CN115975870A - Enterococcus faecium with porcine epidemic diarrhea virus resisting function and application thereof - Google Patents
Enterococcus faecium with porcine epidemic diarrhea virus resisting function and application thereof Download PDFInfo
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Abstract
The invention discloses an Enterococcus faecium (Enterococcus faecium) EFA5 with a function of resisting porcine epidemic diarrhea virus, wherein the preservation number is as follows: GDMCC NO.62904, deposited in the Guangdong province culture Collection of microorganisms at the institute of microbiology, guangdong province academy of sciences, guangzhou, guangdong province. Research shows that the strain has obvious probiotic characteristics, rapid growth and strong acid production capacity, and can be colonized in pig intestines. In addition, the compound has strong capability of resisting porcine epidemic diarrhea virus. The strain can be used as probiotic bacteria for preparing feed or feed additives, and has a wide application scene in preparing medicaments for resisting porcine epidemic diarrhea viruses.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to enterococcus faecium with a porcine epidemic diarrhea virus resisting function and application thereof.
Background
Intestinal diseases are a common problem in modern pig farms, but diarrhea diseases are relatively easy to occur in piglet stages and comprise bacterial diarrhea, viral diarrhea, nutritional diarrhea and diarrhea caused by parasites, wherein the viral diarrhea has high incidence rate, rapid transmission, wide transmission range, high death rate and unobvious treatment effect, the prevention is mainly performed, in particular to Porcine Epidemic Diarrhea Virus (PEDV) which continuously erupts in 2010 till now, the epidemic diarrhea is acute highly infectious diseases and mainly presents clinical symptoms such as diarrhea, vomiting, dehydration and the like, the death rate of some pig farms in part of large-scale pig farms is high and even can reach over 80%, the death rate of some pig farms is an important cause of enteritis and death of newborn piglets, and the economic loss is caused to the breeding industry.
The prevention effect is better than the treatment effect for diseases, especially viral diseases, but PEDV is not exceptional, and PEDV has strict cell tropism and host specificity. However, due to viral genetic variation, effective vaccine protection is affected; antiviral drugs generally have severe toxic side effects and are limited in use amount and effect. Therefore, there is a continuing need for effective methods of controlling viral diseases, and probiotics are receiving widespread attention for their non-toxic, non-drug resistant, residue-free, antibacterial, antiviral, growth-promoting, green and safe advantages.
Enterococcus urinarius has obvious probiotic characteristics, and most strains are safe and reliable, so the enterococcus urinarius becomes one of the probiotic strains which are published in 1989 and can be directly fed by words, and is also a probiotic strain which is allowed to be used in related documents of feed additive variety catalog of Ministry of agriculture in China, and a plurality of pharmaceutical companies and enterprises prepare the probiotic preparation for adjusting the intestinal microecology of human or animals. Enterococcus faecium can regulate the balance of intestinal flora, reduce the infection of pathogenic bacteria in the intestinal tract, treat diarrhea and enteritis, and enhance the immune function of a host, and can generate organic acid, hydrogen peroxide, bacteriocin and the like through normal metabolism so as to inhibit and kill pathogenic bacteria and viruses. Related researches show that the enterococcus faecium can inhibit the replication of the porcine transmissible gastroenteritis virus and the porcine influenza A virus, but no report is found on the porcine epidemic diarrhea virus.
Disclosure of Invention
Based on the strain, the enterococcus faecium with the function of resisting the porcine epidemic diarrhea virus and the application thereof are provided, and the strain can be used for preparing feeds, feed additives and medicines for preventing PEDV, so that a new direction is provided for preventing and treating the PEDV.
In a first aspect of the present invention, there is provided an Enterococcus faecium (Enterococcus faecium) EFA5 strain having a function of resisting porcine epidemic diarrhea virus, wherein the collection number is: GDMCC NO.62904 is deposited at the Guangdong province culture Collection of microorganisms at 2022, 10 months and 19 days, and the deposit is the institute of microorganisms of Guangdong province academy of sciences, guangzhou, guangdong province.
