging system. The RAPD fingerprint of strain K18 is shown below. Figure 5 As shown.

[0082] 2.3.4.2 rep-PCR fingerprinting

[0083] 1) Primer sequence: 5′-CTACGGCAAGGCGACGCTGACG-3′ (as shown in SEQ ID NO:5).

[0084] 2) The reaction system for rep-PCR is shown in Table 2.

[0085] Table 2 Reaction system of rep-PCR

[0086] Reactive components volume r Taq DNA polymerase 0.2μL 10×Ex Taq DNA Buffer 2μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 2μL DNA template 2μL sterile double-distilled water 12.8 μL

[0087] 3) Electrophoresis

[0088] The DL2000 DNA Marker was used as a result control. Amplification results were detected at 100 V for 80 min. The rep-PCR fingerprint of strain K18 is shown below. Figure 6 As shown.

[0089] In summary, based on the colony morphology, carbon source metabolism, and molecular biological identification results of strain K18, the applicant has determined that strain K18 is a novel *Lactobacillus rhamnosus*, and named it *Lactobacillus rhamnosus* K18. Lacticaseibacillus rhamnosus K18).

[0090] Example 3: Safety evaluation of Lactobacillus rhamnosus K18

[0091] Preparation of Lactobacillus rhamnosus K18 suspension:

[0092] Purified *Lactobacillus rhamnosus* K18 colonies were inoculated into fresh MRS liquid medium and cultured at 37°C for 24 h. Then, 1% (V / V) of the inoculum was added to MRS liquid medium and cultured at 37°C for another 24–48 h. 1 mL of fresh *Lactobacillus rhamnosus* K18 culture was centrifuged at 5000 rpm for 5 min, and the fermentation supernatant and bacterial cells were collected separately. The bacterial cells were washed twice with PBS buffer (pH 7.4), and the bacterial concentration was adjusted to 5 × 10⁻⁶ cells / mL with PBS buffer. 7 CFU / mL (OD) 600 The absorbance value is approximately 0.4, resulting in a bacterial suspension.

[0093] 3.1 Hemolytic test

[0094] Weigh out all components of TBS basal medium (17.0 g tryptone, 3.0 g soybean peptone, 5.0 g sodium chloride, 2.5 g anhydrous potassium dihydrogen phosphate, 2.5 g glucose, 1000.0 ml distilled water), dissolve them, and autoclave at 121℃ for 15 min. When the medium cools to 50℃, add 5% sterile defibrinated sheep blood and mix well. Pour the mixture into plates. Streak the test strain onto the prepared blood cell plates and incubate at 37℃. Observe whether the test bacteria exhibit hemolysis after 24-48 h.

[0095] The results showed that Lactobacillus rhamnosus K18 could grow, but there was no change in the blood cell plate, indicating that Lactobacillus rhamnosus K18 does not produce hemolysin and cannot lyse blood cells.

[0096] 3.2 Antibiotic tolerance test

[0097] Antibiotic preparation: Ampicillin, erythromycin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, and clindamycin were all prepared into stock solutions of 2048 μg / mL and stored at -20℃ for later use. Before use, the stock solutions were serially diluted 2-fold with MRS liquid medium to prepare the working solutions, with concentrations ranging from 1 to 1024 μg / mL (11 gradients).

[0098] The minimum inhibitory concentration (MIC) of antibiotics against Lactobacillus rhamnosus K18 was determined by the micro-broth dilution method.

[0099] (1) Add MRS liquid culture medium without antibiotics to the first column of a 96-well plate as a negative control. Add 190 μL of MRS liquid culture medium containing different concentrations of antibiotics to the second to 12th columns. Then, inoculate 10 μL of the above inoculum into each well, make 3 parallel wells, and use 1 well without bacterial culture as a blank.

[0100] (2) Add 50 μL of paraffin oil to cover and prevent moisture evaporation.

[0101] (3) After incubating the 96-well plate at 37℃ for 24 hours, remove it and measure the OD. 600 The MIC values ​​of antibiotics against the bacterial strains were calculated using the results over 24 hours.

