CN117070413A - Lactobacillus paracasei BY5 and application thereof - Google Patents
Lactobacillus paracasei BY5 and application thereof Download PDFInfo
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- CN117070413A CN117070413A CN202311057873.1A CN202311057873A CN117070413A CN 117070413 A CN117070413 A CN 117070413A CN 202311057873 A CN202311057873 A CN 202311057873A CN 117070413 A CN117070413 A CN 117070413A
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- lactobacillus paracasei
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- mastitis
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The application discloses lactobacillus paracasei BY5 and application thereof, and belongs to the technical field of microorganisms. The strain is preserved in the microorganism strain collection of Guangdong province at the 7 th month 12 of 2023, the preservation address is the No. 59 building 5 of the 100 th university of Mitsui in Guangzhou city, and the preservation number is GDMCC NO:63639. the probiotics preparation used in the application does not contain antibiotics, has no drug residue and drug resistance, improves the biological safety of cow breeding, and accords with the current concept of green ecological breeding. The application is a functional probiotics specially developed for the problem of cow mastitis, has higher palatability, and can effectively improve the feeding rate of cows. The application can effectively prevent and treat the occurrence of cow recessive mastitis, reduce the incidence of the mastitis and improve the milk quality.
Description
Technical Field
The application relates to the technical field of microorganisms, in particular to lactobacillus paracasei BY5 and application thereof.
Background
Cow mastitis is the first of three diseases of cows, is a complex disease, and is the result of the combined action of pathogenic microorganisms, environmental factors, management factors, cows themselves, genetic factors and the like. The disease not only causes serious loss to the breeding industry, but also brings great threat to food safety. The prevention and treatment difficulty of the mastitis is very large, the stock quantity of the dairy cows is 2.2 hundred million worldwide, the average incidence rate of the foreign mastitis is about 25% -60%, and the clinical incidence rate of the domestic dairy cows is about 33.41%. The loss caused by mastitis is up to 350 hundred million dollars each year, and the loss of the lactation yield reduction and the medical expense expenditure caused by the mastitis is about 1200-3600 yuan/head each year in China.
With the long-term use and unscientific utilization of antibiotics, the problem of bacterial drug resistance is more and more serious, and great hidden trouble is brought to the production and life of human beings and the health of human beings and animals. To address this problem and to search for "subtractive anti-tiepin" strategies, the study of probiotic products as "subtractive anti-tiepin" products is rapidly evolving. Probiotics are living microorganisms that, when administered in sufficient amounts, have a beneficial effect on host health. At present, the probiotics preparation is taken as a non-toxic, pollution-free and side-effect-free green environment-friendly product, and is increasingly valued by the breeding industry. The probiotics preparation is not only the need of producing pollution-free animal products, but also one of the necessary conditions for enhancing the capability of animal products in China to break through the green barrier in international trade and promoting the sustainable development of the feed industry and the livestock breeding industry. However, the probiotics preparation is used as a safe, efficient and environment-friendly novel product, and has few reports on preventing and treating recessive mastitis and subclinical mastitis of dairy cows. Therefore, the screening of the probiotic bacterial strain for preventing and treating the recessive mastitis and the subclinical mastitis of the dairy cows and the research and development of related probiotic products have important practical significance and assist the healthy development of dairy cow industry in China.
Disclosure of Invention
The application aims to provide lactobacillus paracasei BY5 and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present application provides the following solutions:
the application provides a lactobacillus paracasei BY5 (Lacticaseibacillus paracasei), which is preserved in the microorganism strain preservation center of Guangdong province at the 7 th month 12 day of 2023, and the preservation address is the building 5 No. 59 of the 100 th university of Mitsui, guangzhou, and the preservation number is GDMCC NO:63639.
the application also provides a microbial agent, which comprises the lactobacillus paracasei BY5.
The application also provides application of the lactobacillus paracasei BY5 or the microbial agent in preparing medicines for preventing and treating cow mastitis.
The application also provides application of the lactobacillus paracasei BY5 or the microbial agent in preparing a product for inhibiting adhesion of pathogenic bacteria.
Preferably, the pathogenic bacteria include E.coli, salmonella and Staphylococcus aureus.
Preferably, the product comprises an agent or a medicament.
The application also provides application of the lactobacillus paracasei BY5 or the microbial agent in preparing medicines for treating diseases caused BY escherichia coli.
The application also provides application of the lactobacillus paracasei BY5 or the microbial agent in preparing medicines for treating diseases caused BY staphylococcus aureus.
