Lactobacillus paracasei IB2 and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to lactobacillus paracasei IB2 and application thereof. The strain is preserved in the Guangdong Microbial Culture Collection Center on January 2, 2024, the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC NO: 64237. The probiotic preparation used in the invention does not contain antibiotics and drug residues, so that the safety of dairy cow breeding and dairy products is improved. The functional probiotics are developed for preventing and treating recessive mastitis of dairy cows, and can regulate the gastrointestinal function of the dairy cows and effectively improve the feed intake of the dairy cows. The dairy cow subclinical mastitis can be effectively prevented and treated, the morbidity of the dairy cow subclinical mastitis is reduced, and the cow milk quality is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a strain of Lactobacillus paracasei IB2 and application thereof. Background Art

[0002] Cow mastitis is a complex multifactorial disease, which is the result of the combined effects of pathogenic microorganisms, environmental factors, management factors, cow factors and genetic factors. Cow mastitis ranks first among the three major diseases of dairy cows (mastitis, endometritis and hoof disease). This disease not only causes serious losses to the dairy farming industry, but also threatens food safety. It is one of the important diseases that seriously endanger the dairy industry. There are 220 million dairy cows in the world, and about 1 / 3 of them suffer from mastitis. The average incidence of mastitis abroad is about 25% to 60%. For example, in Japan, the average incidence is 45.1%, and in Western European countries, the incidence is 25%. The Lanzhou Animal Husbandry and Veterinary Drug Research Institute of the Chinese Academy of Agricultural Sciences has conducted a survey on 10,371 adult dairy cows in 32 large-scale dairy farms in 22 cities in my country. Among them, the incidence of clinical mastitis is 33.41% (9.7% to 55.6%), and the average positive rate of latent mastitis is 73.91%. Mastitis in dairy cows causes a decrease in milk production, the discarding of diseased milk, and even the permanent loss of milk secretion due to suppuration, gangrene, and atrophy of the udder area, which leads to the culling of dairy cows, causing great losses to the dairy industry. In my country, the number of dairy cows culled due to mastitis accounts for 9% to 10% of the total culling rate each year.

[0003] In the prevention and treatment of clinical mastitis and latent mastitis, antibiotics are still the first choice for treating mastitis in dairy cows. However, due to the long-term use or unscientific use of antibiotics, the problem of bacterial resistance is becoming more and more serious, posing a huge hidden danger to human and animal health. For this reason, the government has set relevant restrictions on the use of antibiotics, so other treatment methods (such as Chinese herbal medicine treatment and probiotic preparations, etc.) have received attention from breeding companies and researchers. In order to solve the problem of "reducing antibiotics and replacing antibiotics", probiotic products are developing rapidly as "reducing antibiotics and replacing antibiotics" products. Probiotics refer to living microorganisms that have beneficial effects on host health when given in sufficient amounts. At present, probiotic preparations, as a new type of safe, efficient and environmentally friendly product, have been rarely reported in the prevention and treatment of clinical mastitis and latent mastitis. Therefore, screening probiotic strains that can prevent and treat clinical mastitis and latent mastitis in dairy cows and developing related probiotic products have broad application prospects, important practical significance, and help the healthy development of my country's dairy industry. Summary of the invention

[0004] To solve the above problems, the present invention provides a strain of Lactobacillus paracasei IB2 (Lacticaseibacillus paracasei IB2), which was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 2, 2024, with a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC NO: 64237.

[0005] The present invention also provides application of the Lactobacillus paracasei IB2 in preparing products for inhibiting pathogenic bacteria.

[0006] Furthermore, the pathogenic bacteria include Escherichia coli, Mycoplasma bovis, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus uberis and Streptococcus agalactiae.

[0007] On the other hand, the present invention also provides a product for preventing and treating latent mastitis in dairy cows, the raw materials of which include at least one of the above-mentioned strains and / or the supernatant of the strains.

[0008] The invention also provides a feed additive for preventing and treating latent mastitis in dairy cows, the ingredients of which include the above product.

[0009] The present invention has the following beneficial effects:

[0010] 1. The probiotic preparation used in the present invention does not contain antibiotics and drug residues, thereby improving the safety of dairy cow breeding and conforming to the current concept of green and healthy dairy cow breeding.