[0102] The results showed that the Lactobacillus rhamnosus K18 provided by the present invention was sensitive to common antibiotics such as ampicillin, erythromycin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, and clindamycin, and did not exhibit drug resistance, demonstrating good biosafety.

[0103] Example 4: Tolerance of Lactobacillus rhamnosus K18 to simulated porcine gastric and intestinal fluids

[0104] 4.1 Preparation of bacterial culture

[0105] The cryopreserved Lactobacillus rhamnosus K18 strain was streaked into MRS solid medium and cultured at 37°C for 24-48 hours. After being subcultured once in MRS liquid medium, Lactobacillus rhamnosus K18 was inoculated into fresh MRS liquid medium at a 5% inoculum and cultured at 40°C with shaking for 24-48 hours to obtain fresh bacterial culture.

[0106] 4.2 Preparation of simulated porcine gastric juice

[0107] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, and 2g of NaCl, add them to 1000mL of distilled water, adjust the pH to 3.0 with dilute hydrochloric acid, and sterilize at 121℃ for 15min. Then, before use, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and place in a 37℃ water bath shaker for 1h to simulate the body temperature of a pig.

[0108] 4.3 Preparation of simulated pig intestinal fluid

[0109] Weigh out 5g of peptone, 2.5g of yeast extract, 1g of glucose, 6.8g of KH2PO4, and 3.0g of porcine bile salts, respectively. Add them to 190mL of 0.2mol / L NaOH solution, and bring the volume to 1000mL. Adjust the pH to 7.5±0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 121℃ for 15 min. Before use, add 10g of porcine pancreatic enzyme, shake well to dissolve, and incubate in a 37℃ water bath for 1 hour to simulate porcine body temperature.

[0110] 4.4 Test Methods

[0111] Take 2 mL of fresh bacterial culture, centrifuge at 5000 rpm for 5 min to collect bacterial cells, wash the cells three times with physiological saline, and then resuspend them in 2 mL of physiological saline as inoculum. Take 1 mL of inoculum and add it to 9 mL of simulated pig gastric fluid that has been warmed for 1 h. Place the mixture in a 37℃ water bath shaker at 200 rpm for 4 h. Take 1 mL samples at 0 h and 4 h to detect the viable bacterial count. Then take 1 mL of simulated pig intestinal fluid digested for 4 h and add it to 24 mL of simulated pig intestinal fluid. Place the mixture in a 37℃ water bath shaker (200 rpm) for 12 h. Take 1 mL samples to detect the viable bacterial count. The LOG (CFU / mL) values ​​of the viable bacterial count of this strain after treatment with simulated pig gastric and intestinal fluids are shown in Table 3.

[0112] Table 3. Viable bacterial counts after simulated digestion of pig gastric and intestinal fluids.

[0113] strains Before digestion After simulating digestion by pig gastric juice Simulated digestion of pig intestinal fluid Lactobacillus rhamnosus K18 7.85±0.03 7.71±0.05 7.52±0.04

[0114] As can be seen from Table 3, after digestion with simulated pig gastric and intestinal fluids, the viable count of Lactobacillus rhamnosus K18 decreased only slightly, indicating that this strain has strong tolerance to simulated pig gastric and intestinal fluids and can play a positive probiotic role in the intestine.

[0115] Example 5: Determination of the antioxidant capacity of Lactobacillus rhamnosus K18

[0116] 5.1 Determination of DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) scavenging ability

[0117] Take 1 mL of the *Lactobacillus rhamnosus* K18 suspension described in Example 3, add 1 mL of 0.4 mM freshly prepared DPPH radical solution, mix well, and incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the sample at a wavelength of 517 nm. 样品 The test was performed in triplicate. The control group consisted of an equal volume of PBS solution and a DPPH·ethanol mixture, with an equal volume of bacterial suspension and ethanol mixture used as a blank to zero the test. The clearance rate was calculated using the following formula: Clearance rate % = [1 - (A 样品 -A 空白 ) / A 对照 ×100%. See Table 4 for specific results.