The 16S rRNA gene sequence of the Enterococcus faecium (Enterococcus faecium) EFA5 is shown in SEQ ID NO. 1. The strain has the following microbiological characteristics: bacterial colonies of the strain EFA5 on a culture medium are milky white, the center of the bacterial colonies is smooth and convex, and the edge of the bacterial colonies is rough; the strain is in a short rod shape under a lens, gram stain is positive, and no spore exists; the growth is rapid, and the stable period can be reached after the growth is carried out for 18 hours on the culture medium; has stronger acid production capacity, and the pH value of the culture medium is close to 4.0 from 5.4 within 20 hours; has good acid resistance and bile salt resistance; has stronger adhesion capability to the epithelial cells of the small intestine of the pig.
The enterococcus faecium EFA5 is obtained by separating feces of healthy Luchuan sows and weaned piglets.
The enterococcus faecium EFA5 has an inhibition effect on porcine epidemic diarrhea viruses, the inhibition rate of the fermented supernatant of the enterococcus faecium EFA5 on PEDV is indirectly detected by a CCK8 method, the inhibition rate of the fermented supernatant of the enterococcus faecium EFA5 on the PEDV is found to be very strong at a cell level, and the inhibition rate of the fermented supernatant with 1/4 dilution on the PEDV is about 0.6; TCID of the experimental group to which the fermentation supernatant was added was found by examining the influence of the enterococcus faecium EFA5 fermentation supernatant on the PEDV titer 50 Is 10 -3.50 0.1ml of TCID of control without addition of fermentation supernatant 50 Is 10 -4.33 0.1ml; by determining the effect of the PEDV fermentation supernatant on the copy number of the virus M gene, after 24 hours of culture, the copy number of the virus M gene of the probiotic fermentation supernatant treatment group was found to be significantly lower than that of the control group.
In a second aspect of the invention there is provided a feed or feed additive comprising enterococcus faecium EFA5 or a fermentation product thereof.
In a third aspect of the invention, the application of enterococcus faecium EFA5 in the preparation of a medicament for resisting porcine epidemic diarrhea virus is provided.
In some of these embodiments, the medicament contains enterococcus faecium EFA5 and/or a fermentation product thereof.
In some of these embodiments, the pharmaceutical dosage form is a tablet, capsule, powder, pill, granule, or solution.
In some of these embodiments, the medicament comprises enterococcus faecium EFA5 and a pharmaceutically acceptable excipient.
Compared with the prior art, the invention has the following advantages:
the enterococcus faecium EFA5 provided by the invention has obvious probiotic characteristics, can grow rapidly, has strong acid production capacity, can be colonized in the porcine intestinal tract, and experimental determination shows that the enterococcus faecium EFA5 screened by the invention can effectively inhibit the replication of Porcine Epidemic Diarrhea Virus (PEDV) and has strong antiviral capacity. The enterococcus faecium with the function of resisting the porcine epidemic diarrhea virus disclosed by the invention can be used as a probiotic for preparing feed or feed additives, and has a wide application scene in preparing medicaments for resisting the porcine epidemic diarrhea virus.
Drawings
FIG. 1 is a morphological diagram of an enterococcus faecium EFA5 colony;
FIG. 2 is a gram stain microscopic image of enterococcus faecium EFA5 under a 100-fold oil microscope;
FIG. 3 is a diagram of the genetic evolution analysis of enterococcus faecium EFA 5S rRNA;
FIG. 4 is a graph of the growth of enterococcus faecium EFA 5;
FIG. 5 is a graph of acid production by enterococcus faecium EFA 5;
FIG. 6 is a graph of the results of an acid and bile salt tolerance assay for enterococcus faecium EFA 5;
FIG. 7 is a graph showing the adhesion of enterococcus faecium EFA5 to IPEC-J2 cells;
FIG. 8 is a normal morphology map of vero cells;
FIG. 9 is a graph showing the results of PEDV infection of vero cells to produce cytopathic effects;
FIG. 10 is a graph showing the results of the cytotoxicity assay of enterococcus faecium EFA5 fermentation supernatant;
FIG. 11 is a graph showing the results of measuring the PEDV inhibition ratio of the E.faecium EFA5 fermentation supernatant;
FIG. 12 is a graph showing the results of measurement of the effect of enterococcus faecium EFA5 fermentation supernatant on PEDV titer;
FIG. 13 is a graph showing the results of measurement of the influence of the supernatant from the enterococcus faecium EFA5 fermentation on the copy number of the PEDV M gene.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, 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 invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present invention, "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity indicating the technical feature indicated. Also, "first," "second," "third," "fourth," etc. are used for non-exhaustive enumeration of description purposes only and should not be construed as a closed limitation to the number.
