CN108373983B - Pediococcus pentosaceus CCFM1012, fermented food thereof and application of pediococcus pentosaceus CCFM1012 in preparation of medicines for antagonizing campylobacter jejuni infection - Google Patents
Pediococcus pentosaceus CCFM1012, fermented food thereof and application of pediococcus pentosaceus CCFM1012 in preparation of medicines for antagonizing campylobacter jejuni infection Download PDFInfo
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- CN108373983B CN108373983B CN201810214462.1A CN201810214462A CN108373983B CN 108373983 B CN108373983 B CN 108373983B CN 201810214462 A CN201810214462 A CN 201810214462A CN 108373983 B CN108373983 B CN 108373983B
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Abstract
The invention relates to pediococcus pentosaceus CCFM1012, fermented food thereof and application thereof in preparing a medicine for antagonizing campylobacter jejuni infection. The pediococcus pentosaceus CCFM1012 can obviously reduce the colonization rate of campylobacter jejuni in a mouse infected by campylobacter jejuni and the transcriptional activity of virulence genes flaA, cadF, cdtB, cdtC and dnaJ of the campylobacter jejuni, effectively relieves physiological damage caused by campylobacter jejuni infection, and can be used for preparing dairy products, bean products and fruit and vegetable products for preventing campylobacter jejuni infection. Can also be used for preparing additives which can be added into the feed of poultry and livestock, is used for reducing the infection and carrying of campylobacter jejuni in the poultry and the livestock, and has very wide application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to pediococcus pentosaceus CCFM1012, fermented food thereof and application thereof in preparing a medicine for antagonizing campylobacter jejuni infection.
Background
Campylobacter jejuni (Campylobacter jejuni) is a gram-negative bacterium. Campylobacter jejuni is widely distributed in nature, can be transmitted by animals, food, water, milk and the like, and can be planted in intestinal tracts of various wild animals, poultry and livestock. Human infection can be caused by exposure to poultry and livestock, ingestion of uncooked or contaminated chicken, beef, incompletely sterilized milk, and contaminated water. In recent years, the infection rate of Campylobacter jejuni is generally on the rise around the world. In some developed countries, the number of diarrhea cases caused by campylobacter jejuni infection even exceeds that of salmonella and shigella, and becomes the most common pathogenic bacteria for diarrhea. In developing countries, campylobacter jejuni is the most common pathogenic bacterium of infectious diarrhea in infants. After a person is infected with campylobacter jejuni, gastroenteritis, diarrhea, fever and abdominal colic are caused most frequently, and people with low immunity can further cause a series of complications such as cholecystitis, peritonitis, meningitis, septicemia, osteomyelitis and the like. The most serious complication caused by Campylobacter jejuni is Guillian-Barre Syndrome (GBS), which causes axonal damage and irreversible nerve damage, leading to paralysis of respiratory muscles and death.
At present, antibiotics are commonly used in clinical treatment of campylobacter jejuni infection, but the use of the antibiotics can bring about the drug resistance of campylobacter jejuni and intestinal bacteria to the antibiotics, and the excessive use of the antibiotics can cause the residues of the antibiotics in vivo. An edible strain which has antagonism to campylobacter jejuni is screened out from nature, which is beneficial to enriching the strategy of relieving campylobacter jejuni infection and improving the efficiency of treating diseases caused by campylobacter jejuni infection.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, as one aspect of the invention, the invention overcomes the defects in the prior art, and provides Pediococcus pentosaceus (CCFM 1012) which is deposited in the Guangdong province microorganism strain collection center in 2018, 2 and 11 days, wherein the deposit address is Guangzhou city, Jie 100, college 59, building 5, Guangdong province microorganism research institute, and the deposit number is GDMCC No. 60331.
As another aspect of the present invention, the present invention overcomes the disadvantages of the prior art and provides a fermented food.
In order to solve the technical problems, the invention provides the following technical scheme, wherein: the fermented food is prepared by fermenting pediococcus pentosaceus CCFM1012, and the fermented food comprises solid food, liquid food and semi-solid food.
As a preferred method of the fermented food product of the present invention, wherein: the fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of pediococcus pentosaceus CCFM1012 in preparing probiotics for in vivo colonization.