[0118] Table 4 DPPH free radical scavenging rate

[0119] strains Clearance Standard deviation Lactobacillus rhamnosus K18 52.57% 0.28%

[0120] 5.2 Determination of hydroxyl radical (HRS) scavenging ability

[0121] 200 μL of bacterial suspension, 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, and 500 μL of deionized water were mixed thoroughly, and then 100 μL of 3 mM hydrogen peroxide solution was added. The mixture was incubated at 37°C for 15 min, and the absorbance of the bacterial suspension was measured at 510 nm. The scavenging rate of the bacterial suspension on HRS was calculated. Alternatively, an equivalent amount of fermentation supernatant was used instead of the bacterial suspension to determine the scavenging rate of the supernatant on HRS.

[0122] The hydroxyl radical scavenging rate is calculated using the following formula.

[0123] Clearance rate % = (A 样品 -A 控制 ) / (A 空白 -A 控制 ) × 100%.

[0124] Where A 控制 A represents the absorbance of a mixed solution of ferrous sulfate, hydrogen peroxide, and sodium salicylate. 空白 The absorbance is shown in Table 5 for the mixed solution of ferrous sulfate and sodium salicylate. Specific results are shown in Table 5.

[0125] Table 5. Scavenging of HRS free radicals by Lactobacillus rhamnosus K18

[0126] Lactobacillus rhamnosus K18 Clearance Standard deviation Bacterial cells 41.36% 0.15% Fermentation supernatant 53.35% 0.62%

[0127] 5.3 Determination of anti-lipid peroxidation capacity

[0128] Preparation of linoleic acid emulsion: 0.1 mL linoleic acid, 0.2 mL Tween 20, 19.7 mL deionized water.

[0129] Add 1 mL of linoleic acid emulsion and 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of sample. Incubate at 37°C for 1.5 h. Add 0.2 mL of TCA (4%) and 2 mL of TBA (0.8%) to the mixture, incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 rpm / min for 15 min, collect the supernatant, and measure the absorbance at 532 nm, which is A. The control group is 0.5 mL of distilled water instead of the sample, which is A0. Inhibition rate / % = (A0 - A) / A0 × 100%.

[0130] Note: A represents the absorbance of the sample group; A0 represents the absorbance of the control group. See Table 6 for specific results.

[0131] Table 6. Inhibition rate of anti-lipid peroxidation

[0132] Lactobacillus rhamnosus K18 Inhibition rate Standard deviation Bacterial cells 10.74% 0.45% Fermentation supernatant 32.55% 0.12%

[0133] The above results indicate that the Lactobacillus rhamnosus K18 provided by the present invention has strong antioxidant capacity, can effectively scavenge DPPH and HRS free radicals, and has significant anti-lipid peroxidation effect.

[0134] Example 6: Determination of the in vitro cholesterol degradation capacity of Lactobacillus rhamnosus K18

[0135] The cholesterol determination method follows GB / T 5009.128-2003 "Determination of Cholesterol in Food". Accurately weigh 1g of cholesterol, dissolve it in anhydrous ethanol, and bring the volume to 100mL. Filter the solution under aseptic conditions through a 0.22μm microporous membrane for sterilization. Inoculate fresh bacterial culture at a rate of 0.1%, and incubate at 37℃ for 48h. Then, take 0.2mL of the bacterial culture, add 1.8mL of anhydrous ethanol, mix well, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and use the supernatant for cholesterol content determination.

[0136] The results showed that the degradation rate of cholesterol by Lactobacillus rhamnosus K18 provided by the present invention reached 32.15%±0.74%, and the degradation rate of cholesterol containing bile salts also reached 14.45%±0.22%.

[0137] Example 7 Cell adhesion assay of Lactobacillus rhamnosus K18

[0138] 7.1 Preparation of bacterial suspension

[0139] Lactobacillus rhamnosus K18 was cultured in MRS liquid medium to the stationary phase, washed three times with phosphate-buffered saline (PBS, pH 7.4), resuspended in cell culture medium, and the bacterial concentration was adjusted to reach 1 × 10⁻⁶ cells / day. 8 CFU / mL, for later use.