[0011] 2. The present invention is a functional probiotic specially developed for the problem of latent mastitis in dairy cows. It can regulate the gastrointestinal function of dairy cows and effectively increase the feed intake of dairy cows.

[0012] 3. The present invention can effectively prevent and treat the occurrence of latent mastitis in dairy cows, reduce the incidence of latent mastitis in dairy cows, and improve the quality of cow milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 is the colony morphology of Lactobacillus paracasei IB2;

[0015] Figure 2 is the Gram staining image of Lactobacillus paracasei IB2;

[0016] Figure 3It is the electrophoresis diagram of the PCR product of Lactobacillus paracasei IB2, wherein lane M is a 2000 bp DNA marker, and lane 1 is the PCR specific product of strain IB2 of the present invention;

[0017] Figure 4 This is the antibacterial graph of Lactobacillus paracasei IB2 inhibiting Staphylococcus aureus ATCC6538. DETAILED DESCRIPTION

[0018] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The culture medium used in the embodiment of the present invention includes:

[0024] Sterile PBS: Weigh 8.0 g NaCl, 0.2 g KCl, 0.27 g KH2PO4, and 1.42 g Na2HPO4 and dissolve them in 800 mL deionized water in sequence. After stirring thoroughly to dissolve, add hydrochloric acid to adjust the pH to 7.2, add deionized water to make up to 1 L, sterilize at 121°C for 20 min, and store at room temperature for later use.

[0025] MRS liquid culture medium: peptone 10.0 g, yeast extract powder 5.0 g, glucose 20.0 g, K2HPO4 2.0 g, triammonium citrate 2.0 g, anhydrous sodium acetate 5.0 g, MnSO4 0.058 g, MgSO4 0.29 g, Tween 80 1.0 mL. Dissolve the above ingredients in 800 mL of deionized water in turn, adjust the pH to 7.2, and make the volume to 1000 mL. Sterilize under high pressure at 121°C for 20 min.

[0026] MRS solid culture medium: Add 1.5% agar powder to MRS liquid culture medium and sterilize by high pressure.

[0027] Example 1 Isolation and identification of strains

[0028] 1.1 Isolation of strains

[0029] The present invention separates and screens Lactobacillus paracasei IB2 from yogurt brewed by herdsmen in Qinghai Province using an ancient method. The specific separation and purification method of IB2 is as follows: weigh 10 g of yogurt, add it into a conical flask containing 90 mL of sterile PBS and a number of sterile glass beads, vortex and mix thoroughly, take 1 mL of the suspension, and continuously dilute it to 10 with PBS. -4 , 10 -5 , 0.1mL was spread on the MRS plate, and cultured anaerobically at 37℃ for 24h, and single colonies of different shapes and sizes were picked and purified continuously on the MRS plate. Lactic acid bacteria were picked from the MRS plate and numbered accordingly, and streaked and purified on the MRS plate; after each isolate was purified, a single colony was picked and cultured anaerobically at 37℃ in MRS for 24h, centrifuged and discarded the supernatant, and the strains were preserved in liquid nitrogen containing 15% glycerol-MRS preservation solution for later use.

[0030] 1.2 Morphological observation of strains

[0031] After 24 hours of anaerobic culture of strain IB2 on MRS plate, its colony morphology was round, grayish white, with smooth and moist surface, neat edges and bulges ( Figure 1 ). Strain IB2 was observed under an oil microscope to be a Gram-positive bacillus, with a blue-purple body and no spores ( Figure 2 ).

[0032] 1.3 Molecular biological identification of strains

[0033] The genome of IB2 isolate was extracted as a PCR amplification template, and the bacterial identification 16S rDNA universal primers 27F and 1492R were used for PCR amplification. The reaction system was 25 μL. The PCR amplification procedure was as follows: pre-denaturation at 94°C for 3 min; 94°C for 45 s; 55°C for 45 s; 72°C for 70 s; repeat 30 cycles; 72°C for 8 min; the amplified product was identified by 1.5% agarose gel electrophoresis, and the product size was about 1500 bp ( Figure 3 ). The PCR product was sent to Qingke Jersey Biological Co., Ltd. for sequencing, and the sequencing results were submitted to NCBI for Blast comparison analysis. The sequence of the isolate IB2 was 100% homologous to that of Lactaseibacillusparacasei. The strain was submitted and deposited in Guangdong Microbiological Culture Collection Center on January 2, 2024, with the deposit number GDMCC NO:64237.