The application also provides application of the lactobacillus paracasei BY5 or the microbial agent in preparing medicines for treating diseases caused BY salmonella.
The application also provides application of the lactobacillus paracasei BY5 or the microbial agent in preparing medicines for treating diseases caused BY common proteus.
Based on the technical scheme, the application has the following technical effects:
1. the probiotics preparation used in the application does not contain antibiotics, has no drug residue and drug resistance, improves the biological safety of cow breeding, and accords with the current concept of green ecological breeding.
2. The application is a functional probiotics specially developed for the problem of cow mastitis, has higher palatability, and can effectively improve the feeding rate of cows.
3. The application can effectively prevent and treat the occurrence of cow recessive mastitis, reduce the incidence of the mastitis and improve the milk quality.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a colony morphology of Lactobacillus paracasei BY5;
FIG. 2 is a gram-stain microscopic image of Lactobacillus paracasei BY5;
FIG. 3 is a PCR amplification diagram of Lactobacillus paracasei BY5, wherein lane M is a DNAMaroer of 2000bp, and lane 1 is the strain BY5 of the present application;
FIG. 4 is a diagram showing the inhibition of mycoplasma bovis BY Lactobacillus paracasei BY5;
FIG. 5 is a diagram showing the inhibition of Staphylococcus aureus ATCC6538 BY Lactobacillus paracasei BY5.
Detailed Description
Various exemplary embodiments of the application will now be described in detail, which should not be considered as limiting the application, but rather as more detailed descriptions of certain aspects, features and embodiments of the application.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the application. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present application. The specification and examples of the present application are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The technical scheme of the application is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.
The culture medium used in the embodiment of the application comprises the following components:
sterile PBS: 8.0g of NaCl, 0.2g of KCl and Na are weighed 2 HPO 4 1.42g，KH 2 PO 4 Adding 0.27g into 800mL of deionized water, fully stirring and dissolving, adding deionized water to a volume of 1L, dripping hydrochloric acid to adjust the pH value to 7.4, sterilizing at 121 ℃ for 20min under high pressure, and preserving at room temperature for later use.
MRS medium: 10.0g of peptone, 10.0g of beef extract powder, 5.0g of yeast extract powder, 20.0g of glucose, 1.0mL of Tween 80 and K 2 HPO 4 ·3H 2 O2.0 g, anhydrous sodium acetate 5.0g, triammonium citrate 2.0g, mgSO 4 ·7H 2 O 0.29g，MnSO 4 ·H 2 O0.058 g, adding the above components into distilled water, fixing volume to 1000mL, adjusting pH to 7.2, and autoclaving at 121deg.C for 20min.
MRS solid medium: 1.5% agar powder was added to MRS broth.
EXAMPLE 1 isolation and identification of strains
1.1 isolation of strains
The lactobacillus paracasei BY5 (Lacticaseibacillus paracasei BY 5) is separated and screened from the self-brewed yoghurt of herdsman in Qinghai province county. The specific separation and purification method comprises the following steps: 1g of yoghourt is weighed, 4 sterile glass beads are added into 9mL of sterile PBS, and after vortex and full mixing, the yoghourt is taken1mL of filtrate was serially diluted to 10 with PBS -4 ，10 -5 0.1mL of the extract is coated on an MRS plate, anaerobic culture is carried out at 37 ℃ for 24 hours respectively, and single colonies with different morphological sizes are picked up and continuously purified on the MRS plate.
Picking lactobacillus from MRS plate, numbering, and respectively scribing and purifying on MRS plate; after each isolate was purified, a single colony was selected and cultured anaerobically in MRS at 37℃for 24 hours, and the bacterial solution was preserved for use.
1.2 morphological observations of strains
Strain BY5 appeared off-white on MRS plates, with round colonies, smooth and moist surface, clean edges, and ridges (fig. 1). The strain BY5 was observed under an oil microscope as gram-positive bacillus, without spores, and the thallus was blue-violet (FIG. 2).