As another aspect of the invention, the invention overcomes the defects in the prior art and provides the application of pediococcus pentosaceus CCFM1012 in preparing the medicine for antagonizing campylobacter jejuni infection.
As a preferable scheme of the application of the pediococcus pentosaceus CCFM1012 in the preparation of in vivo field planting probiotics, the method comprises the following steps: the Pediococcus pentosaceus CCFM1012 can resist gastric acid and bile salt, inhibit the growth of the Campylobacter jejuni, reduce the in vivo colonization amount of the Campylobacter jejuni, reduce the expression level of virulence genes flaA, cadF, cdtB, cdtC and dnaJ of the Campylobacter jejuni, and relieve physiological damage caused by infection of the Campylobacter jejuni.
As another aspect of the present invention, the present invention overcomes the disadvantages of the prior art and provides the use of the fermented food product of claim 2 or 3 for the preparation of a functional food product for antagonizing Campylobacter jejuni infection.
As a preferable embodiment of the use of the fermented food product of the present invention for producing a functional food product antagonistic to campylobacter jejuni sensation, wherein: the fermented food can inhibit the growth of the campylobacter jejuni, reduce the in vivo colonization rate of the campylobacter jejuni, reduce the expression level of virulence genes flaA, cadF, cdtB, cdtC and dnaJ of the campylobacter jejuni, and relieve physiological damage caused by campylobacter jejuni infection.
As another aspect of the present invention, the present invention overcomes the disadvantages of the prior art and provides a feed additive, wherein: the feed additive is liquid, powder or granules containing pediococcus pentosaceus CCFM1012, and the feed comprises feed of poultry and livestock.
As a preferable scheme of the application of the feed additive in the preparation of medicines for antagonizing campylobacter jejuni infection, the feed additive provided by the invention comprises the following components: the feed additive can reduce Campylobacter jejuni infection and carrying of poultry and livestock.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention has the beneficial effects that: the pediococcus pentosaceus CCFM1012 has good gastric acid resistance and cholate resistance, has a strong inhibiting effect on the growth of Campylobacter jejuni, the size of a bacteriostatic circle in an Oxford cup experiment can reach 15.65 +/-0.47 (mm), and simultaneously has good adhesion capacity on intestinal epithelial cells, the adhesion index can reach 15.7 +/-2.1, the single bacterium can prolong the service life of nematodes after the Campylobacter jejuni is infected, the planting amount of the Campylobacter jejuni in a mouse infected by the Campylobacter jejuni and the transcriptional activity of virulence genes flaA, cadF, cdtB, cdtC and dnaJ of the Campylobacter jejuni can be remarkably reduced, and the physiological damage caused by the Campylobacter jejuni infection can be effectively relieved.
The pediococcus pentosaceus CCFM1012 can be used for preparing dairy products, bean products and fruit and vegetable products for preventing campylobacter jejuni infection. Can also be used for preparing additives which can be added into the feed of poultry and livestock, is used for reducing the infection and carrying of campylobacter jejuni in the poultry and the livestock, and has very wide application prospect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a schematic diagram showing the effect of the strain on fecal occult blood of mice infected with Campylobacter jejuni;
FIG. 2 is a schematic diagram showing the change of the colonization amount of Campylobacter jejuni in three days and five days after the intervention of the strain on the infection of mice by Campylobacter jejuni;
FIG. 3 is a schematic diagram of a pathological section of colon tissue after intervention of the strain in mice infected with Campylobacter jejuni;
FIG. 4 is a diagram showing pathological scoring of colon tissues after intervention of the present strain in Campylobacter jejuni infected mice;
FIG. 5 is a schematic diagram showing the change of the transcription activity of campylobacter jejuni virulence genes flaA, cadF, cdtB, cdtC and dnaJ in mouse intestinal tracts after the strain intervenes infection of mice by campylobacter jejuni.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The Pediococcus pentosaceus CCFM1012(Pediococcus pentosaceus) is preserved in Guangdong province microbial strain preservation center in 2018, 2 and 11 months, and the preservation address is microbial research institute of Guangdong province, No. 59 building, No. 5 building, No. 59 college of Middleyao, Guangzhou city, and the preservation number is GDMCC No. 60331.