As used herein, the term "and/or", "and/or" includes any one of two or more of the associated listed items, as well as any and all combinations of the associated listed items, including any two of the associated listed items, any more of the associated listed items, or all combinations of the associated listed items.
In the present invention, the technical features described in the open type include a closed technical solution including the listed features, and also include an open technical solution including the listed features.
In the following examples of the invention, the experimental materials and reagent sources used are as follows:
the porcine enterococcus faecium (Limosilactibacillus reuteri) EFA5 is preserved in a strain preservation center with the preservation number as follows: GDMCC NO.62904.
Porcine small intestinal epithelial (IPEC-J2) cells and african green monkey kidney (Vero) cells were stored by the laboratory.
The PEDV-JS2013 strain was presented by the Shanghai veterinary institute.
Other such agents or materials, unless otherwise specified, are commercially available.
Example 1 isolation and identification of enterococcus faecium
1.1 Strain isolation
Collecting feces of healthy Luchuan sow and weaned piglet in certain pig farm of Guangxi Guigang Yangxiang Bingxiang Bingquan, taking 1g feces sample, adding 99ml sterile normal saline in sterile environment, shaking up fully, diluting feces sample in gradient of 10 -3 、10 -4 、10 -5 、10 -6 . MRS medium (Kyoto loop Kai Biotechnology Co., ltd.; 027315) was sterilized and then inverted, after it was solidified, 100. Mu.L of each sample dilution with different gradients was applied to the plate, numbered and recorded, and then placed in an incubator at 37 ℃ for anaerobic culture for 48 hours. And (3) selecting a colony with a typical shape on a plate to an MRS culture medium for streak purification, selecting a single colony after purification, inoculating the single colony into the MRS liquid culture medium, carrying out anaerobic culture at 37 ℃ for 48h, and storing 25% glycerol in a ultralow temperature refrigerator at-80 ℃ to obtain the strain EFA5.
1.2 identification of the Strain
And (3) carrying out gram staining on the separated strains, and selecting the strains which are rod-shaped or short rod-shaped under the microscope, gram-positive and spore-free for further identification. Bacterial colonies of the strain EFA5 on an MRS culture medium are milky white, the center of the bacterial colonies is smooth and convex, the edge of the bacterial colonies is rough, and the morphology of the bacterial colonies is shown in figure 1; the strain is short rod-shaped under the lens, gram-positive, has no spore, and the thallus morphology is shown in figure 2.
The bacterial strain EFA5 was subjected to genomic DNA extraction using a bacterial DNA extraction kit (Servicebio, china), followed by PCR amplification of the 16S gene using Ex Taq DNA Polymerase kit (TaKaRa, china). 16S rRNA gene amplification was performed on the strain using 27F (5 'AGAGTTTGATCCTGGCTCAG 3') and 1492R (5 'TACGGTTACCTTGTTGACTT 3') universal primers. The amplification conditions were: pre-denaturation at 95 ℃ for 5min; 35 cycles of 95 ℃ 30s,56 ℃ 30s,72 ℃ 1min30s, and 72 ℃ annealing and extending for 10min. After pipetting 5. Mu.L of the PCR product and detecting the product by 1% (w/v) agarose gel electrophoresis, the remaining PCR product was used as a sample and sent to Shanghai Producer for sequencing. The sequence was aligned to the NCBI database (https:// blast. NCBI. Nlm. Nih. Gov) and phylogenetic trees were established using MEGA 5.1 software. As shown in FIG. 3, the established phylogenetic tree shows that the strain has the closest homology with Enterococcus faecium ATCC 19434 (ID: NR _ 115764.1), enterococcus faecium NBRC 100486 (ID: NR _ 113904.1) and 16Sr RNA of Enterococcus faecium DSM 20477 (ID: NR _ 114742.1) in GenBank of NCBI, and the strain is determined to be Enterococcus faecium and named as Enterococcus faecium EFA5. And Enterococcus faecium (Enterococcus faecium) EFA5 is preserved in No. 10/25 in 2022 in Guangdong province microorganism culture collection center with the preservation number of GDMCC NO.62904, the survival state is survival, the preservation address is Microbiol institute of Guangdong academy of sciences in Guangdong province, guangzhou, the postal code is 510075, and the contact telephone is 020-87137633.