1.3 molecular biological characterization of strains
The bacterial identification 16S rDNA universal primers 27F and 1492R are adopted for PCR amplification, 1/4 of the purified single colony is selected as a PCR amplification template, and the reaction system is 25 mu L. The PCR amplification procedure was as follows: 95 ℃ for 5min;95 ℃ for 45s;55 ℃,45s;72 ℃,70s; repeating 30 cycles; 72 ℃ for 8min; amplification products were identified by 1% agarose gel electrophoresis and the product size was approximately 1500bp (FIG. 3). The PCR product was sequenced by the Optimazethapyr BioCo., ltd. And the sequencing result was submitted to NCBI for Blast comparison analysis. Wherein the sequence homology of the isolate BY5 sequence with Lacticaseibacillus paracasei is 100%. The strain was submitted and deposited at the Cantonese province microorganism strain collection at 7.12 of 2023 under accession number GDMCC NO. 63639.
Example 2 evaluation of safety of Lactobacillus paracasei BY5
2.1 hemolysis test
Lactobacillus paracasei BY5 is continuously passaged for three times in an MRS liquid culture medium, a small amount of culture solution is dipped in an inoculating loop to be streaked on a Columbia blood agar culture medium, after anaerobic culture is carried out for 24 hours at 37 ℃, whether hemolysis loops appear around colonies is observed, and a staphylococcus hemolyticus (Staphylococcus haemolyticus, obtained BY separation in the laboratory) is used as a positive control. Bacterial production of cytolysin has three manifestations on platelets: if grass green circles appear, alpha-hemolysis is carried out; if a transparent circular ring is formed around the colony, beta-hemolysis is carried out; if there is no hemolytic ring, it is gamma-hemolysis, also called no hemolysis. The results showed that Lactobacillus paracasei BY5 did not exhibit alpha-hemolysis and beta-hemolysis on the blood plates, and the strain exhibiting gamma-hemolysis was primarily considered as a safe strain.
2.2 drug resistance Gene detection test
DNA extraction: the bacterial suspension cultured overnight after three successive passages is centrifuged, the bacterial cells are collected, and bacterial DNA is extracted according to the method provided by the bacterial genome DNA extraction kit.
The extracted DNA was used as a template, and the kanamycin resistance gene, the gentamicin resistance gene, the tetracycline resistance gene and the vancomycin resistance gene of BY5 were amplified BY PCR to synthesize primers (Table 1). PCR reaction System (25. Mu.L): LA Taq premix enzyme 12.5. Mu.L, 1. Mu.L each of the upstream and downstream primers, 1. Mu.L of DNA template, ddH 2 O 9.5μL。
TABLE 1 primer sequences for drug resistance genes
The test results show that BY5 is negative to all 4 drug resistance genes, which indicates that the BY5 drug resistance gene transfer possibility is low.
2.3 drug sensitivity test
BY5 was tested for drug sensitivity using a paper sheet agar diffusion method (Kirby-Bauer test, K-B method). Uniformly coating bacterial suspension on the surface of an MRS flat plate respectively by using a sterile cotton stick, standing at room temperature for 10-20min, attaching a drug-sensitive tablet on the surface of flat agar by using sterile forceps, respectively carrying out anaerobic culture at 37 ℃ for 24h, measuring the bacteriostasis circle of the drug-sensitive tablet by using a vernier caliper, taking Escherichia coli (ATCC 25922) as a quality control strain, and judging the drug-sensitive result by referring to the standard of the American clinical laboratory standardization institute (clinical and laboratory standards institute, CLSI) as follows: sensitivity (S), moderate sensitivity (I) and resistance (R). The antibiotics used in the test were 8 kinds of 11 kinds, including beta-lactam Ampicillin (Ampicillin, 10. Mu.g/tablet), amoxicillin (Amoxicillin, 20. Mu.g/tablet), cefotaxime (Cefotaxime Sodium, 30. Mu.g/tablet); aminoglycoside Kanamycin (Kanamycin, 30 μg/tablet), streptomycin (Streptomycin, 10 μg/tablet); quinolone Norfloxacin (Norfloxacin, 10 μg/tablet); macrolide Erythromycin (15 μg/tablet); tetracyclines (tetracyclines, 30 μg/tablet); glycopeptides Vancomycin (Vancomycin, 30 μg/tablet); chloramphenicol (chlormphenicol, 30 μg/tablet); lincomamine Clindamycin (Clindamycin, 2 μg/tablet).
TABLE 2 BY5 results of sensitivity to antibiotics
The results show that Lactobacillus paracasei BY5 is resistant to the aminoglycosides kanamycin and streptomycin.