[0140] 7.2 Culture of Caco-2 cells

[0141] Caco-2 cells were removed from the liquid nitrogen tank, revived, and passaged to expand the cell number to the required level. The Caco-2 cells were then seeded into six-well plates containing 10% fetal bovine serum-containing cell culture medium and built-in cell spreaders, with approximately 2 × 10⁶ cells per well. 6 Cells, place the six-well plate in a carbon dioxide incubator for 24 hours.

[0142] 7.3 Adhesion Test

[0143] The Caco-2 monolayers that had adhered to the wells of a six-well plate were washed three times with PBS buffer, and the prepared *Lactobacillus rhamnosus* K18 suspension was added. The plate was then incubated in a CO2 incubator for 1 hour. Cell slides were washed three times repeatedly with PBS buffer to remove any unadhered bacteria. The slides were fixed with anhydrous methanol for 20 minutes, air-dried, and Gram-stained. Twenty random fields (100 cells) were examined under a 100x oil immersion microscope to observe the lactic acid bacteria adhering to each cell. The average number of lactic acid bacteria adhering to each cell was calculated.

[0144] Statistical analysis showed that the adhesion amount of Lactobacillus rhamnosus K18 to Caco-2 cells was 11.25±0.82 CFU / cell, indicating that this strain has a strong adhesion ability to Caco-2 cells.

[0145] Example 8 Cytotoxicity test of Lactobacillus rhamnosus K18

[0146] 8.1 Preparation of bacterial suspension

[0147] Lactobacillus rhamnosus K18 was cultured in MRS liquid medium to the stationary phase, washed three times with phosphate-buffered saline (PBS, pH 7.4), and the bacterial concentration was adjusted to reach 5 × 10⁻⁶. 7 CFU / mL (OD) 600 The absorbance value is approximately 0.4. It is inactivated in a 70°C water bath for 20 minutes and then put into use.

[0148] 8.2 Cytotoxicity test

[0149] Resuscitate Caco-2 cells and seed them into 24-well culture plates containing 10% fetal bovine serum cell culture medium at a seeding density of 2 × 10⁶ cells / well. 5 Cells / well, cultured for 24 h. Inactivated strain K18 was added to the cells at an MOI (Multiplicity of Infection) of 10, and a blank control group without bacterial infection was set up. Cells were cultured for another 24 h. MTT solution was added to each well to a final concentration of 0.3 mg / ml, and the cells were incubated in a CO2 incubator for 3 h. The supernatant was carefully discarded, and 500 μL of DMSO was added to each well of a 24-well plate. The cells were incubated at 37°C for 30 min to fully dissolve the purple crystals. The absorbance was measured at 490 nm.

[0150] Test results as follows Figure 7 As shown, compared with the control group, Lactobacillus rhamnosus K18 had no significant effect on the proliferation activity of Caco-2 cells, showed no cytotoxicity, and had good safety.

[0151] Example 9: Detoxification effect of Lactobacillus rhamnosus K18 on other mycotoxins

[0152] The mycotoxins used in this embodiment include: zearalenone (ZEN), vomitoxin (DON), fumonisin B1 (FB1), and trichothecenone (T-2).

[0153] Using the method described in Example 1.2.1, the detoxification rate of Lactobacillus rhamnosus K18 against the above four mycotoxins was detected and calculated using a mycotoxin detection kit. The results are shown in Table 7.

[0154] Table 7. Detoxification effect of Lactobacillus rhamnosus K18 on mycotoxins

[0155] Toxin types Detoxification rate (%) Zearalenone (ZEN) 87.24±0.36 DON (vomiting toxin) 85.45±0.78 Fumonisin B1 (FB1) 61.73±0.72 Trichothecene (T-2) 82.21±0.54

[0156] As shown in Table 7, Lactobacillus rhamnosus K18 has a significant inhibitory effect on a variety of molds that can produce mycotoxins, with an inhibition rate of over 80%.

[0157] Example 10: Inhibitory effect of Lactobacillus rhamnosus K18 on other pathogenic fungi and bacteria.