The sequencing result of the 16S rRNA gene of the EFA5 strain is shown in SEQ ID NO. 1:
ACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAATCAAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTTGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTTTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTTGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTTTAGCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTTTTAAAGCTTCTTTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACCCCGCCCGTCACACCCCGAGAGTTTGTAACCCCCGAAGTCGGTGAGGTAACCT
example 2 enterococcus faecium EFA5 probiotic Properties study
2.1 study of growth characteristics and acid production characteristics of enterococcus faecium EFA5
After activated and cultured for 12h by an MRS liquid culture medium, the activated strain is inoculated into a plurality of 10mL sterile centrifuge tubes filled with 9mL MRS liquid culture medium according to the inoculation amount of 1 percent, and is subjected to static culture at 37 ℃. And taking out one piece every three hours from 0h to 24h, measuring the optical density value of the piece at the OD value of 600nm by using a specific instrument, and drawing a growth curve by taking the OD value as a vertical coordinate and the time as a horizontal coordinate. The results show that the strain grows rapidly, the strain can enter a stationary phase within 18 hours, and the growth curve is shown in figure 4.
The acid-producing capacity of the culture broth was measured by the same inoculation procedure, and the culture broth was taken out after 4h, 8h, 12h, 16h, 20h and 24h of culture, the pH of the culture broth was measured by a pH meter, two parallel samples each time were averaged, and the measured pH was used for plotting. As shown in figure 5, the EFA5 strain has strong acid production capacity, the initial pH of the culture solution is 5.7, and the pH of the culture solution is reduced to 4.2 after 24 hours of culture, which indicates that the strain has good acid production capacity, and can reduce the pH value of the intestinal tract and adjust the intestinal homeostasis.
2.2 enterococcus faecium EFA5 acid tolerance and bile salt tolerance Studies
Respectively adjusting the pH value of the MRS culture solution to 2.0,3.0 and 4.0 by using 1M HCl, sterilizing for 15min by using high-pressure steam at 121 ℃, and preserving the temperature to a proper temperature for later use; adding 0%, 0.1%, 0.3% and 0.5% (w/v) of choline into MRS liquid culture medium, shaking sufficiently to dissolve choline completely, sterilizing, and keeping the temperature to a proper temperature for use. Taking out EFA5 strain from refrigerator at-80 deg.C, activating, taking 100 μ l of the bacterium solution, inoculating to 900 μ l of MRS broth with pre-adjusted pH or added with acetylcholine, marking, culturing in incubator for 12h, and setting control group. OD values of the bacterial liquid were measured at 0h and 12h after the culture, respectively. As shown in FIG. 6, the bacterial strain has good acid resistance and bile salt resistance, the OD value of the bacterial liquid of the bacterial strain in the environment with the pH value less than or equal to 3 is lower, and the OD value of the bacterial liquid is basically the same as that of the control group in the environment with the pH value of 4 or the concentration of 0%, 0.1%, 0.3% and 0.5%. The above results show that: the isolate has certain tolerance to acid environment, and the survival rate of the isolate is hardly influenced in the high-concentration bile salt environment, which shows that the isolate has good stress resistance and can survive in the gastrointestinal tract.
