CN109136126B - Pediococcus pentosaceus for preventing diarrhea of weaned piglets - Google Patents
Pediococcus pentosaceus for preventing diarrhea of weaned piglets Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
Abstract
The invention belongs to the technical field of biology, and provides pediococcus pentosaceus and application thereof in preventing diarrhea of weaned piglets. The Pediococcus pentosaceus strain L1 has been deposited in China general microbiological culture Collection center (CGMCC) in 2017, 5 months and 4 days, has a collection number of CGMCC No.14092, and is classified and named as Pediococcus pentosaceus. The pediococcus pentosaceus L1 is used for preventing diarrhea of weaned pigs, and the specific method is that the sterile normal saline containing the pediococcus pentosaceus L1 is infused into the weaned pigs, 10mL is taken every day, and 7 days are continued. The invention has the advantages of remarkably reducing the diarrhea rate of weaned piglets, improving the survival rate and the growth performance of the piglets and the economic benefit of pig breeding enterprises, and having very wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, particularly relates to the field of pig breeding in animal science, and particularly relates to pediococcus pentosaceus for preventing diarrhea of weaned piglets.
Background
China is the first country for pig breeding and pork consumption in the world, and the pig industry occupies a leading position in the agricultural industry and economy. However, China is not a strong pig-raising country, and has a gap compared with the level of pig-raising in developed pig-raising countries, and some space still needs to be improved. The piglets are the core of modern pig raising production, and the health condition of the piglets has extremely important influence on the later growth and development. In recent years, piglet diarrhea is a high-incidence disease which appears under the current intensive pig raising condition and is a main reason for the growth obstruction and high mortality rate of piglets. Colibacillary diarrhea is a common symptom of diarrhea in piglets, and is mainly caused by pathogenic bacteria such as enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC), and enteropathogenic Escherichia coli (EPEC). According to statistics, about 50% of piglets died every year are related to escherichia coli infection, and the piglets are clinically manifested as yellow scour, white scour, septicemia and the like of the piglets, which affects the growth and development of the piglets, reduces the feed conversion rate, and even causes the death of the piglets to cause larger economic loss.
In the production process of modern live pigs, piglets are generally weaned early. After weaning, food is changed from breast milk to feed, and weaning stress is aggravated due to environmental change, so that the immunity of piglets is reduced, the diarrhea rate and the death rate are increased, and huge loss is brought to the breeding industry. In order to improve the economic benefit of breeding, antibiotics are widely applied to livestock and poultry breeding as growth promoters. However, the long-term use of antibiotics in large quantities destroys the micro-ecological balance of the organism, causes the generation of variation of bacteria and drug-resistant bacteria, and remains in meat products, thus threatening the health of human beings. The European Union has completely prohibited any antibiotics from being added into the feed in 2006, Japan, America and China have issued relevant laws and regulations to limit or prohibit the use of antibiotics in the feed, and the complete limit of the use of antibiotics as livestock and poultry growth promotion additives is a necessary trend of the development of animal husbandry. Therefore, safe, efficient and green feed antibiotic substitutes capable of preventing piglet diarrhea are developed, and the feed antibiotic substitutes have remarkable economic, social and ecological benefits.
The microbial feed additive is a microbial preparation product which improves the animal health level by improving the ecological balance of animal intestinal flora, promoting the digestion and absorption of nutrient substances and enhancing the immune function of livestock. Compared with antibiotics, the microbial feed additive has the advantages of safety, no toxicity, no residue, no drug resistance, environmental friendliness and the like, and is a substitute with the most development potential of feed antibiotics. The species of the strain, namely the probiotics, is the basis for the function of the microbial feed additive and the primary guarantee of the product safety, and all countries in the world have clear regulations and strict management on the microbial feed additive. The food and drug administration and the feed administration society of America published 42 probiotics for direct feeding in 1989, and the Ministry of agriculture in China published 16 safe microorganisms for application in the feed industry in 2000. The probiotics currently applied to animal production mainly comprise strains such as lactobacillus, bacillus, saccharomycetes and the like.