[0158] The pathogenic fungi used in this embodiment include: Aspergillus flavus BNCC 142801, Fusarium graminearum BNCC 113713, and Fusarium moniliforme BNCC 340687. Potato dextrose agar medium was used, and the fungi were cultured at 28°C for 5-7 days.

[0159] The pathogenic bacteria used in this embodiment include: Escherichia coli (CMCC 44102, EC 7.1, EC 6.1, EC 8.1, EC 1.1), Salmonella (ATCC 14028, SE 1.1, SE 6.1, SE 5.1, SE 3.1), Staphylococcus aureus (ATCC 29213, SA 4.1, SA 5.1, SA 7.1, SA 8.1), and Clostridium perfringens CP 2.1. Nutrient broth medium was used, and the cultures were incubated at 37°C for 16-24 hours.

[0160] Lactobacillus rhamnosus K18 was inoculated into MRS liquid medium at a rate of 1% and incubated at 37°C for 24 hours.

[0161] The inhibition rate of Lactobacillus rhamnosus K18 against Aspergillus flavus, Fusarium graminearum, and Fusarium moniliforme was evaluated using the double-layer plate method in Example 1.2.2. The results are shown in Table 8.

[0162] Table 8. Inhibitory effect of Lactobacillus rhamnosus K18 on molds

[0163] Pathogenic molds Antibacterial rate (%) Aspergillus flavus 89.78±0.45 Fusarium graminearum 83.30±0.66 Fusarium moniliforme 81.42±0.58

[0164] As shown in Table 8, Lactobacillus rhamnosus K18 has a significant inhibitory effect on a variety of molds that can produce mycotoxins, with an inhibition rate of over 80%.

[0165] The inhibition ability of Lactobacillus rhamnosus K18 against Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens was evaluated by the diameter of the inhibition zone using the double-layer plate method. The results are shown in Table 9.

[0166] Table 9. Inhibitory effect of Lactobacillus rhamnosus K18 on pathogenic bacteria

[0167] Pathogenic bacteria Diameter of the inhibition zone (mm) Escherichia coli CMCC 44102 21.25±0.23 Escherichia coli EC 7.1 18.32±0.41 Escherichia coli EC 6.1 20.70±0.45 Escherichia coli EC 8.1 20.22±0.19 Escherichia coli EC 1.1 19.21±0.81 Salmonella ATCC 14028 20.65±0.34 Salmonella SE 1.1 14.43±0.54 Salmonella SE 6.1 32.20±0.57 Salmonella SE 5.1 20.19±0.33 Salmonella SE 3.1 18.75±0.78 Staphylococcus aureus ATCC 29213 21.24±0.94 Staphylococcus aureus SA 4.1 18.31±0.38 Staphylococcus aureus SA 5.1 19.32±0.89 Staphylococcus aureus SA 7.1 16.85±0.74 Staphylococcus aureus SA 8.1 18.45±0.85 Clostridium perfringens CP 2.1 22.74±0.52

[0168] As shown in Table 9, Lactobacillus rhamnosus K18 has a significant inhibitory effect on a variety of pathogens, including Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens, with an inhibition zone diameter as high as 22.74 mm, achieving unexpected technical results.

[0169] Example 11: Effects of Lactobacillus rhamnosus K18 on the prevention of mycorrhizal diseases and growth performance in large-scale broiler chicken farming.

[0170] One hundred and eighty healthy 21-day-old white-feathered broilers with similar initial weights were randomly divided into three groups: Group I (fed a basal diet), Group II (fed a slightly moldy diet / kg + 10... 11 Groups K18 and III (fed with slightly moldy feed) were divided into three groups with three replicates, each containing 20 birds. There were no significant differences in initial body weight among the replicates (P>0.05). Chickens were raised on the ground, fed mixed feed, and provided free access to feed and water. The experimental site and animals were provided by Gaomi Chicken Farm in Weifang City, Shandong Province. The experiment lasted 21 days. Preventative immunizations were administered according to the farm's standard immunization program, and other management practices were consistent with those of the chicken farm. Morbidity and mortality rates were observed and recorded in each group; the results are shown in Table 10.