[0034] Example 2 Safety Evaluation of Lactobacillus paracasei IB2

[0035] 2.1 Hemolytic test

[0036] Lactobacillus paracasei IB2 was subcultured three times in MRS liquid medium. A small amount of culture solution was dipped in an inoculation loop and streaked on Columbia blood agar medium. After anaerobic culture at 37°C for 24 hours, the hemolysis ring was observed around the colony. Staphylococcus aureus (isolated and preserved in this laboratory) was used as a positive control. According to the ability of bacteria to dissolve red blood cells, it can be divided into the following three types of hemolysis and phenomena: α hemolysis: hemolysin is produced, incomplete hemolysis, and there is a 1-2mm, narrow, translucent, grass-green hemolysis ring around the colony on the blood agar plate. β hemolysis: complete hemolysis, there is a 2-4mm wide, clearly defined, colorless and transparent hemolysis ring around the colony on the blood agar plate. γ hemolysis: no hemolysin is produced, and there is no hemolysis ring around the colony on the blood agar plate. The results showed that Lactobacillus paracasei IB2 did not show α-hemolysis and β-hemolysis on the blood plate, belonging to γ ​​hemolysis. It is preliminarily believed that strain IB2 is a safe strain.

[0037] 2.2 Drug resistance gene detection test

[0038] DNA extraction: After strain IB2 was cultured anaerobically in MRS liquid medium for 24 hours, the cells were centrifuged and the bacterial cells were collected. The bacterial genomic DNA was extracted according to the method provided by the bacterial genomic DNA extraction kit. The extracted bacterial genomic DNA was used as a template, and PCR amplification was performed using specific primers for kanamycin resistance gene, gentamicin resistance gene, tetracycline resistance gene and vancomycin resistance gene (Table 1). The PCR reaction system (25 μL) was: 12.5 μL LATaq premixed enzyme, 1 μL of upstream and downstream primers, 1 μL DNA template, and 9.5 μL ddH2O. The amplified product was identified by 1.5% agarose gel electrophoresis, and the size of its specific product is shown in Table 1. The PCR results showed that IB2 was negative for all four drug resistance genes, indicating that IB2 did not contain the above four drug resistance genes.

[0039] Table 1 Primer sequences of drug resistance genes

[0040]

[0041] 2.3 Drug sensitivity test

[0042] The Kirby–Bauer test (KB method) was used to determine the drug sensitivity of strain IB2. Dip a sterile cotton swab in the IB2 bacterial suspension diluted to an appropriate concentration, spread it evenly on the surface of the MRS plate, and place it at room temperature for 10-20 minutes. Use sterile tweezers to place the drug-sensitive sheet on the surface of the MRS agar plate. After anaerobic culture at 37°C for 24 hours, use a vernier caliper to measure the inhibition zone of the drug-sensitive sheet. Escherichia coli (ATCC25922) was used as a quality control strain. The drug sensitivity results were determined according to the standards of the American Clinical Laboratory Standards Institute. The results were: sensitive (S), moderately sensitive (I), and resistant (R). The antibiotics used in this study included 11 antibiotics from 8 categories, including amoxicillin (20 μg / tablet), β-lactams ampicillin (10 μg / tablet), cefotaxime sodium (30 μg / tablet); quinolone norfloxacin (10 μg / tablet); macrolide erythromycin (15 μg / tablet); aminoglycosides streptomycin (10 μg / tablet), kanamycin (30 μg / tablet); chloramphenicol (30 μg / tablet); tetracycline (30 μg / tablet); lincosamides clindamycin (10 μg / tablet); and glycopeptide vancomycin (30 μg / tablet). The results showed that, except for moderate sensitivity to kanamycin, Lactobacillus paracasei IB2 was sensitive to the other 10 antibiotics and had no resistance (Table 2).