Pediococcus, Streptococcus, Pediococcus, is a gram-positive bacterium that is one of lactic acid bacteria because it can ferment glucose to produce lactic acid. In 2006, the Ministry of agriculture 658 of China announced "catalog of varieties of feed additives" listed Pediococcus pentosaceus in the list of microorganisms that can be used as permitted. Therefore, the Pediococcus pentosaceus which has the functions of inhibiting the proliferation of pathogenic bacteria such as enterotoxin-producing Escherichia coli and the like, resisting gastric acid and intestinal juice environment, being sensitive to antibiotics, effectively adhering to intestinal mucosa epithelial cells and the like, remarkably reducing the diarrhea rate of weaned piglets, improving the survival rate and the growth performance is obtained by separation, and has wide application prospect in the field of live pig breeding.
Disclosure of Invention
The invention provides a pediococcus pentosaceus strain, which is named as pediococcus pentosaceus L1. The pediococcus pentosaceus L1 has the beneficial effects of inhibiting the proliferation of main pathogenic bacteria of piglet diarrhea, resisting gastric acid and intestinal juice environment, being sensitive to antibiotics, effectively adhering to porcine intestinal mucosa epithelial cells and the like, and can remarkably reduce the diarrhea rate of weaned piglets and improve the survival rate and the growth performance of the piglets after being administrated.
Technical scheme
The technical scheme of the invention is as follows:
the Pediococcus pentosaceus strain L1 for preventing diarrhea of weaned piglets is preserved in China general microbiological culture Collection center (CGMCC) at 5, 4 and 2017 with the preservation number of CGMCC No.14092 and is classified and named as Pediococcus pentosaceus. China general microbiological culture Collection center address: west road No.1, north chen, chaoyang district, beijing, zip code: 100101. the pediococcus pentosaceus has the beneficial functions of inhibiting the proliferation of pathogenic bacteria such as enterotoxigenic escherichia coli and the like, resisting gastric and intestinal environments, being sensitive to antibiotics, effectively adhering to intestinal mucosa epithelial cells and the like, and can obviously reduce the diarrhea rate of weaned piglets and improve the survival rate and the growth performance by feeding the weaned piglets through drenching.
The invention has the following positive effects:
(1) reducing the diarrhea rate of weaned piglets.
(2) Improve survival rate and growth performance.
(3) Reducing antibiotic dependence.
Drawings
FIG. 1 is a graph showing the results of the test of the gastrointestinal environmental tolerance of piglets by Pediococcus pentosaceus L1.
Detailed Description
The following further describes a specific embodiment of the present invention with reference to the drawings and technical solutions.
Example 1 isolation and identification of Pediococcus pentosaceus L1
And (3) adopting an MRS selection culture medium to separate suspected single lactobacillus colonies from the pickle, and performing molecular biological identification by using a 16SrDNA sequence analysis method. The amplified 16S rDNA fragment sequences were aligned at NCBI using the BLAST program and showed greater than 99% homology to pediococcus pentosaceus. Based on the criterion that a 16S rDNA variable region sequence homology of more than 97.5% can be regarded as the same species, the strain is identified as Pediococcus pentosaceus and named Pediococcus pentosaceus L1. The 16S rDNA sequence has been submitted to GenBank with the sequence number KP 792278.
Example 2 Pediococcus pentosaceus L1 has bacteriostatic effect on main intestinal pathogenic bacteria causing piglet diarrhea
The inhibition of growth and propagation of enterotoxigenic Escherichia coli (ETEC), enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic Escherichia coli (EPEC) by Pediococcus pentosaceus L1 was investigated using agar punch diffusion. ETEC K88ac+And EHEC O157: H7 were purchased from the chinese veterinary medical institute, EPEC1, ETEC1, ETEC3 and ETEC4 were all isolated from diarrheal piglet feces by the applicant as primary isolates. Inoculating Pediococcus pentosaceus L1 in MRS broth, anaerobically culturing at 37 deg.C for 24 hr, and regulating thallus number to 10 with MRS broth8CFU/mL; centrifuging at 4 deg.C and 5000rpm for 10min, and collecting supernatant; filtering the supernatant with 0.22 μm microporous membrane, and adjusting pH to 6.2 with sodium hydroxide; 20 μ L of the extract was added at a concentration of 1X 108CFU/mL indicator bacterium liquid (EHEC O157: H7, EPEC1, ETEC K88 ac)+ETEC1, ETEC3 and ETEC4) were added to LB solid medium sterilized at high temperature and high pressure and cooled to about 45 deg.C, to thereby obtain a productAfter the mixture is intensively mixed, the mixture is quickly poured into a plate, after the culture medium is solidified, an oxford cup is used for punching a hole (the diameter is 8mm) on the culture medium, 90 mu L of culture solution supernatant of the pediococcus pentosaceus L1 is sucked and injected into the small hole, the lactobacillus rhamnosus LGG is used as a reference, the sterilized MRS liquid culture medium is used as a blank control, after the blank control is diffused for 4h at room temperature, the blank control is placed in a thermostat at 37 ℃ for culturing for 24h, and the diameter of a bacteriostatic ring is measured. The antibacterial activity results of pediococcus pentosaceus L1 on main intestinal pathogenic bacteria of piglet diarrhea are shown in table 1, and the results show that pediococcus pentosaceus L1 can obviously inhibit the growth of ETEC, EHEC and EPEC, and the antibacterial activity is superior to that of reference probiotic strain Lactobacillus rhamnosus LGG.