2.3 enterococcus faecium EFA 5-adherent porcine small intestine epithelial cells
Enterococcus faecium (Lactobacillus reuteri) EFA5 was inoculated into MRS broth, anaerobically cultured at 37 ℃ for 12 hours, centrifuged at 4000rpm at 4 ℃ for 10min, collected, washed 2 times with sterile PBS solution, and finally resuspended in DMEM (Gibco, USA) at a concentration of 1X 107cfu/mL. The porcine small intestine epithelial cells IPEC-J2 preserved in a laboratory are recovered and then subcultured, the small intestine epithelial cells IPEC-J2 in the logarithmic phase are taken for trypsinization, and the cell concentration is adjusted to about 1 multiplied by 105/mL by a DMEM medium. Placing cell slide in 6-well plate, inoculating cells into 6-well plate, inoculating 2mL per well, inoculating 5% (v/v) CO at 37 deg.C 2 Culturing overnight in the incubator, removing the culture medium after the cell slide adheres to the wall, and washing for 2 times by using PBS solution. Then 1mL of the above DMEM-resuspended EFA5 bacterial solution was added to each well, covered with a 6-well plate lid and shaken gently, placed at 37 ℃ in 5% (v/v) CO 2 Culturing for 2h in the incubator, taking out 6-well plate, discarding culture medium, washing with sterilized PBS solution for 5 times, and removing non-adhered bacteria. Then fixed with 4% paraformaldehyde overnight, stained with 0.5% crystal violet, and observed under an oil microscope for enterococcus faecium EFA5 adhering to IPEC-J2 cells. Randomly selecting the visual fields under the oil lens, wherein the visual fields are finally uniformly distributed, calculating the adhesion quantity of the lactic acid bacteria on the surfaces of all IPEC-J2 cells in each visual field, calculating an average value to represent the adhesion capacity of the strain, and taking a picture by using imaging software matched with a computer, wherein the result shows that the strain EFA5 has stronger adhesion performance to the IPEC-J2, as shown in figure 7. Calculated, an average of 8.75 bacteria were adhered to each cell. The above results show that the strain is applied to the small intestine of pigsThe skin cells have good adhesion capability and have the potential of being planted in the intestinal tracts of pigs.
Example 3 evaluation of enterococcus faecium EFA5 against porcine epidemic diarrhea Virus
3.1 cell culture
Vero cells were cultured in DMEM (Gibco, USA) containing 10% FBS (Gibco, USA), passaged when they grew into adherent monolayer cells, and digested with trypsin at 0.25% for about 1 min. The method comprises the following specific steps: discarding an old culture solution of adherent cells, washing twice by using a PBS solution, and removing a residual old culture medium; adding 1mL of digestive juice (0.25% pancreatin) into the flask, gently shaking the flask to allow the digestive juice to flow over all cell surfaces; under microscope observation, when the cell retraction process becomes round or the intercellular space increases, the digestion is stopped immediately (the digestion solution is poured off, a small amount of fresh culture solution containing serum is added, and the digestion is stopped); sucking the culture solution in the bottle by using a pipette, repeatedly blowing and beating the digested cells to enable the digested cells to be detached from the wall and dispersed to form cell suspension; subpackaging the cell suspension into new culture bottles, and supplementing a certain amount of fresh culture solution containing serum to make the total volume reach 5ml; gently shaking to make cells uniformly distributed, covering the bottle cap, and placing back to the cell incubator for culture. The morphology of normal Vero cells is shown in FIG. 8, and it can be seen that the cells are uniformly distributed, compact in density, clear in boundary and good in state.
3.2PEDV Virus propagation and titer determination
(1) Propagation of viruses
Inoculating the digested Vero cells into a T25 cell culture flask, incubating at 37 deg.C and 5% in CO2 incubator, and performing detoxification until the cells grow into a monolayer. Mixing 50 μ l of PEDV virus solution (JS 2013) preserved at-80 deg.C with 1ml of serum-free virus inoculation culture medium (10 ug EDTA-free pancreatin per ml DMEM), inoculating into a culture bottle with a single-layer Vero cell, incubating at 37 deg.C and 5% CO2 constant temperature culture box, supplementing 4ml virus inoculation culture medium after 1h, and culturing at 37 deg.C and 5% CO2 constant temperature culture box for 24-48h; the cell state was observed every day, and virus cell proliferation solution was harvested when lesions appeared in more than 80% of the cells. Repeatedly freezing and thawing the virus cell proliferation solution for 3 times, centrifuging at 4000rpm for 10min, collecting supernatant virus solution, and storing at-80 deg.C for use. PEDV infected Vero cells produced lesions as shown in FIG. 9, the cell boundaries were blurred in the lesion area, the cells fused and crushed, and the cell debris floated on the culture medium.