2.4 in vitro safety test
Healthy female 21-day-old Kunming (KM) mice were selected for 30, fed adaptively for 1 week, fed freely with water, and randomly divided into 10 animals/group, 3 groups respectively: normal control, low dose, high dose. Mice in each experimental group are fed with basic ration, normal groups are filled with 0.2mL of gastric physiological saline per day, the experimental groups are filled with lactobacillus paracasei BY5 which is the strain of the application, and the specific experimental design is shown in Table 3. The test lasts for 14 days, and the growth condition, the mental state, the fur, the urination and the like of the mice are observed every day, so that records are made. After the test is finished, the weight gain rate of the mice is calculated, and whether the viscera have lesions or not is observed in an anatomic mode.
TABLE 3 safety test design for mice
The results are shown in Table 3, and the mice in each group were normal in feeding, mental, behavioural and both, without obvious differences, indicating that the strain of the present application had no effect on the growth status of the mice.
TABLE 4 weight gain in mice (%)
After dissection, the organs of each group of mice are carefully observed, and the heart, liver, spleen, kidney and lung of the test mice are free from macroscopic lesions, which indicates that the Lactobacillus paracasei BY5 is safe for the mice.
Example 3 probiotic properties of strains
3.1 acid and bile salt resistance test
BY5 was activated BY three successive passages, inoculated in PBS with different pH=3.0 and 0.3% (w/v) pig bile salt at an inoculum size of 2%, vortexed, mixed well, placed at 37℃for stationary culture, sampled at 0h and 3h, diluted to a suitable gradient, counted on MRS plates, and anaerobically cultured at 37℃for 24h.
The calculation formula of strain tolerance is: survival (%) =n 3 /N 0 ×100％，
Wherein: n (N) 3 The number of viable bacteria is 3 hours; n (N) 0 The number of viable bacteria is 0 h; the number of viable bacteria is expressed in lg CFU/mL.
The results show that BY5 has higher survival rate in acid and bile salt environments, and the survival rate of BY5 is 75.12 percent after being respectively tolerant for 3 hours under the conditions of pH=3.0 and above, and the survival rate of BY5 is lower under the condition of 0.3 percent bile salt.
3.2 bacteriostasis test
The fresh bacterial suspension of Lactobacillus paracasei BY5 was centrifuged (4 ℃,6000rpm,10 min) and the supernatant was filtered through a 0.22 μm filter, in this example the 12 pathogenic strains were: escherichia coli ATCC43888, yersinia enterocolitica ATCC23715, staphylococcus aureus ATCC6538, proteus vulgaris ATCC29905, and salmonella ATCC14028 were purchased from chinese veterinary medicine monitoring institute; mycoplasma bovis 08m is given away by animal science research institute of Ningxia national academy of sciences; escherichia coli LXR1, escherichia coli LXR2, klebsiella pneumoniae SQ3A, streptococcus uberis NM1, streptococcus agalactiae BY1, and Klebsiella pneumoniae SQ5A were isolated from this laboratory. The concentration of the pathogenic strain used for 12 bacteria inhibition was adjusted to 1X 10 with sterile PBS 6 CFU/mL, uniformly coating on NA plate with sterile cotton swab, standing at room temperaturePunching (aperture.+ -. 8mm, depth.+ -. 4.5 mm) after 10-20min, sealing bottom with 1% agar, sucking 150 μl of filtrate, adding into the hole, standing at room temperature for 2 hr, culturing at 37deg.C, and taking out (about 18 hr) when clear inhibition zone appears. The diameter (mm) of the inhibition zone is measured by a vernier caliper, and the inhibition activity of the probiotics is represented by the diameter of the inhibition zone.
The results show that the Lactobacillus paracasei BY5 has different degrees of inhibition on 12 common pathogenic bacteria. As shown in Table 5, the strain can inhibit the growth of various pathogenic bacteria such as Escherichia coli, staphylococcus aureus, salmonella, proteus vulgaris, etc., has strong inhibition effect on mycoplasma bovis, and the inhibition effect on Staphylococcus aureus ATCC6538 is shown in FIG 5, wherein the diameter of the inhibition zone is 17.5mm (FIG 4).
TABLE 5 Lactobacillus paracasei BY5 antibacterial ability assay (mm)
3.3 experiments to inhibit pathogenic adhesion
Culturing Caco-2 cells: RPMI-1640 medium containing 10% fetal bovine serum (fetal bovine serum, FBS) was used as cell culture medium and subculture medium, caco-2 cells were cultured at 37℃with 5% CO 2 Culturing in a constant temperature incubator, and culturing for 1d, and transferring after 3d (digestion with pancreatin at the time of transfer, 1:5 transfer).
