Lactobacillus rhamnosus for preventing and treating mycosis of livestock and poultry and application of lactobacillus rhamnosus

Abstract

The invention belongs to the technical field of probiotic screening and application, and particularly relates to lactobacillus rhamnosus for preventing and treating livestock and poultry mycosis and application of the lactobacillus rhamnosus. The lactobacillus rhamnosus is separated from intestinal tracts of healthy chickens and is preserved in China Center for Type Culture Collection in Wuhan University in China on February 27, 2025, and the preservation number of the lactobacillus rhamnosus is CCTCC NO: M2025309. The strain has remarkable inhibition and detoxification effects on various moulds and toxins thereof, can be added into animal medicines, animal feeds, feed additives and feed leavening agents, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic screening and application, and particularly relates to a Lactobacillus rhamnosus for preventing and treating mycosis in livestock and poultry and its application. Background Art

[0002] The pollution of molds and their toxins is very common in livestock and poultry farming and has become an important factor affecting the healthy development of the breeding industry. The sources of mycotoxins are extensive, including feed, bedding, and transfer of breeding chickens, etc., which cause livestock and poultry to be easily invaded by mycotoxins during the growth process.

[0003] Due to the widespread existence of mycotoxins, the prevalence of mycosis in livestock and poultry is relatively high. Research shows that more than 80% of raw materials and feeds are contaminated with molds, more than 80% of chicken farms are contaminated with molds and toxins, and more than 80% of chicken flocks are infected with molds and toxins. Mycotoxins can cause damage to multiple organs such as the liver, kidneys, and immune system of livestock and poultry, causing symptoms such as immunosuppression, growth retardation or stagnation, substandard body weight, and decreased egg production rate. It is extremely easy to secondary diseases such as colibacillosis, serositis, and respiratory diseases, seriously affecting the health and production performance of livestock and poultry, and directly leading to an increase in breeding costs and a decrease in the quality of livestock and poultry products. Mycotoxins may also remain in livestock and poultry products, thus threatening human health and increasing the risk of food safety.

[0004] Traditional means of preventing mold contamination are mainly physical and chemical methods, and these two methods cannot completely and effectively control toxins or will cause great damage to feed nutrients. At present, the biological method of removing mycotoxins has received more and more attention due to its high efficiency and non-toxic side effects.

[0005] Lactic acid bacteria are considered an ideal substitute for antibiotics. After being used in livestock and poultry diets, they can have a positive impact on the health and growth of animals. A large amount of research data shows that lactic acid bacteria can promote animal growth, improve gastrointestinal function; inhibit the reproduction of harmful bacteria, regulate and maintain the balance of gastrointestinal flora; tightly bind to intestinal mucosal cells through adhesion substances, colonize on the surface of the intestinal mucosa, repair the intestinal flora barrier, and cure intestinal diseases; they can also stimulate specific immune responses and improve the body's immunity, etc.

[0006] Lactic acid bacteria mainly reduce mycotoxins comprehensively through ways such as acid production, bacteriostasis, decomposition, and adsorption. However, most microorganisms cannot degrade mycotoxins, and only strains that have been carefully screened through long-term research and targeted have the function of degrading mycotoxins. Therefore, it is of great significance to screen lactic acid bacteria that can not only effectively degrade mycotoxins but also have good bacteriostatic functions. Summary of the Invention

[0007] The object of the present invention is to provide a Lactobacillus rhamnosus strain for preventing and treating mycosis in livestock and poultry and its application. This strain is isolated from the healthy chicken intestine, has a significant inhibitory and detoxifying effect on common molds and their toxins in livestock and poultry breeding, and has important application value for preventing and treating mycosis in livestock and poultry.

[0008] On the one hand, the present invention provides a Lactobacillus rhamnosus strain, named Lactobacillus rhamnosus K18 ( Lacticaseibacillus rhamnosus K18), which was deposited on February 27, 2025 at the China Center for Type Culture Collection of Wuhan University, Wuhan, China, and its deposit number is CCTCC NO: M2025309.

[0009] The MALDI-TOF ribosomal protein molecular weight map of the Lactobacillus rhamnosus K18 strain is as Figure 3 shown; its Riboprinter fingerprint map is as Figure 4 shown; its RAPD fingerprint map is as Figure 5 shown; its rep-PCR fingerprint map is as Figure 6 shown.

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

[0011] On the one hand, the present invention provides the application of the Lactobacillus rhamnosus K18 strain in feed production.

[0012] The present invention also provides the application of the Lactobacillus rhamnosus K18 strain in the preparation of a medicine for preventing or treating mycosis in livestock and poultry.

[0013] The mycosis mentioned above is a disease caused by mold infection, and mainly causes harm to livestock and poultry through mycotoxins, the metabolites of molds.

[0014] The mold mentioned above is one or more of Penicillium viridicatum, Aspergillus flavus, Fusarium graminearum and Fusarium moniliforme; The mycotoxin mentioned above is one or more of aflatoxin, ochratoxin, zearalenone, vomitoxin, T-2 toxin.