(2) Viral titer determination
Calculation of Virus Titers (TCID) in PEDV Virus solution Using Reed-Muench method 50 And/ml) comprises the following specific steps:
inoculating the digested Vero cells to a 96-well plate, incubating at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator until monolayer cells grow out; PEDV virus solution was serially diluted 10-fold to 10-fold with inoculation medium in 2ml sterile centrifuge tubes -6 (ii) a Inoculating diluted virus solution into 96-well plate with single-layer Vero cells, inoculating one vertical row of each dilution for 8 wells, inoculating 100ul virus amount into each well, inoculating virus-free inoculation culture medium in 7 th and 8 th vertical rows as control, and culturing at 37 deg.C and 5% CO 2 Incubation in a constant temperature incubator at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator for 2-5 days; cytopathic (CPE) status was observed and recorded daily and TCID of PEDV virus fluid was calculated according to the Reed-Muerch method 50 。
As a result, the TCID of the virus was found 50 Is 10 -4.33 /0.1ml。
3.3 preparation of enterococcus faecium EFA51 fermentation supernatant
Activating enterococcus faecium EFA5, inoculating into MRS broth, standing at 37 deg.C for 10 h, centrifuging at 10000r/min for 5min, adjusting supernatant pH to 7.0 with NaOH, filtering with 0.22 μm filter, and storing at-20 deg.C in refrigerator.
3.4 cytotoxicity assay of enterococcus faecium EFA51 fermentation supernatant
The cell suspension was seeded in a 96-well plate and cultured until a monolayer of cells grew out. Taking the fermentation supernatant of enterococcus faecium EFA51 to perform gradient dilution by 2 times, and selecting different gradient dilutions of 1, 1/2, 1/4, 1/8, 1/16 and 1/32. Treatment groups were added 100ul of fermentation supernatants at different dilutions per well, 5% CO at 37 ℃ in 5 replicates per dilution 2 Incubating in an incubator, and simultaneously setting a cell control and a virus control;observing cytopathic condition every day, and measuring cell activity and cytotoxicity by CCK-8 method when cell CPE of virus control group reaches above 80 = (control group OD) 450 Treatment group OD 450 ) Control group OD 450 X 100%. As shown in FIG. 10, the fermentation supernatant stock was highly toxic to the cells, while the 1/4 dilution of the fermentation supernatant was not toxic to the cells.
3.5CCK8 method for indirectly detecting inhibition rate of enterococcus faecium EFA5 fermentation supernatant on PEDV
The cell suspension was seeded in a 96-well plate and cultured until a monolayer of cells grew out. Inoculating enterococcus faecium EFA5 fermentation supernatant into 96-well plate cells, culturing for 90min, washing with DMEM, infecting the cells with PEDV with MOI =0.01, placing in an incubator for adsorption for 90min, washing, adding cell maintenance liquid, and continuing culturing. A virus control group and a normal cell control group are simultaneously set. Virus control: PEDV with MOI =0.01 was used to infect Vero cells, which were placed in an incubator for 90min and washed with PBS, and cell maintenance medium was added to continue the culture. Viral inhibition rate = (probiotic treated group OD) 450 Viral control group OD 450 ) V (cell control OD) 450 Viral control group OD 450 ) X 100%. As shown in FIG. 11, the inhibition rate of the enterococcus faecium EFA5 fermentation supernatant on PEDV at the cell level can reach 60%, and the inhibition effect on viruses is gradually weakened along with the increase of the dilution degree in a dose-dependent manner.
3.6 Effect of enterococcus faecium EFA5 fermentation supernatants on PEDV titre
The experiment is divided into a treatment group and a control group, and the specific operation is as follows:
treatment group: spreading the digested Vero cells in a 96-well plate, incubating at 37 deg.C, 5% 2 Culturing in a constant temperature incubator until monolayer cells grow out; discarding the old culture medium, washing with PBS, adding 100 μ l of bacterial metabolite per well at 37 deg.C, 5% CO2, incubating for 90min; continuously diluting PEDV virus solution 10 times to 10-6 with DMEM, inoculating one vertical row at each dilution, inoculating 100ul virus amount in each well, inoculating nontoxic DMEM culture medium in 7 th and 8 th vertical rows as control, and determining CO content at 37 deg.C and 5% 2 Incubating for 1h in a constant-temperature incubator; carefully aspirate the virus incubation and DMEM media with a pipette, adding 10 per well0ul DMEM cell maintenance solution, 37 ℃, 5% 2 Culturing for about 72 hours in a constant temperature incubator.
Control group: the bacterial metabolites were replaced with DMEM, 100. Mu.l per well, incubated for 90min, and the rest of the treatment was the same.
Cytopathic (CPE) status was observed and recorded daily until no new lesions were produced and the TCID50 of the PEDV virus fluid was calculated according to the Reed-Muerch method.