The pediococcus pentosaceus CCFM1012 has the following biological properties:
(1) the characteristics of the thallus are as follows: gram-positive, spherical cell, diameter of 0.8-1.0 μm, no flagellum, no spore;
(2) colony characteristics: the colony is milky white, the edge is neat, spherical, convex and non-transparent, and the surface is moist and smooth;
(3) growth characteristics: the minimum growth temperature of the strain is 15 ℃, the maximum growth temperature is 45 ℃, the strain grows optimally at the temperature of 35-37 ℃, the optimum growth pH is 6.5, and the strain enters a stable period after being cultured for 18 hours;
(4) has good acid resistance and bile salt resistance;
(5) the growth of campylobacter jejuni can be obviously inhibited in vitro;
(6) has better adhesion capacity to intestinal epithelial cells;
(7) the pediococcus pentosaceus can obviously improve the service life of nematodes infected with campylobacter jejuni in a caenorhabditis elegans infection model;
(8) pediococcus pentosaceus can effectively reduce the colonization amount of campylobacter jejuni in a mouse infected by campylobacter jejuni;
(9) the Pediococcus pentosaceus can obviously inhibit the transcriptional activity of virulence factors such as campylobacter jejuni flaA, cadF, cdtB, cdtC, dnaJ and the like in a mouse infected by campylobacter jejuni.
The extraction method of the pediococcus pentosaceus CCFM1012 comprises the following steps:
separating and screening lactic acid bacteria:
(l) Collecting a plurality of fecal samples of healthy chickens, and enriching the samples in a culture medium containing sorbitol GM17 at 35 ℃ for 12 h;
(2) performing gradient dilution on the enriched sample, then coating the enriched sample on a GM17 solid plate added with 0.02% of olcresol purple, and culturing for 24-48 h;
(3) selecting single bacterial colony with obvious color changing circle and according with the basic morphology of lactobacillus, carrying out plate streaking purification, and screening and separating out lactobacillus;
(4) and culturing the single colony in a liquid GM17 culture solution for 24h, performing gram staining, and selecting gram-positive bacteria for subsequent tests.
(II) preliminary identification of lactic acid bacteria: caldolytic ring assay
(l) Culturing the lactic acid bacteria obtained by screening in the step (I) in a liquid sorbitol GM17 culture solution for 24h, and then centrifuging l mL of culture at 8000rpm for 2 min;
(2) with 0.05M KH2PO4Washing the solution twice;
(3) resuspending the resulting pellet and streaking on sorbitol GM 17-0.75% CaCO3Culturing for 24 hours on the solid culture medium;
(4) selecting bacterial colonies which are obvious in calcium-dissolving ring, round in convex surface, fine, dense and white in color and sterile filaments, and preliminarily determining to be cocci when the bacteria are spherical after gram staining and observed by a microscope.
(III) molecular biological identification of lactic acid bacteria:
(l) Extracting a single-bacterium genome:
A. culturing the lactic acid bacteria obtained by screening in the step (II) overnight, taking l mL of the overnight-cultured bacterial suspension into a 1.5mL centrifuge tube, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain thalli;
B. purging the thalli with l mL of sterile water, centrifuging at 10000rpm for 2min, and removing the supernatant to obtain the thalli;
C. adding 200 μ LSDS lysate, and water-bathing at 80 deg.C for 30 min;
D. adding 200 μ L of phenol-chloroform solution into the thallus lysate, wherein the phenol-chloroform solution comprises Tris saturated phenol, chloroform and isoamylol at a volume ratio of 25:24:1, mixing, centrifuging at 12000rpm for 5-10min, and collecting 200 μ L of supernatant;
E. adding 400 μ L of glacial ethanol or glacial isopropanol into 200uL of supernatant, standing at-20 deg.C for 1h, centrifuging at 12000rpm for 5-10min, and discarding the supernatant;
F. adding 500 μ L70% (volume percentage) of glacial ethanol, resuspending the precipitate, centrifuging at 12000rpm for 1-3min, and discarding the supernatant;
drying in an oven at G.60 ℃ or naturally airing;
H.50μLddH2re-dissolving the precipitate with O for PCR;
(2)16S rDNA PCR
A. bacterial 16S rDNA 50 μ LPCR reaction:
10×taq buffer, 5. mu.L; dNTP, 5. mu.L; 27F, 0.5 μ L; 1492R, 0.5 μ L; taq enzyme, 0.5. mu.L; template, 0.5 μ L; ddH2O，38μL。
PCR conditions:
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
(3) preparing 1% agarose gel, mixing the PCR product with 10000 × loading buffer, loading the sample by 5 μ L, running at 120V for 30min, and performing gel imaging;
(4) and (3) sequencing the PCR product of the 16S rDNA, searching and comparing similarity of the obtained sequence result in GeneBank by using BLAST, selecting a newly discovered strain identified as belonging to pediococcus pentosaceus as a sequencing result, and preserving at-80 ℃ for later use.