[0171] Growth performance was assessed: Animals were weighed at 21 and 42 days of age, with a 12-hour fast before weighing, but water was provided continuously. Feed intake, remaining feed, and feed loss were recorded at each stage. Initial body weight, final body weight, feed intake, and remaining feed were accurately recorded for each stage during the experiment. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (F / G) were calculated. The results are shown in Table 11.

[0172] The calculation formula is as follows:

[0173] Average daily weight gain = (average weight at the end of the trial - average weight at the beginning of the trial) / number of days in the trial.

[0174] Average daily feed intake = total feed consumption during the trial period / (number of trial days × number of chickens).

[0175] Feed conversion ratio = average daily feed intake / average daily weight gain.

[0176] Table 10. Inhibitory effect of Lactobacillus rhamnosus K18 on fungal diseases in broiler chickens.

[0177] Group Incidence rate mortality rate Group I 0 0 Group II 6.67% 3.33% Group III 53.33% 31.67%

[0178] As shown in Table 10, compared with Group III broiler chickens fed only with slightly moldy diet, Group II broiler chickens fed with Lactobacillus rhamnosus K18 had significantly lower morbidity and mortality rates. This demonstrates that Lactobacillus rhamnosus K18 provided by this invention can effectively inhibit the occurrence of fungal diseases in broilers.

[0179] Table 11 Effects of Lactobacillus rhamnosus K18 on the growth performance of broiler chickens

[0180] project Group I Group II Group III Average daily weight gain (ADG / g) 42.34±0.92 45.25±1.21 35.56±0.82 Average daily feed intake (ADF / g) 80.12±0.74 81.67±0.82 72.11±0.69 Material weight ratio F / G 1.89±0.06 1.80±0.05 2.03±0.02

[0181] As shown in Table 11, compared with Group III broiler chickens fed only with slightly moldy diet, Group II broiler chickens fed with Lactobacillus rhamnosus K18 simultaneously showed an increase of 27.25% in average daily weight gain and 13.26% in average daily feed intake, while a decrease of 11.33% in feed conversion ratio. This demonstrates that the Lactobacillus rhamnosus K18 provided by this invention can significantly improve the growth performance of broiler chickens, achieving unexpected technical effects.

[0182] In summary, the *Lactobacillus rhamnosus* (Lactobacillus casei) provided by this invention... acticaseibacillus rhamnosus Strain K18 significantly inhibits the growth of common molds and bacteria in livestock and poultry farming. It is tolerant of the gastrointestinal environment and exhibits strong adhesion to intestinal epithelial cells, providing favorable conditions for its successful arrival, colonization, and probiotic effects in the intestines. It possesses strong antioxidant and cholesterol-degrading capabilities, significantly reducing the incidence and mortality of mycotic diseases in broiler chickens, increasing average daily weight gain, reducing feed conversion ratio, and improving growth performance. This strain is non-cytotoxic, has good safety profile, and can be widely used in animal pharmaceuticals, animal feed, feed additives, and feed fermentation agents, showing broad application prospects.

Claims

1. A Lactobacillus rhamnosus (L. Lacticaseibacillus rhamnosus ) K18, characterized in that, The Lacticaseibacillus rhamnosus K18 has been preserved in the China Center for Type Culture Collection of Wuhan University on February 27, 2025, and the preservation number is CCTCC NO: M2025309.

2. The Lacticaseibacillus rhamnosus K18 of claim 1 is applied in feed production.

3. The Lacticaseibacillus rhamnosus K18 of claim 1 is applied in the preparation of a medicine for preventing or treating mycosis of livestock and poultry.

4. The use according to claim 3, wherein the compound is ###0002### The mycosis is a disease caused by mycotic infection, mixed mycotic and bacterial infection, or mycotic secondary bacterial infection.

5. The use according to claim 4, wherein the compound is ###0002### The mycotic is any one or more of Penicillium viridicatum, Aspergillus flavus, Fusarium graminearum, and Fusarium moniliforme.

6. The Lacticaseibacillus rhamnosus K18 of claim 1 is applied in the preparation of a medicine for preventing or treating bacterial infection of livestock and poultry.

7. Use according to claim 6, wherein The bacteria are any one or more of Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens.

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