As a result, as shown in fig. 12, the EFA5 fermentation supernatant pretreatment group reduced the virus titer very significantly (about 10-fold) compared to the control group.
3.7 Effect on viral M Gene copy number
(1) Sample preparation
Vero cell suspension was seeded in 12-well plates and cultured until a monolayer of cells grew out. Inoculating 1ml of probiotic metabolite to cells, co-culturing at 37 ℃ for 90min in a cell culture box, washing with PBS, infecting the cells with PEDV (PEDV) with MOI =0.01, adsorbing for 1h in the culture box, washing with PBS, adding a cell maintenance solution, and continuing culturing. And simultaneously, a virus control group is set. Each set was 3 replicates. And (3) when the cytopathic effect of the virus control group reaches about 80 percent, collecting the virus, putting the 12-hole cell culture plate into a refrigerator at minus 80 ℃ for repeated freeze thawing three times, and centrifugally collecting supernatant to obtain the sample to be detected.
(2) RT-qPCR method for detecting copy number of PEDV M gene in each sample
I. Extraction of Total RNA from viral supernatants
The total RNA of the virus liquid is extracted by an RNA fast200RNA kit (Dieckmann, china), and is stored in a refrigerator at the temperature of 80 ℃ below zero for later use.
Reverse transcription of
The total RNA obtained was reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, china) in the following system:
a. genomic DNA removal (16 μ l system):
reaction procedures are as follows: 42 ℃ for 2min.
b. First strand cDNA Synthesis reaction solution (20. Mu.l system) was prepared:
reaction procedure: 15min at 50 ℃; at 85 ℃ for 2min.
Real-time quantification of PEDV M Gene Using qPCR
Real-time fluorescence quantification of reverse transcription products was performed using AceQ Universal SYBR qPCR Master Mix kit (Vazyme, CHINA) in the following reaction system:
reaction procedure: 5min at 95 ℃; 10s at 95 ℃, 30s at 60 ℃, and 40 cycles;
the detection results are shown in fig. 13, and after 12h and 24h of virus infection, the copy numbers of the virus M genes of the probiotic fermentation supernatant pretreatment group are both significantly lower than those of the control group. Taking 24h after infection as an example, the copy number of the virus gene in the control group is 4.8 multiplied by 10 8 While the EDA5 fermentation supernatant pretreatment group had a viral gene copy number of 1.1X 10 8 . The results show that the virus content in Vero cells can be obviously reduced by the enterococcus faecium EFA5 fermentation supernatant, so that the enterococcus faecium EFA5 screened by the method can effectively inhibit the replication of porcine epidemic diarrhea viruses and has strong antiviral capacity.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (8)
1. An Enterococcus faecium (Enterococcus faecium) EFA5 with the function of resisting porcine epidemic diarrhea virus, and the preservation number is as follows: GDMCC NO.62904 is deposited at the microbial cultures Collection of Guangdong province, and the deposition address is the institute of microbiology, guangdong province academy of sciences, guangzhou, guangdong province.
2. The enterococcus faecium EFA5 having the function of resisting the porcine epidemic diarrhea virus according to claim 1, wherein the enterococcus faecium EFA5 is separated from the feces of healthy Luchuan sows and weaned piglets.
3. The enterococcus faecium EFA5 having the anti-porcine epidemic diarrhea virus function of claim 1, wherein the enterococcus faecium EFA5 has an inhibitory effect on porcine epidemic diarrhea virus.
4. A feed or feed additive comprising enterococcus faecium EFA5 and/or a fermentation product thereof according to any one of claims 1 to 3.
5. Use of enterococcus faecium EFA5 according to any one of claims 1 to 3 for the preparation of a medicament against porcine epidemic diarrhea virus.
6. The use of enterococcus faecium EFA5 in the preparation of a medicament for the treatment of porcine epidemic diarrhea virus according to claim 5, wherein said medicament comprises enterococcus faecium EFA5 and/or a fermentation product thereof.
7. Use according to claim 5, wherein the pharmaceutical dosage form is a tablet, capsule, powder, pill, granule or solution.
8. The use of claim 5, wherein the medicament comprises enterococcus faecium EFA5 and/or a fermentation product thereof, and a pharmaceutically acceptable excipient.
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