Example 1: pediococcus pentosaceus CCFM1012 tolerance to simulated gastrointestinal fluids
The method comprises the steps of streaking and inoculating cryopreserved pediococcus pentosaceus CCFM1012 in an MRS solid culture medium, carrying out aerobic culture for 48h at 37 ℃, carrying out subculture for 2-3 times by using the MRS culture medium, taking the pediococcus pentosaceus CCFM1012 culture medium, centrifuging for 5min at 8000r/min, collecting thalli, re-suspending in artificial simulated gastric juice (MRS culture medium containing 1% pepsin and pH 2.5) at pH 2.5 (1:1), mixing, carrying out anaerobic culture at 37 ℃, sampling at the beginning (0h), 1h, 2h and 3h respectively, carrying out pouring culture by using the MRS agar culture medium, carrying out plate colony counting, measuring the number of viable bacteria and calculating the survival rate of the viable bacteria. The survival rate is the ratio of the number of viable bacteria in the culture medium to the number of viable bacteria at 0h, and is expressed as%.
Taking a culture solution of pediococcus pentosaceus CCFM1012, centrifuging at 8000r/min for 5min, collecting thalli, suspending in (1:1) artificial simulated intestinal fluid (MRS culture medium containing 0.3% of bovine bile salt, 1% of trypsin and having pH of 8.0), aerobically culturing at 37 ℃, sampling at 0h, 1h, 2h, 3h and 4h respectively, pouring and culturing by using MRS agar culture medium, counting plate colonies, measuring the number of viable bacteria and calculating the survival rate. The survival rate is the ratio of the number of viable bacteria at the time of sampling the culture medium to the number of viable bacteria at 0h, and is expressed as%. As shown in tables 1 and 2, it can be seen that pediococcus pentosaceus CCFM1012 has good tolerance to both simulated gastric and intestinal fluids.
TABLE 1 tolerance of Pediococcus pentosaceus CCFM1012 in simulated gastric fluid
TABLE 2 tolerance of Pediococcus pentosaceus CCFM1012 in artificially simulated intestinal fluid
Example 2: pediococcus pentosaceus CCFM1012 in vitro inhibition of campylobacter jejuni growth
The method comprises the steps of taking out pediococcus pentosaceus CCFM1012, pediococcus pentosaceus H29M-8M and lactobacillus rhamnosus LGG strains in a refrigerator at the temperature of-80 ℃, streaking in an MRS plate, culturing for 48H at the temperature of 37 ℃, selecting a single colony in an MRS liquid tube, culturing for 18H at the temperature of 37 ℃, inoculating in a new MRS liquid culture medium by 2% of volume, culturing for 18H at the temperature of 37 ℃, culturing for one generation again according to the same mode, centrifuging a lactobacillus suspension for 8min at the temperature of 4 ℃ at 8000r/min, sucking a supernatant, performing filtration sterilization by using a 0.22 mu M water system filter membrane to obtain a lactobacillus fermentation supernatant, and adjusting the pH of a fermentation liquid to be 6.5 by using 1mol/L NaOH.