[0043] Table 2 Results of IB2 antibiotic sensitivity test

[0044]

[0045] 2.4 In vitro safety tests

[0046] Select 30 healthy male C57BL / 6J mice of 21 days old, adaptive feeding for 1 week, free access to food and drinking water, randomly divided into 3 groups, 10 / group, including control group, low-dose group, high-dose group (Table 3). Each experimental group mouse is fed with basal diet, and the control group is gavaged with PBS 0.2mL / only / day every day, and the experimental group is gavaged with strain Lactobacillus paracasei IB2 of the present invention, and specific gavage dosage is shown in Table 3. The test lasts for 30 days, and the mental state, growth, coat, stool and other situations of mice are observed every day. After the test, the weight gain rate of mice is calculated, and the viscera are observed to have or not lesions.

[0047] Table 3 Design of mouse safety study

[0048]

[0049] The results are shown in Table 4. During the test, the feeding, spirit, behavior and stool of each group of mice were normal, with no significant difference, indicating that the strain of the present invention had no effect on the growth of mice. After the dissection, the organs of each group of mice were carefully observed. There were no visible lesions in the heart, liver, spleen, kidney and lung of the test mice, indicating that Lactobacillus paracasei IB2 of the present invention is safe for mice.

[0050] Table 4 Weight gain rate of mice (%)

[0051]

[0052] Example 3 Study on the probiotic properties of strains

[0053] 3.1 Acid and bile salt resistance test

[0054] The strain IB2 was subcultured and activated three times in succession, inoculated at a 2% inoculum size in PBS (pH = 3.0) containing 0.3% (w / v) porcine bile salts, vortexed and placed in static anaerobic culture at 37°C, samples were taken at 0h and 3h, and the diluted samples were counted on MRS plates and cultured anaerobically at 37°C for 24h.

[0055] The calculation formula of strain tolerance is: survival rate (%) = N3 / N0×100%

[0056] Where: N3 is the number of viable bacteria at 3 hours; N0 is the number of viable bacteria at 0 hours; the number of viable bacteria is expressed in CFU / mL.

[0057] The experimental results showed that the survival rate of strain IB2 was higher in acid and bile salt environments. After incubation for 3 hours at pH = 3.0, the survival rate of strain IB2 was 75.12%; after incubation for 3 hours at 0.3% bile salt, the survival rate was 61.50%. These results show that IB2 has strong acid and bile salt resistance.

[0058] 3.2 Antibacterial test

[0059] The strain IB2 was subcultured twice, inoculated into MRS liquid medium at 3% inoculum, placed at 37°C for 24h, centrifuged at 6000rpm for 10min at 4°C, and the supernatant was filtered and sterilized with a 0.22μm filter for later use. The 14 pathogenic strains in this embodiment are: Escherichia coli ATCC43888, Proteus vulgaris ATCC29905, Staphylococcus aureus ATCC6538 and Salmonella typhimurium ATCC14028 purchased from the China Veterinary Drug Administration; Mycoplasma bovis (08m strain and NX1 strain) was donated by Dr. Guo Yanan from the Institute of Animal Science, Ningxia Academy of Agriculture and Forestry Sciences; Escherichia coli (LXR1 strain, LXR4 strain and YT1 strain), Klebsiella pneumoniae (SQ3 strain and YT2 strain), Streptococcus uberis (NM1 strain and ZJ2 strain), Streptococcus agalactiae BY1 were all isolated and obtained by this laboratory. The concentration of the 14 pathogenic strains used for antibacterial treatment was adjusted to 1 × 10 6 CFU / mL, and evenly spread on the NA plate with a sterile cotton swab, let it stand for 10-20 minutes at room temperature, then punch holes (aperture ± 7mm, depth ± 4.5mm), seal the bottom with 1% agar, and draw about 150μL of filtrate into the hole, let it stand at room temperature for 2 hours, then culture at 37℃, and take it out when a clear inhibition zone appears (about 18 hours). Use a vernier caliper to measure the diameter of the inhibition zone (mm), and the size of the inhibition zone diameter represents the antibacterial ability of probiotics.