TABLE 1 antibacterial Activity of Pediococcus pentosaceus L1 against the major intestinal pathogens of piglet diarrhea
Note: "-" indicates no bacteriostatic effect; "+" indicates a zone of inhibition radius <3 mm; "+ +" indicates a 3-6mm radius zone of inhibition; "+ + + +" indicates a radius of the zone of inhibition > 6 mm.
Example 3 gastric fluid and intestinal fluid tolerance of Pediococcus pentosaceus L1
Inoculating Pediococcus pentosaceus L1 into MRS liquid culture medium with pH of 3.0, culturing at 37 deg.C for 4h, and counting viable bacteria by plate counting method at 0, 1, 2, 3, 4 hr; subsequently, pediococcus pentosaceus L1 was inoculated into MRS liquid medium containing 0.3% swine bile salt, cultured at 37 ℃ for 4 hours, viable cell count was calculated by plate counting method at 5, 6, 7, and 8 hours, and gastric juice and intestinal juice tolerance of pediococcus pentosaceus L1 was tested. As shown in FIG. 1, compared with the reference strain Lactobacillus rhamnosus LGG, the L1 has stronger gastrointestinal environment tolerance, the number of L1 of the L3578 is not reduced in the whole detection time range (8h), and the advantage of gastrointestinal environment tolerance is prominent.
Example 4 antibiotic susceptibility of Pediococcus pentosaceus L1
In the using process of the probiotics, if the probiotics carry resistance genes, the resistance genes can be transferred to pathogenic bacteria, so that the drug resistance of the pathogenic bacteria is enhanced. Therefore, the evaluation of antibiotic sensitivity is an important index for the study of the probiotic properties of lactic acid bacteria. The sensitivity of pediococcus pentosaceus L1 to 7 commonly used antibiotics was examined by a paper diffusion method, and the results are shown in Table 2, in which pediococcus pentosaceus L1 is sensitive to ampicillin, streptomycin, erythromycin, tetracycline and chloramphenicol, moderately sensitive to kanamycin, and resistant to vancomycin. Studies have shown that resistance to vancomycin by Pediococcus pentosaceus L1 is innate and generally does not transfer to other strains. Thus, the results of the above study show that pediococcus pentosaceus L1 is sensitive to commonly used antibiotics.
TABLE 2 sensitivity of Pediococcus pentosaceus L1 to antibiotics
Example 5 porcine intestinal epithelial cell adhesion Capacity of Pediococcus pentosaceus L1
Inoculating Pediococcus pentosaceus L1 in MRS broth, anaerobically culturing at 37 deg.C for 18h, 4 deg.C and 5000rpm, centrifuging for 10min, removing supernatant, collecting thallus, and resuspending in DMEM high-sugar culture medium to reach thallus concentration of 5 × 107CFU/mL is ready for use. Porcine intestinal epithelial cells (cell density 9X 10) cultured to confluence with IPEC-J25cell/mL), 2mL of 5X 10 was added7CFU/mL Pediococcus pentosaceus L1-DMEM high-sugar medium mixture, CO2Culturing at 37 ℃ for 1h in an incubator, removing the culture medium, washing with PBS for three times to remove the non-adhered lactic acid bacteria, adding 1mL of PBS containing 0.1% Triton X-100, acting at 37 ℃ for 30min to lyse IPEC-J2 cells, coating the lysate in an MRS agar plate after gradient dilution with PBS, culturing at 37 ℃ for 48h, and calculating the number of the lactic acid bacteria adhered to the IPEC-J2 cells. The adhesion index was calculated as compared with the reference strain LGG according to the following formula: adhesion index (%) (number of adhered bacteria/number of cells) × 100. The results of the ability of Pediococcus pentosaceus L1 to adhere to porcine intestinal epithelial cells are shown in Table 3, wherein the adhesion index of Pediococcus pentosaceus L1 on the cell surface of IPEC-J2 is 14.4%, and the difference is not significant compared with that of the reference strain LGG, which shows that Pediococcus pentosaceus L1 can effectively adhere to the porcine intestinal epithelial cellsIn the porcine intestinal epithelial cells, the probiotic function is exerted.