[0015] The present invention also provides the application of the Lactobacillus rhamnosus K18 strain in the preparation of a medicine for preventing or treating mixed mycosis and bacterial infection in livestock and poultry, secondary bacterial infection of mycosis or bacterial infection diseases; The bacteria mentioned above are any one or more of Escherichia coli, Salmonella, Staphylococcus aureus or Clostridium perfringens.

[0016] The present invention also provides a probiotic preparation, which contains at least one of viable bacteria, inactivated cells, extracellular metabolites or intracellular extracts of the Lactobacillus rhamnosus K18 strain.

[0017] The Lactobacillus rhamnosus K18 provided by the present invention can significantly inhibit the growth of common molds in livestock and poultry breeding. The inhibition rates of the live bacteria fermentation broth containing Lactobacillus rhamnosus K18 against Penicillium viridicatum, Aspergillus flavus, Fusarium graminearum, and Fusarium moniliforme all reach over 80%.

[0018] The Lactobacillus rhamnosus K18 provided by the present invention also has a strong inhibitory effect on the growth of common bacteria in livestock and poultry breeding such as Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens, and the diameter of the inhibition zone is as high as 22.74 mm.

[0019] The Lactobacillus rhamnosus K18 provided by the present invention has strong antioxidant and cholesterol-degrading abilities. The scavenging rate of DPPH free radicals is 52.57%. The scavenging rates of its bacterial cells and supernatant against hydroxyl free radicals are 41.36% and 53.35% respectively, and the anti-lipid peroxidation inhibition rates are 10.74% and 32.55% respectively, and the cholesterol degradation rate reaches 32.15%.

[0020] The Lactobacillus rhamnosus K18 provided by the present invention has strong tolerance to simulated pig gastrointestinal fluid and can smoothly reach the intestine to play a probiotic role.

[0021] The adhesion index of the Lactobacillus rhamnosus K18 provided by the present invention to Caco-2 cells 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.

[0022] The Lactobacillus rhamnosus K18 provided by the present invention can significantly reduce the incidence and mortality of mycosis in white - feather broilers and improve their growth performance. Compared with the white - feather broilers in Group III fed with slightly moldy feed alone, the average daily gain and average daily feed intake of the white - feather broilers in Group II fed with Lactobacillus rhamnosus K18 at the same time increased by 27.25% and 13.26% respectively, and the feed - to - gain ratio decreased by 11.33%.

[0023] The Lactobacillus rhamnosus K18 provided by the present invention has no toxic effect on the body, is sensitive to common antibiotics, has good biosafety, and can be added to animal drugs, animal feeds, feed additives, and feed fermenters, and has broad application prospects. Description of the Drawings

[0024] Figure 1 It is the colony map and Gram - staining map of strain K18; among them, A is the colony map and B is the Gram - staining map; Figure 2 It is the API test result map of strain K18; Figure 3 It is the MALDI - TOF ribosomal protein fingerprint map of strain K18; Figure 4 is the Riboprinter fingerprint of strain K18; Figure 5 is the RAPD fingerprint of strain K18; Figure 6 is the rep-PCR fingerprint of strain K18; Figure 7 is the cytotoxicity graph of strain K18 against Caco-2 cells. Detailed implementation

[0025] The present invention screened a Lactobacillus rhamnosus strain that has significant inhibitory and detoxifying effects on Penicillium viridicatum, ochratoxin, and other pathogenic molds and mycotoxins of mycosis, and has very important application value for preventing and treating mycosis in livestock and poultry.

[0026] All aspects of the screening process of Lactobacillus rhamnosus K18 provided by the present invention are carried out in accordance with regulatory requirements. Through polyphasic taxonomic identification, Lactobacillus rhamnosus K18 is a novel strain of Lactobacillus rhamnosus.

[0027] The applicant deposited Lactobacillus rhamnosus K18 on February 27, 2025, at the China Center for Type Culture Collection of Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M2025309.

[0028] The screening method described in the present invention is not limited to that described in the examples, and any known method that can achieve the screening purpose can be used. The screening description in the examples is only an illustration of the present invention, not a limitation on the protection scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, or conditions of the present invention belongs to the scope of the present invention.

[0029] The following describes the present invention in detail with specific examples.

[0030] The test materials used in the examples of the present invention are all conventional test materials in the art and can be obtained through commercial channels.

[0031] Among them, the deposit number of Aspergillus flavus ( Aspergillus flavus ) is BNCC 142801, the deposit number of Penicillium viridicatum ( Penicillium viridicatum ) is BNCC 337558, the deposit number of Fusarium graminearum ( Fusarium graminearum ) is BNCC 113713, and the deposit number of Fusarium moniliforme ( Fusarium verticillioides ) is BNCC 340687, all purchased from Beina Biology.

[0032] Escherichia coli ( Escherichia coli) was deposited under the accession number CMCC 44102 and was purchased from the China National Center for Medical Culture Collections; Salmonella enteritidis ( Salmonella enteritidis ) was deposited under the accession number ATCC 14028, and Staphylococcus aureus ( Staphylococcus aureus ) was deposited under the accession number ATCC 29213 and was purchased from the China National Center for Industrial Culture Collections; 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 were all clinically isolated pathogenic bacteria and were stored by the company.