[0060] The results showed that Lactobacillus paracasei IB2 had different degrees of inhibitory effects on 14 common pathogens (Table 5). The strain could inhibit the growth of 14 pathogens including Escherichia coli, Mycoplasma bovis, Staphylococcus aureus, Salmonella typhimurium, Proteus vulgaris, Klebsiella pneumoniae, Streptococcus uberis, and Streptococcus agalactiae, and had a stronger inhibitory effect on Mycoplasma bovis 08m, Escherichia coli LXR4, and Streptococcus uberis NM1 (Table 5).

[0061] Table 5 Determination of antibacterial ability of Lactobacillus paracasei IB2 (mm)

[0062]

[0063] 3.3 Adhesion assay of IB2

[0064] The strain IB2 and the control strain (Lactobacillus rhamnosus GG, kindly donated by Professor Si Hongwei of Tennessee State University, USA) were labeled with fluorescein isothiocyanate (FITC) solution. The concentration of the bacterial solution was adjusted to 2×10 8 CFU / mL and measure its fluorescence intensity when the absorption wavelength is 485nm and the emission wavelength is 530nm, which is the initial relative fluorescence value R0.

[0065] 0.5 mL of IB2 bacterial suspension or Lactobacillus rhamnosus GG was added to the monolayer cells, respectively. The cells were incubated for 2 h and rinsed 4 times with sterile PBS. After 5 min of trypsin digestion, the fluorescence intensity of the cell suspension was measured, which was the relative fluorescence value R2 after adhesion.

[0066] Adhesion rate of strain = (R2 / R0) × 100%

[0067] The adhesion rate of IB2 to epithelial cells Caco-2 was 13.72%, slightly higher than that of the reference strain Lactobacillus rhamnosus GG, but the difference was not significant (Table 6).

[0068] Table 6 Adhesion rate of IB2

[0069] strain Adhesion rate (%) IB2 13.72±0.43 Lactobacillus rhamnosus GG 12.68±0.54

[0070] 3.4 Study on the characteristics of IB2 in inhibiting pathogenic bacteria adhesion to Caco-2 cells

[0071] Fluorescently label Escherichia coli and Salmonella at a concentration of 2 × 10 8 CFU / mL. Adjust the concentration of Lactobacillus paracasei IB2 suspension to 2×10 8 CFU / mL.

[0072] Competition test: 0.25 mL of Lactobacillus paracasei IB2 (or Lactobacillus rhamnosus GG) and pathogenic bacteria were added to the monolayer cells in the test wells and mixed evenly; 0.5 mL of pathogenic bacteria was added to the blank wells. After incubation for 2 hours, the cells were washed 4 times with sterile PBS.

[0073] Rejection test: Add 0.5 mL of Lactobacillus paracasei IB2 (or Lactobacillus rhamnosus GG) to the monolayer cells in the test wells, and add an equal volume of cell culture medium to the blank wells. After incubation for 1 hour, discard the liquid in the wells and rinse with sterile PBS 3 times. Add 0.5 mL of pathogenic bacteria to each well, incubate for 1 hour, and rinse with sterile PBS 4 times.

[0074] Displacement test: Add 0.5 mL of pathogenic bacteria to the monolayer cells and incubate for 1 hour. Discard the liquid in the wells and rinse 3 times with sterile PBS. Add 0.5 mL of Lactobacillus paracasei IB2 (or Lactobacillus rhamnosus GG) to the test wells and add an equal volume of cell culture medium to the blank wells. After incubation for 1 hour, rinse 4 times with sterile PBS.

[0075] After rinsing, the cells were digested with trypsin for 5 min, then digested with 1 mL of cell culture medium, and the fluorescence intensity was measured.

[0076] The inhibition rate of Lactobacillus paracasei IB2 on the adhesion of pathogenic bacteria to Caco-2 (%) = 1-(1-R T / R B )×100%

[0077] Among them, R T is the relative fluorescence value of the cell suspension in the test strain well, R B is the relative fluorescence value of the cell suspension in the blank well.