TABLE 3 Pediococcus pentosaceus L1 ability to adhere to porcine intestinal epithelial cells
Note: the data from the same group are marked with the same lower case letters to indicate that the difference is not significant (P <0.05)
Example 6 Effect of Pediococcus pentosaceus L1 on growth Performance and diarrhea of ETEC-infected weaned piglets
The experiment adopts 30 Dudu multiplied by long multiplied by big ternary hybrid weaned piglets (weaning day age 21d), and after the daily ration of the fed piglets is excessive for 7d, the piglets are randomly distributed to a blank control group, an ETEC group and a pediococcus pentosaceus + ETEC group. The treatment for each group was as follows:
a. blank control group: on days 1-8, 10mL of sterile normal saline was continuously administered.
ETEC group: continuously administering 10mL of sterile normal saline on days 1-7, and administering 10mL of 10-concentration saline on day 89ETEC K88ac of CFU/mL+And (5) bacteria.
c. Pediococcus pentosaceus + ETEC group: continuously drenching 10mL of L1 containing Pediococcus pentosaceus on days 1-7 (10)9CFU/mL) of 10mL of 10-strength saline, administered on day 89ETEC K88ac of CFU/mL+And (5) bacteria.
The experimental period is 14 days, the piglets are fed freely and drunk freely, the initial weight and the final weight of the piglets are weighed, the daily feed intake and the diarrhea condition of the piglets are recorded, and the daily gain, the daily feed intake, the feed conversion ratio and the diarrhea rate are calculated.
Diarrhea rate (%) - (total diarrhea days per group piglet)/(number of piglets × number of test days) ] × 100
The influence of pediococcus pentosaceus L1 on the growth performance and diarrhea of ETEC-infected weaned piglets is shown in Table 4, the daily gain of pediococcus pentosaceus + ETEC-infected piglets is increased by 2.72% (P > 0.05) and 6.32% (P > 0.05), respectively, the feed-meat ratio is reduced by 0.65% (P > 0.05) and 4.94% (P > 0.05), and the diarrhea rate is reduced by 30.75% (P <0.05) and 61.70% (P <0.05), respectively, compared with the blank control group and the ETEC group. The result shows that the pediococcus pentosaceus L1 can obviously reduce the diarrhea rate of ETEC infected weaned pigs and has the tendency of improving the growth performance of the piglets.
TABLE 4 influence of Pediococcus pentosaceus L1 on growth Performance and diarrhea Rate of ETEC-infected weaned piglets
Note: the data in the same row are marked with different lower case letters to indicate significant difference (P < 0.05).
Claims (1)
1. The Pediococcus pentosaceus strain for preventing diarrhea of weaned piglets is characterized by being deposited in China general microbiological culture Collection center (CGMCC) at 5, 4 and 2017 with the preservation number of CGMCC No.14092 and classified and named as Pediococcus pentosaceus (Pediococcus pentosaceus).
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| CN113462605B (en) * | 2020-07-21 | 2023-03-21 | 云南省畜牧兽医科学院 | Pediococcus pentosaceus368 microbial inoculum and preparation method and application thereof |
| CN111793586B (en) * | 2020-08-13 | 2022-05-20 | 石河子大学 | Breast milk source pediococcus pentosaceus and application thereof |
| CN111961625B (en) * | 2020-08-26 | 2022-11-04 | 湖南师范大学 | Pediococcus pentosaceus SMM914 and screening method and application thereof |
| CN113528381B (en) * | 2021-06-30 | 2022-09-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | An antagonistic escherichia coli O157: h7 pediococcus pentosaceus and application thereof |
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