[0033] Example 1 Isolation and Screening of Lactobacillus rhamnosus K18 1.1 Primary Screening Prepare MRS agar medium, adjust the pH to 6.2 - 6.5, and autoclave at 121 °C for 15 min.

[0034] Sample source: 16 samples of cecal contents from healthy white - feather broilers in a farm in Laixi, Qingdao, Shandong Province. The collection time was January 11, 2024.

[0035] Take 1 g of chicken intestinal contents, dilute it with sterile normal saline and put it into a sterile sample bag, and mix well with a homogenizer; take 100 μL of the mixed solution and dilute it serially, spread it on MRS agar medium, and culture it anaerobically at 37 °C for 48 h. Wait for single colonies to grow on the plate and then conduct microscopic examination. According to the colony morphology and microscopic examination results, the applicant screened a total of 63 potential lactic acid bacteria, which were named K01, K02, ……, K17, K18,......, K63 respectively.

[0036] 1.2 Re - screening 1.2.1 Screening of Lactic Acid Bacteria with High - Efficiency Detoxification of Ochratoxin OTA Cultivate the 63 lactic acid bacteria obtained from the primary screening in MRS liquid medium until the cell concentration of the bacteria reaches 2.0×10 10 CFU / mL. Take 1 mL of fresh bacterial liquid, centrifuge it at 8000 rpm for 5 min, discard the supernatant, wash the bacterial cell precipitate twice with PBS, and then resuspend it in PBS solution containing OTA. Set 2 parallels for each strain, and use the PBS solution containing OTA without inoculation as the blank control. After culturing at 37 °C for 2 h, centrifuge it at 8000 rpm for 5 min, and collect the supernatant for detection. According to the method provided by the ELISA kit, determine the concentration of OTA in the supernatant and calculate the detoxification rate of the strain to OTA.

[0037] 1.2.2 Screening of Lactic Acid Bacteria That Inhibit Penicillium viridicatum The double-layer plate method was used to determine the inhibition of Penicillium viridicatum by 20 strains of lactic acid bacteria with a relatively high OTA detoxification rate. First, pour the MRS solid medium into a petri dish as the lower layer plate. After solidification, use an inoculation loop to dip the activated lactic acid bacteria broth in the logarithmic phase and draw 2 parallel lines, 2 - 3 cm long, on the plate. Incubate at 37 °C for 48 h until bacterial colonies grow. Pour the PDA medium containing 10 6 spores / mL of Penicillium viridicatum into the plate and cover the lower layer medium containing the lactic acid bacteria colonies. Incubate at 30 °C for 3 - 5 days, measure the size of the inhibition zone around the lactic acid bacteria growth strip, and calculate the inhibition rate.

[0038] Inhibition rate = [(control colony diameter - treated colony diameter) / control colony diameter] × 100%.

[0039] The results showed that among the 20 strains of Lactobacillus with a relatively high detoxification rate of ochratoxin, strain K18 had the highest inhibition rate against Penicillium viridicatum, reaching 91.21%, indicating that strain K18 had the strongest inhibitory effect on Penicillium viridicatum.

[0040] Example 2 Strain Identification 2.1 Colony and Bacterial Cell Morphology Identification Inoculate strain K18 on MRS agar medium and incubate anaerobically at 37 °C for 24 h. It can be seen that the single colony of K18 is white, with a moist and smooth surface, circular in shape, opaque, and the colony diameter is about 0.5 - 2 mm; the bacterial cells are short rod-shaped under the microscope, single, paired or in chains, do not form spores, are Gram-positive, non-motile, and have no flagella. The single colony of K18 and its culture state under the optical microscope are shown in Figure 1.

[0041] 2.2 Carbon Source Metabolism Test Use API 50CHL reagent to verify the carbon source metabolism performance of strain K18. API 50CHL reagent can be used to identify the differences between strains at the genus or species level. The experimental method and result analysis are specifically referred to the API 50CHL kit instruction manual. The analysis results showed that the ID value of strain K18 and Lactobacillus rhamnosus was 99.8%, and the carbohydrate metabolism activities were the same. The API test results are shown in Figure 2 .

[0042] 2.3 Molecular Biology Identification 2.3.1 16s rDNA Gene Sequence Analysis 1) Genomic DNA Extraction Refer to the operation of Tiangen Bacterial Genomic DNA Extraction Kit (Catalog No.: DP302).

[0043] 2) 16s rDNA Gene Amplification The primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2 as follows: 27F: 5′-AGAGTTTGATCCTGGCTCA-3′ (shown in SEQ ID NO:1); 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (shown in SEQ ID NO:2).

[0044] The 16s rDNA sequence of strain K18 obtained by sequencing is SEQ ID NO:3, which is as follows:

[0045] The 16S rDNA sequence with SEQ ID NO:3 was aligned in the NCBI database, and it was determined that strain K18 was Lactobacillus rhamnosus.