[0078] As shown in Table 7, both Lactobacillus paracasei IB2 and Lactobacillus rhamnosus GG can inhibit the adhesion of Escherichia coli and Salmonella typhimurium to Caco-2 cells by competition, exclusion and displacement, and mainly inhibit the adhesion of Escherichia coli and Salmonella typhimurium by competing for adhesion sites. The competitive adhesion rate of Lactobacillus paracasei IB2 to Escherichia coli is 42.36%, and the competitive adhesion rate to Salmonella typhimurium is 53.34%.

[0079] Table 7 Lactobacillus paracasei IB2 inhibits pathogenic bacteria from adhering to Caco-2 cells

[0080]

[0081] Example 4 Clinical application of Lactobacillus paracasei IB2 in preventing and treating latent mastitis in dairy cows

[0082] 4.1 Test methods

[0083] This experiment was conducted in a dairy farm in Inner Mongolia. Thirty lactating cows with similar lactation conditions and subclinical mastitis detected by LMT were selected and randomly divided into a control group, an IB2 group, and a compound probiotic group, with 10 cows in each group (each cow had 4 milk quarters, a total of 40 milk quarters), and the numbers were recorded. The control group was fed normal feed, and the IB2 group was supplemented with probiotics IB2 (3×10 10 CFU / head), the composite probiotic group was supplemented with composite probiotic preparation (Lactobacillus paracasei IB2, Bacillus subtilis TA1, Lactobacillus paracasei BY5, 2×10 10 CFU / head). The test period was 7 days, and LMT was tested every day. The mental state, milk quality, and severity of latent mastitis of the 30 dairy cows were judged before and after the test. During the test, the basic diet of each group was exactly the same, and they were free to drink water, eat food, and exercise.

[0084] 4.2LMT determination criteria

[0085] Before testing, clean and disinfect the udder areas of the tested cows, discard the first few handfuls of milk from the four udder areas of each tested cow, squeeze the milk from the four udder areas of each tested cow into the four small chambers of the diagnostic tray in turn, pour out the excess milk so that about 2 mL of milk remains in each small chamber, add 2 mL of LMT detection reagent into each small chamber, shake the diagnostic tray in concentric circles, and make judgments according to the judgment criteria shown in Table 8.

[0086] Table 8 LMT judgment criteria

[0087]

[0088] 4.3 Prevention and control results

[0089] As shown in Tables 9 and 10, no cows died in the farm during the prevention and treatment of latent mastitis in dairy cows. The effective rate of latent mastitis prevention and treatment in the IB2 group reached 82.5%, and the recovery rate of the milk area was 82.5% after feeding the IB2 probiotic preparation; the effective rate of latent mastitis prevention and treatment in the composite group reached 90.0%, and the recovery rate of the milk area was 90.0% after feeding the composite probiotic preparation. In addition, it can be seen from Table 9 that for both weak positive and positive latent mastitis types, the IB2 group and the composite probiotic group showed good prevention and treatment effects, but for the strong positive latent mastitis type, the treatment effect of the composite probiotic group was better than that of the IB2 group.

[0090] Table 9 Detection results of latent mastitis in dairy cows

[0091]

[0092] Table 10 Results of probiotic preparations for preventing and treating negative mastitis in dairy cows

[0093] Group Number of breast areas Number of effective breast areas Efficiency Number of breast areas recovered Breast area recovery rate Control group 40 / / / / IB2 Group 40 33 82.5％ 33 82.5％(33 / 40) Composite Group 40 36 90.0％ 36 90.0％(36 / 40)

[0094] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Lactobacillus paracasei IB2 (Lacticaseibacillusparacasei IB2), characterized in that The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on January 2, 2024. The storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the storage number is GDMCC NO: 64237.

2. the application of Lactobacillus paracasei IB2 as claimed in claim 1 in the product of preparing inhibition of pathogenic bacteria.

3. The use according to claim 2, characterized in that: The pathogenic bacteria include Escherichia coli, Mycoplasma bovis, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus uberis and Streptococcus agalactiae.

4. A product for preventing and treating latent mastitis in dairy cows, characterized in that: The raw materials include at least one of the strain described in claim 1 and / or the supernatant of the strain.

5. A feed additive for preventing and treating latent mastitis in dairy cows, characterized in that: The composition comprises the product as claimed in claim 4.

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