Pathogen marker: the freshly cultured E.coli (Escherichia coli, ATCC 43888), staphylococcus aureus (Staphylococcus aureus, ATCC 6538), salmonella typhimurium (Salmonella typhimurium, ATCC 14028) suspensions were centrifuged (4 ℃,4000rpm,5 min), washed three times successively with sterile PBS, the cells were resuspended with FITC solution which was left at room temperature for 30min after the last washing was completed, incubated in dark with shaking at 37℃for 2h, centrifuged (4 ℃,4000 rpm) after the incubation was completed and the strains were washed 3 times with PBS, unbound FITC-labeled solution was washed off, resuspended with RPMI-1640 cell culture medium and the concentration of the bacterial solution was adjusted to 2X 10 8 CFU/mL, relative fluorescence intensity values (RFU) of the strain at 485nm (absorption wavelength) and 530nm (emission wavelength) were measured and recorded as adhesionThe relative fluorescence intensity value R0 of the precursory strain.
Preparation of probiotic bacterial suspension: after the lactobacillus paracasei BY5 strain is activated for 3 times continuously, the lactobacillus paracasei BY5 strain is centrifuged (4 ℃ C., 4000rpm,10 min) to collect thalli, and after the lactobacillus paracasei BY5 strain is washed for three times BY using the sterile PBS, the concentration of the thalli is regulated to 2 multiplied BY 10 BY using the RPMI-1640 cell culture solution 8 CFU/mL。
Competition test: the following test was carried out under light-shielding conditions. Setting a test hole and a blank control hole, adding 0.3mL of probiotic bacterial suspension and 0.3mL of marked pathogenic bacterial suspension into the test hole, uniformly mixing, adding 0.3mLRPMI-1640 cell culture solution and 0.3mL of marked pathogenic bacterial suspension into the blank control group, uniformly mixing, simultaneously setting 3 repeated holes for each probiotic bacterial strain, and setting 3 repeated holes at 37 ℃ and 5% CO 2 Incubate in incubator dark for 2h.
Rejection test: the following test was carried out under light-shielding conditions. Setting test holes and blank control holes, firstly adding 0.6mL of probiotic bacterial suspension into the test holes, adding 0.6mL of RPMI-1640 cell culture solution into the blank control group, setting 3 repeated holes for each probiotic bacterial strain at 37 ℃ and 5% CO 2 After incubation for 1h in incubator, washing with PBS; next, 0.6mL of the labeled pathogenic bacteria suspension was added to each well and incubated for 1h in the dark.
Displacement test: the following test was carried out under light-shielding conditions. Test wells and blank wells were set, first 0.6mL of the labeled pathogenic bacterial suspension was added per well, 37℃and 5% CO 2 Washing with PBS after dark incubation for 1h in an incubator; secondly, adding 0.6mL of probiotic bacterial suspension into a test group, adding 0.6mL of RPMI-1640 cell culture solution into a blank control group, setting 3 repeated holes for each probiotic bacterial strain, and setting 5% CO at 37 DEG C 2 Incubate in incubator for 1h.
After the test, washing three times with PBS, adding 0.3mL pancreatin to each well to digest for 5min, and stopping digestion with 0.5mL RPMI-1640 cell culture solution; the relative fluorescence intensity values (RFU) of the cell suspensions were determined under the same wavelength conditions.
The adhesion inhibition rate (%) of the strain to pathogenic bacteria is calculated by the formula: (1-R/R0). Times.100%, wherein: r: relative fluorescence intensity values of the experimental group; r0: relative fluorescence intensity values for the blank.
The preparation (1 mL) of the Fluorescein Isothiocyanate (FITC) marking solution comprises the following steps: taking out FITC stored at 4 ℃ and standing for 30min at room temperature, weighing 0.5g of FITC, and dissolving in 1mL of dimethyl sulfoxide (DMSO), thus preparing 500mg/mL of solution; mu.L of the above solution was pipetted into 999. Mu.L of sterile PBS, i.e.a fluorescent labeling solution with a working concentration of 500. Mu.g/mL.
The adhesion test result of the inhibition pathogenic bacteria shows that the lactobacillus paracasei strain can competitively, replaceably and repulsively inhibit the adhesion of three pathogenic bacteria, wherein the replaceability inhibition rate of the lactobacillus paracasei strain to staphylococcus aureus is up to 29.33%, and the competitive inhibition rate of the lactobacillus paracasei strain to staphylococcus aureus is 25.72%. The lactobacillus paracasei BY5 strain can effectively inhibit adhesion of staphylococcus aureus through competition and substitution, so as to prevent cow mastitis caused BY the staphylococcus aureus.