[0046] 2.3.2 Detection of ribosomal protein expression of the strain by MALDI-TOF-MS Fresh bacterial liquid was inoculated into MRS liquid medium at an inoculation amount of 0.1%. After culturing at 37 °C and 150 rpm for 48 h, the bacterial cells were collected, washed 4 times with sterile water, and the surface moisture was air-dried. Then, a small amount of fresh bacterial cells was evenly coated on the target plate in the form of a thin film. 1 μL of lysis solution was added to cover the sample. After air-drying, 1 μL of matrix solution was added to cover the sample. After air-drying, the sample target was placed in a mass spectrometer for identification. The co-crystalline film formed by the sample and the matrix was irradiated with a laser to ionize the proteins in the sample. The ions were accelerated through the flight tube under the action of an electric field of 10 - 20 KV, and the molecular weights of the proteins were detected according to the different flight times to reach the detector. The protein fingerprint was obtained using the Autofms 1000 analysis software Autof Analyzer v1.0. The main ion peaks of strain K18 were: m / z 2945.129, 4447.585, 5349.271, 5887.628, 7573.299, etc. The identification results are as Figure 3 shown.

[0047] 2.3.3 Riboprinter fingerprint A single well-purified colony was picked up from the agar medium plate with a bacterium picker and placed into a sample tube with buffer. It was stirred with a hand-held stirrer to suspend it in the buffer. Then, the sample holder was inactivated in a heater and placed into the Riboprinter system. After DNA preparation, membrane transfer, imaging detection, and data processing of the sample, the Riboprinter fingerprint of strain K18 was obtained ( Figure 4 ).

[0048] 2.3.4 Identification of RAPD and rep-PCR fingerprints 2.3.4.1 Identification of RAPD fingerprint 1) Primer sequence: 5′- GAGGGTGGCGGTTCT-3′ (as shown in SEQ ID NO:4).

[0049] 2) The RAPD reaction system is shown in Table 1.

[0050] Table 1 RAPD reaction system table Reaction 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 3) Electrophoresis Prepare a 1.5% agarose gel plate, use DL2000 DNA Marker as the result control, perform electrophoresis at a constant voltage of 100 V for 80 min, and finally detect the electrophoresis pattern using a gel imaging system. The RAPD fingerprint pattern of strain K18 is as Figure 5 shown.

[0051] 2.3.4.2 rep-PCR fingerprint pattern 1) Primer sequence: 5′-CTACGGCAAGGCGACGCTGACG-3′ (as shown in SEQ ID NO:5).

[0052] 2) The reaction system of rep-PCR is shown in Table 2.

[0053] Table 2 Reaction system table of rep-PCR Reaction components Volume r Taq DNA polymerase 0.2 μL 10× Ex Taq DNA Buffer 2 μL Primer (10 uM) 1 μL dNTPs (2.5 mM) 2 μL DNA template 2 μL Sterile double-distilled water 12.8 μL 3) Electrophoresis Use DL2000 DNA Marker as the result control. Detect the amplification result at a voltage of 100 V and an electrophoresis time of 80 min. The rep-PCR fingerprint pattern of strain K18 is as Figure 6 shown.

[0054] In summary, combining the colony morphological characteristics, carbon source metabolism and molecular biology identification results of strain K18, the applicant determined that strain K18 is a novel Lactobacillus rhamnosus, and named it Lactobacillus rhamnosus K18 ( Lacticaseibacillus rhamnosus K18).

[0055] Example 3 Safety evaluation of Lactobacillus rhamnosus K18 Preparation of Lactobacillus rhamnosus K18 bacterial suspension: Pick the purified colonies of Lactobacillus rhamnosus K18 and inoculate them into fresh MRS liquid medium, and culture at 37°C for 24 h; then inoculate them into MRS liquid medium at an inoculation amount of 1% (V / V), and continue to culture at 37°C for 24 - 48 h; take 1 mL of fresh bacterial liquid of Lactobacillus rhamnosus K18, centrifuge at 5000 rpm for 5 min, and collect the fermentation supernatant and bacteria respectively; rinse the bacteria twice with PBS buffer solution at pH 7.4, and then adjust the concentration of the bacteria to 5×10 7 CFU / mL (OD 600 absorbance value is about 0.4) to obtain the bacterial suspension.

[0056] 3.1 Hemolytic experiment Weigh various components of the TBS basal medium (17.0 g of tryptone, 3.0 g of soybean peptone, 5.0 g of sodium chloride, 2.5 g of anhydrous potassium dihydrogen phosphate, 2.5 g of glucose, 1000.0 ml of distilled water). After dissolving, autoclave at 121 °C for 15 min. When the medium cools to 50 °C, add 5% sterile defibrinated sheep blood, mix well, and pour plates. Streak the test strain on the prepared blood cell plates and incubate in a 37 °C incubator. Observe whether there is hemolysis of the test bacteria after 24 - 48 h.

[0057] The results showed that Lactobacillus rhamnosus K18 could grow and there was no change in the blood cell plates, indicating that Lactobacillus rhamnosus K18 did not produce hemolysin and could not lyse blood cells.

[0058] 3.2 Antibiotic tolerance experiment Antibiotic preparation: Ampicillin, erythromycin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, and clindamycin were all prepared into a stock solution of 2048 μg / mL and stored at -20 °C for later use. When in use, the stock solution was serially diluted 2-fold with MRS liquid medium into working solutions, and the gradient dilution concentrations were 1 - 1024 μg / mL for a total of 11 gradients.

[0059] The minimum inhibitory concentration (MIC) value of antibiotics against Lactobacillus rhamnosus K18 was determined by the microbroth dilution method.

[0060] (1) Add MRS liquid medium without antibiotics to the first column of the 96-well plate in sequence as a negative control. Add 190 μL of MRS liquid medium containing different concentrations of antibiotics to the second to twelfth columns in sequence, and then inoculate 10 μL of the above inoculum into each well, making 3 parallel wells, and leaving 1 well without bacterial solution as a blank.

[0061] (2) Add 50 μL of paraffin oil to cover and prevent water evaporation.

[0062] (3) Take out the 96-well plate after incubating at 37 °C for 24 h, measure the OD 600 value, and use the results of 24 h to statistically analyze the MIC value of the antibiotics against the strain.

[0063] The results showed that Lactobacillus rhamnosus K18 provided by the present invention was sensitive to common antibiotics such as ampicillin, erythromycin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, and clindamycin, had no drug resistance, and had good biosafety.

[0064] Example 4 Tolerance of Lactobacillus rhamnosus K18 to simulated porcine gastric juice and intestinal juice 4.1 Bacterial solution preparation The lyophilized Lactobacillus rhamnosus K18 strain was streaked on MRS solid medium and cultured at 37 °C for 24 - 48 h. After subculturing once in MRS liquid medium, Lactobacillus rhamnosus K18 was inoculated into fresh MRS liquid medium at an inoculum size of 5% and cultured with shaking at 40 °C for 24 - 48 h to obtain fresh bacterial liquid.

[0065] 4.2 Preparation of simulated porcine gastric juice Weigh 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose, and 2 g of NaCl respectively, add 1000 mL of distilled water, adjust the pH to 3.0 with dilute hydrochloric acid, and sterilize at 121 °C for 15 min. Then, add 3.2 g of porcine gastric mucosa pepsin before use, shake well to dissolve, and place in a 37 °C water bath shaker for 1 h to simulate porcine body temperature.

[0066] 4.3 Preparation of simulated porcine intestinal juice Weigh 5 g of peptone, 2.5 g of yeast extract, 1 g of glucose, 6.8 g of KH2PO4, and 3.0 g of porcine bile salts respectively, add 190 mL of 0.2 mol / L NaOH solution, make up the volume to 1000 mL, adjust the pH to 7.5 ± 0.1 with dilute hydrochloric acid or sodium hydroxide solution, and sterilize at 121 °C for 15 min. Then, add 10 g of porcine pancreatin before use, shake well to dissolve, and place in a 37 °C water bath shaker for 1 h to simulate porcine body temperature.

[0067] 4.4 Test method Take 2 mL of fresh bacterial liquid, centrifuge at 5000 rpm for 5 min to collect the bacterial cells. Wash the bacterial cells 3 times with physiological saline and then resuspend them in 2 mL of physiological saline to obtain the inoculum. Take 1 mL of the inoculum and add it to 9 mL of simulated porcine gastric juice that has been pre-warmed for 1 h. Place it in a 37 °C water bath shaker and shake at 200 rpm for 4 h. Take 1 mL of samples at 0 h and 4 h respectively to detect the viable cell count. Then, take 1 mL of the simulated porcine intestinal juice after digestion for 4 h and add it to 24 mL of simulated porcine intestinal juice. Place it in a 37 °C water bath shaker (200 rpm) for 12 h, take 1 mL of the sample to detect the viable cell count. The LOG (CFU / mL) values of the viable cell count of this strain after passing through simulated porcine gastric juice and intestinal juice are shown in Table 3.

[0068] Table 3 Viable cell count after digestion with simulated porcine gastric juice and intestinal juice Strain Before digestion After simulated porcine gastric juice digestion After simulated porcine intestinal juice digestion Lacticaseibacillus rhamnosus K18 7.85±0.03 7.71±0.05 7.52±0.04 As can be seen from Table 3, after Lactobacillus rhamnosus K18 was digested with simulated porcine gastric juice and intestinal juice, the viable cell count only decreased slightly, indicating that this strain has strong tolerance to both simulated porcine gastric juice and intestinal juice and can play an active probiotic role in the intestine.