TABLE 6 inhibition of Lactobacillus paracasei BY5 against E.coli, staphylococcus aureus and Salmonella adhesion Caco-2 cells (%)
Example 4 clinical application of Lactobacillus paracasei BY5 in preventing and treating bovine mastitis
4.1 test methods
The experiment is carried out on a certain dairy farm in the inner Mongolian Bayan city, the lactation conditions are selected to be equivalent, 60 dairy cows with recessive mastitis detected BY LMT are randomly divided into a control group, a BY5 group and a compound group, and the number is increased, and each group is 20. The control group is fed with normal feed, and the BY5 group is added with probiotics BY5 (1.5X10) 10 CFU/head), and the compound probiotic preparation (Lactobacillus paracasei BY5, bacillus subtilis TA1, lactobacillus plantarum YJ3 1.2X10 respectively) is added on the basis of the control group 10 CFU/header). The test period was 7d and LMT test was performed daily. And judging the production performance of 60 cows and the mastitis degree of the milk quality before and after the test, wherein the basic ration of each group is completely the same during the test, and the cows and the milk quality are free to drink water.
4.2LMT decision criteria
Cow milk in 4 milk areas of the detected cow is respectively squeezed into 4 cells in the diagnosis tray, redundant milk is poured out, about 2mL of retained milk in each cell is respectively added into the cells, the diagnosis tray is shaken concentrically, and judgment is carried out according to the judgment standard shown in Table 7.
TABLE 7 LMT decision criteria
4.3 control results
As shown in tables 8-9, the farms were free of animal death during the control of cow mastitis. The effective rate of preventing and treating the recessive mastitis of the dairy cows after the BY5 group is fed with the BY5 probiotic preparation reaches 85.0%, and the cure rate is 85.0%; after the compound group is fed with the compound probiotic preparation, the effective rate of preventing and treating the recessive mastitis of the dairy cows reaches 90.0%, and the cure rate is 90.0%. The BY5 single preparation and the BY5 compound preparation have good prevention and treatment effects on cow mastitis.
Table 8 cow stealth mastitis test results
Table 9 probiotic preparation for preventing and treating cow mastitis results
| Group of | Quantity (head) | Effective (head) | Effective rate of | Cure (head) | Cure rate |
| Control group | 20 | / | / | / | / |
| BY probiotics group | 20 | 17 | 85.0％ | 17 | 85.0％ |
| Composite probiotics group | 20 | 18 | 90.0％ | 18 | 90.0％ |
The above embodiments are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application should fall within the protection scope defined by the claims of the present application without departing from the design spirit of the present application.
Claims (10)
1. Lactobacillus paracasei BY5 (Lacticaseibacillus paracasei), which is deposited with the collection of microbiological strains in the cantonese province at 7.12.2023 at floor 5 of the university of first middle road 100 in Guangzhou city under accession number GDMCC NO:63639.
2. a microbial agent comprising lactobacillus paracasei BY5 according to claim 1.
3. Use of lactobacillus paracasei BY5 according to claim 1 or the microbial agent according to claim 2 for the preparation of a medicament for preventing and treating cow mastitis.
4. Use of lactobacillus paracasei BY5 according to claim 1 or a microbial agent according to claim 2 for the preparation of a product for inhibiting the adhesion of pathogenic bacteria.
5. The use of claim 4, wherein the pathogenic bacteria comprise escherichia coli, salmonella, and staphylococcus aureus.
6. The use according to claim 4 or 5, wherein the product comprises an agent or a medicament.
7. Use of the lactobacillus paracasei BY5 according to claim 1 or the microbial agent according to claim 2 for the preparation of a medicament for the treatment of diseases caused BY escherichia coli.
8. Use of the lactobacillus paracasei BY5 according to claim 1 or the microbial agent according to claim 2 for the preparation of a medicament for the treatment of a disease caused BY staphylococcus aureus.
9. Use of the lactobacillus paracasei BY5 according to claim 1 or the microbial agent according to claim 2 for the preparation of a medicament for the treatment of a disease caused BY salmonella.
10. Use of the lactobacillus paracasei BY5 according to claim 1 or the microbial agent according to claim 2 for the preparation of a medicament for the treatment of diseases caused BY proteus vulgaris.
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