[0069] Example 5 Determination of the antioxidant capacity of Lactobacillus rhamnosus K18 5.1 Determination of DPPH (1,1-Diphenyl-2-picrylhydrazyl) Scavenging Ability Take 1 mL of the Lactobacillus rhamnosus K18 bacterial suspension described in Example 3, add 1 mL of freshly prepared 0.4 mM DPPH radical solution, mix well, and place it in the dark at room temperature for 30 min. Then measure the absorbance A of the sample at a wavelength of 517 nm 样品 , and measure it 3 times in parallel. The control group is an equal volume of PBS solution and DPPH·ethanol mixture, and zero is adjusted with an equal volume of bacterial suspension and ethanol mixture. The scavenging rate is calculated according to the following formula: Scavenging rate % = [1 - (A 样品 -A 空白 ) / A 对照 × 100%. The specific results are shown in Table 4

[0070] Table 4 DPPH Radical Scavenging Rate Strain Clearance rate Standard deviation Lacticaseibacillus rhamnosus K18 52.57% 0.28% 5.2 Determination of Hydroxyl Radical (HRS) Scavenging Ability Mix 200 μL of the bacterial suspension, 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, and 500 μL of deionized water, then add 100 μL of hydrogen peroxide solution (3 mM). After incubating in a water bath at 37 °C for 15 min, measure the absorbance of the bacterial suspension at a wavelength of 510 nm, and calculate the scavenging rate of the bacterial suspension against HRS. In addition, use the fermentation supernatant with the same dose instead of the bacterial suspension for the experiment, and measure the scavenging rate of the supernatant against HRS

[0071] The hydroxyl radical scavenging rate is calculated according to the following formula

[0072] Scavenging rate % = (A 样品 -A 控制 ) / (A 空白 -A 控制 ) × 100%

[0073] Where A 控制 is the absorbance of the mixed solution of ferrous sulfate, hydrogen peroxide and sodium salicylate, and A 空白 is the absorbance of the mixed solution of ferrous sulfate and sodium salicylate. The specific results are shown in Table 5

[0074] Table 5 Scavenging of HRS Radical by Lactobacillus rhamnosus K18 Lacticaseibacillus rhamnosus K18 Clearance rate Standard deviation Bacterial cells 41.36% 0.15% Fermentation supernatant 53.35% 0.62% 5.3 Determination of Anti-lipid Peroxidation Ability Preparation of linoleic acid emulsion: 0.1 mL of linoleic acid, 0.2 mL of Tween 20, 19.7 mL of deionized water

[0075] Add 1 mL of an emulsion of linoleic acid, 1 mL of FeSO4 (1%) to 0.5 mL of PBS solution (pH 7.4), then add 0.5 mL of the sample. Incubate in a water bath 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 in a water bath at 100 °C for 30 min, quickly cool, centrifuge at 4000 rpm / min for 15 min, and collect the supernatant. Measure the absorbance at 532 nm, which is A; in the control group, replace the sample with 0.5 mL of distilled water, which is A0. Inhibition rate / % = (A0 - A) / A0 × 100%.

[0076] Note: A is the absorbance of the sample group; A0 is the absorbance of the control group. The specific results are shown in Table 6.

[0077] Table 6 Inhibition rate of anti-lipid peroxidation Lacticaseibacillus rhamnosus K18 Inhibition rate Standard deviation Bacterial cells 10.74% 0.45% Fermentation supernatant 32.55% 0.12% The above results indicate that Lactobacillus rhamnosus K18 provided by the present invention has strong antioxidant ability, can effectively scavenge DPPH and HRS free radicals, and has a significant effect on anti-lipid peroxidation.

[0078] Example 6 Determination of in vitro cholesterol degradation ability of Lactobacillus rhamnosus K18 The cholesterol determination method follows GB / T 5009.128 - 2003 "Determination of Cholesterol in Foods". Accurately weigh 1 g of cholesterol, dissolve it in absolute ethanol, and make up the volume to 100 mL. Filter and sterilize it with a 0.22 μm microporous filter membrane under sterile conditions. Inoculate fresh bacterial liquid according to an inoculation amount of 0.1%, incubate statically at 37 °C for 48 h, then take 0.2 mL of the bacterial liquid, add 1.8 mL of absolute ethanol, mix well, let stand for 10 minutes, centrifuge at 3000 rpm for 5 minutes, and take the supernatant for determination of cholesterol content.

[0079] The results show that the degradation rate of cholesterol by Lactobacillus rhamnosus K18 provided by the present invention reaches 32.15% ± 0.74%, and the degradation rate of cholesterol containing bile salts also reaches 14.45% ± 0.22%.

[0080] Example 7 Cell adhesion test of Lactobacillus rhamnosus K18 7.1 Preparation of bacterial suspension Culture Lactobacillus rhamnosus K18 in MRS liquid medium until the stationary phase, wash it 3 times with phosphate buffer solution (PBS, pH 7.4), resuspend it with cell culture medium, and adjust the concentration of the bacteria to make the number of bacteria reach 1×10 8 CFU / mL, for standby.

[0081] 7.2 Culture of Caco-2 cells Take out Caco-2 cells from the liquid nitrogen tank, resuscitate and subculture them, and expand the number of cultured cells to the required amount. Inoculate Caco-2 cells into a six-well culture plate containing cell culture slides and cell culture medium with 10% calf serum. The number of cells plated in each well is about 2×10 6 cells, and place the six-well plate in a carbon dioxide incubator for 24 h.

[0082] 7.3 Adhesion assay The adherent Caco-2 single cell layer in the six-well plate was washed 3 times with PBS buffer, and the prepared Lactobacillus rhamnosus K18 bacterial suspension was added, and then cultured in a carbon dioxide incubator for 1 h. The cell culture slides were repeatedly washed 3 times with PBS buffer to remove non-adherent bacteria. Fixed with absolute methanol for 20 minutes, taken out the cell culture slides to dry, stained with Gram stain, and observed under an oil immersion microscope at 100× magnification in 20 random fields of view. A total of 100 cells were observed for the adhered lactic acid bacteria, and the number of lactic acid bacteria adhered to each cell was calculated.

[0083] Through statistical analysis, it was found 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.

[0084] Example 8 Cytotoxicity test of Lactobacillus rhamnosus K18 8.1 Preparation of bacterial suspension Culture Lactobacillus rhamnosus K18 in MRS liquid medium until the stationary phase, wash it 3 times with phosphate buffer solution (PBS, pH 7.4), adjust the concentration of bacteria to make the number of bacteria reach 5×10 7 CFU / mL (OD 600 The absorbance value is about 0.4), inactivate it in a water bath at 70 °C for 20 min for standby.

[0085] 8.2 Cytotoxicity assay Resuscitate Caco-2 cells and inoculate them into a 24-well culture plate containing cell culture medium with 10% calf serum. The inoculation density is 2×10 5Cells / well, and the cells were cultured for 24 h. The inactivated strain K18 was added to the cells at a ratio of MOI (Multiplicity of Infection) value of 10, and a blank control group without bacteria was set, and then cultured in an incubator for another 24 h. MTT solution was added to each cell culture well to be detected at a final concentration of 0.3 mg / ml, and incubated in a carbon dioxide incubator for 3 h. The supernatant was carefully discarded, and 500 μl of DMSO was added to each cell culture well of the 24-well plate and incubated at 37 °C for 30 min to fully dissolve the purple crystals. The absorbance value at 490 nm was detected.

[0086] The detection results are as Figure 7 shown. Compared with the control group, Lactobacillus rhamnosus K18 had no significant effect on the proliferation activity of Caco-2 cells, had no cytotoxicity, and had good safety.

[0087] Example 9 Detoxification effect of Lactobacillus rhamnosus K18 on other mycotoxins The mycotoxins used in this example include: zearalenone (ZEN), deoxynivalenol (DON), fumonisin B1 (FB1), and trichothecene (T-2).

[0088] Using the method in Example 1.2.1, a mycotoxin detection kit was used for detection and the detoxification rate of Lactobacillus rhamnosus K18 against the above 4 mycotoxins was calculated. The results are shown in Table 7.

[0089] Table 7 Detoxification effect of Lactobacillus rhamnosus K18 on mycotoxins Toxin type Detoxification rate (%) Zearalenone (ZEN) 87.24±0.36 Deoxynivalenol (DON) 85.45±0.78 Fumonisin B1 (FB1) 61.73±0.72 Trichothecene (T-2) 82.21±0.54 As can be seen from Table 7, Lactobacillus rhamnosus K18 has an obvious inhibitory effect on various molds that can produce mycotoxins, and the inhibition rate exceeds 80%.

[0090] Example 10 Inhibitory effect of Lactobacillus rhamnosus K18 on other pathogenic molds and bacteria The pathogenic molds used in this example include: Aspergillus flavus BNCC 142801, Fusarium graminearum BNCC 113713, Fusarium moniliforme BNCC 340687. Potato dextrose agar medium was used and cultured at 28 °C for 5 - 7 d.

[0091] The pathogenic bacteria used in this example include: Escherichia coli (CMCC 44102, EC 7.1, EC 6.1, EC8.1, 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), Clostridium perfringens CP 2.1. Use nutrient broth medium and culture at 37°C for 16 - 24 h.

[0092] Inoculate Lactobacillus rhamnosus K18 at an inoculation amount of 1% into MRS liquid medium and culture statically at 37°C for 24 h.

[0093] Use the double - layer plate method in Example 1.2.2 to evaluate the antibacterial rate of Lactobacillus rhamnosus K18 against Aspergillus flavus, Fusarium graminearum, and Fusarium moniliforme. The results are shown in Table 8.

[0094] Table 8 Inhibitory ability of Lactobacillus rhamnosus K18 against molds Pathogenic mold Bacteriostatic rate (%) Aspergillus flavus 89.78±0.45 Fusarium graminearum 83.30±0.66 Fusarium moniliforme 81.42±0.58 As can be seen from Table 8, Lactobacillus rhamnosus K18 has an obvious inhibitory effect on various molds that can produce mycotoxins, and the antibacterial rate exceeds 80%.

[0095] Use the double - layer plate method to evaluate the inhibitory ability of Lactobacillus rhamnosus K18 against Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens by the diameter of the inhibition zone. The results are shown in Table 9.

[0096] Table 9 Inhibitory ability of Lactobacillus rhamnosus K18 against pathogenic bacteria Pathogenic bacterium Diameter of 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 As can be seen from Table 9, Lactobacillus rhamnosus K18 has a significant inhibitory effect on various pathogenic bacteria such as Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens. The diameter of the inhibition zone is as high as 22.74 mm, achieving an unexpected technical effect.

[0097] Example 11 Effects of Lactobacillus rhamnosus K18 on the prevention of mycosis and growth performance in large - scale broiler chicken farming Randomly divide 180 healthy 21 - day - old broiler chickens with similar initial weights into 3 groups, namely Group I (fed with basal diet), Group II (fed with slightly moldy diet / kg + 10 11CFU strain K18), Group III (fed a slightly moldy diet). Each group had 3 replicates, with 20 chickens in each replicate. There were no significant differences in the initial weights of the replicates (P>0.05). The floor rearing mode was adopted, and the chickens were fed by mixing feed, with free access to food and water. The experimental site and experimental animals were provided by a chicken farm in Gaomi, Weifang City, Shandong Province. The experimental period was 21 days. Preventive immunization was carried out according to the regular immunization program of the chicken farm, and other breeding management was consistent with that of the chicken farm. The incidence and mortality of chickens in each group were observed and recorded, and the results are shown in Table 10.

[0098] Determination of growth performance: The chickens were weighed once at 21 and 42 days of age. They were fasted for 12 h before weighing, and water was not stopped. The feed supply, remaining feed, and lost feed amounts at each stage were recorded. During the experiment, the initial weight, final weight, feed intake, and remaining feed amount at each stage were accurately recorded, and the average daily gain (ADG), average daily feed intake (ADFI), and feed to gain ratio (F / G) were calculated. The results are shown in Table 11.

[0099] The calculation formulas are as follows: Average daily gain = (average weight at the end of the experiment - average weight at the beginning) / number of experimental days.

[0100] Average daily feed intake = total feed consumption during the experimental period / (number of experimental days × number of chickens).

[0101] Feed to gain ratio = average daily feed intake / average daily gain.

[0102] Table 10 Inhibitory effect of Lactobacillus rhamnosus K18 on mycosis in white - feather broilers Group Incidence Mortality Group I 0 0 Group II 6.67% 3.33% Group III 53.33% 31.67% As can be seen from the results in Table 10, compared with the white - feather broilers in Group III fed a slightly moldy diet alone, the incidence and mortality of the white - feather broilers in Group II fed Lactobacillus rhamnosus K18 at the same time were significantly reduced. Thus, it shows that the Lactobacillus rhamnosus K18 provided by the present invention can effectively inhibit the occurrence of mycosis in broilers.

[0103] Table 11 Effect of Lactobacillus rhamnosus K18 on the growth performance of white - feather broilers Item Group I Group II Group III Average daily 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 Feed to gain ratio F / G 1.89±0.06 1.80±0.05 2.03±0.02 As can be seen from the results in Table 11, compared with the white - feather broilers in Group III fed a slightly moldy diet alone, the average daily gain and average daily feed intake of the white - feather broilers in Group II fed Lactobacillus rhamnosus K18 at the same time increased by 27.25% and 13.26% respectively, and the feed to gain ratio decreased by 11.33%. Thus, it shows that the Lactobacillus rhamnosus K18 provided by the present invention can significantly improve the growth performance of white - feather broilers and achieve unexpected technical effects.

[0104] In summary, the Lactobacillus rhamnosus provided by the present invention ( Lacticaseibacillus rhamnosus)(Strain K18) can significantly inhibit the growth of common molds and bacteria in livestock and poultry farming; it can tolerate the gastrointestinal environment, has a strong adhesion to intestinal epithelial cells, providing favorable conditions for its smooth arrival in the intestine, adhesion and colonization in the intestine, and exerting probiotic effects; it has strong antioxidant and cholesterol-degrading abilities, can significantly reduce the incidence and mortality of mycosis in white - feather broilers, improve the average daily gain of white - feather broilers, reduce the feed - to - weight ratio, and improve growth performance. This strain has no cytotoxicity, good safety, and can be widely used in animal drugs, animal feeds, feed additives, and feed fermenting agents, with broad application prospects.

Claims

1. A Lactobacillus rhamnosus K18, characterized in that, The Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) K18 was deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China on February 27, 2025, and its deposit number is CCTCC NO: M2025309.

2. Use of the Lacticaseibacillus rhamnosus K18 according to claim 1 in feed production.

3. Use of the Lacticaseibacillus rhamnosus K18 according to claim 1 in the preparation of a medicament for preventing or treating mycosis in livestock and poultry.

4. The application according to claim 3, characterized in that, The mycosis is a disease caused by mycotic infection, mixed mycotic and bacterial infection, or secondary bacterial infection caused by mycosis.

5. The application according to claim 4, characterized in that, The mycosis is any one or more of Penicillium viridicatum, Aspergillus flavus, Fusarium graminearum, and Fusarium moniliforme.

6. Use of the Lacticaseibacillus rhamnosus K18 according to claim 1 in the preparation of a medicament for preventing or treating bacterial infection in livestock and poultry.

7. The application according to claim 6, characterized in that, The bacteria are any one or more of Escherichia coli, Salmonella, Staphylococcus aureus, and Clostridium perfringens.

8. A probiotic preparation, characterized in that, The probiotic preparation comprises at least one of viable bacteria, inactivated bacteria, extracellular metabolites, or intracellular extracts of the Lacticaseibacillus rhamnosus K18 according to claim 1.

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