Campylobacter jejuni NCTC11168 strain (purchased from American type culture Collection ATCC) was cultured on two media (i.e., Brookfield agar (product of Qingdao Haibo Biotechnology Co., Ltd.) and brain Heart infusion broth (culture Medium of Oxoid Co.)) at 37 ℃ and 5% O2、10％CO2And 85% N2After subculturing for two generations in the same manner, the cells were centrifuged at 2800r/min for 6min, washed with phosphate buffered saline (PBS, pH 7.2), and resuspended in PBS to a bacterial concentration of 108CFU/mL。
The Campylobacter jejuni bacterial suspension (250. mu.L, 10) was aspirated8CFU/mL)，Uniformly coating the mixture on a Columbia blood agar plate, naturally drying the bacterial solution, placing an Oxford cup, adding 100 mu L of lactobacillus fermentation supernatant (pH 6.5) into the Oxford cup, diffusing at 4 ℃ for 2h, placing the mixture into a three-atmosphere culture box, culturing at 37 ℃ for 48h, and measuring the diameter of a bacteriostatic zone. And (3) detecting the diameter (mm) of a zone of inhibition by taking the added sterilized MRS with the pH value of 6.5 as a negative control and 0.30mg/mL norfloxacin broad-spectrum antibiotic as a positive control.
TABLE 3 measurement results of zone of inhibition diameter
The bacteriostatic effect is shown in table 3, and the result shows that the fermentation supernatant of pediococcus pentosaceus CCFM1012 obviously inhibits the growth of campylobacter jejuni, the diameter of the inhibition zone can reach 15.65 +/-0.47 (mm), and the effect is obviously stronger than that of control pediococcus pentosaceus H29M-8M and lactobacillus rhamnosus LGG.
Example 3: pediococcus pentosaceus CCFM1012 adhesion to intestinal epithelial cells HT-29
The intestinal epithelial cell line HT-29 cells (obtained from the cell bank of the culture Collection of the national academy of sciences) were cultured in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum and 1% streptomycin. HT-29 cells in 5% CO2The cell culture chamber of (1) was used for culturing (37 ℃ C.), and the culture medium was changed 1 time after each 48 hours of culturing, followed by continuous culturing.
Digesting the HT-29 cells grown and fused to 70% -80%, and adjusting the concentration to 2X 105Per mL, sterile coverslips were placed in 6-well cell culture plates, 2mL of cell culture suspension was added per well in 5% CO2Culturing at 37 deg.C in incubator, washing with PBS three times when cell grows to monolayer, adding 1mL of 2 × 10 solution per well8CFU/mL of the serum-free and antibiotic-free RPMI-1640 cell culture bacterial suspension of the lactobacillus, supplementing the RPMI-1640 cell culture solution (without serum and antibiotic) to 2mL, and incubating for 2 h. After incubation, PBS is washed with water IIINext, to remove non-adhering lactic acid bacteria, they were fixed with methanol for 20min, washed three times with PBS, gram-stained, and examined under 100 times of oil microscope. The number of bacteria adhered to each 100 cells was counted by randomly selecting 20 fields as an adhesion index. The results of the adhesion experiments are shown in Table 4.
TABLE 4 adhesion of lactic acid bacteria to HT-29 cell surface
The results in Table 4 show that the pediococcus pentosaceus CCFM1012 has stronger adhesion capability to the intestinal epithelial cells HT-29, the adhesion index can reach 15.7 +/-2.1, the adhesion capability is stronger than that of the control pediococcus pentosaceus H29M-8M and the Lactobacillus rhamnosus LGG, and the pediococcus pentosaceus CCFM1012 with stronger adhesion capability can effectively prevent pathogenic microorganisms from contacting and adhering to intestinal mucosa cells after intestinal colonization, so that intestinal diseases caused by the intestinal pathogens can be prevented.
Example 4: effect of Pediococcus pentosaceus CCFM1012 on longevity of C.elegans infected with Campylobacter jejuni
Preparation of E.coli (Escherichia coli) OP 50: inoculating Escherichia coli OP50 into liquid culture medium, performing shake culture, shaking and mixing uniformly when OD600 is 1.0-1.2, sucking bacterial liquid, dripping on caenorhabditis elegans growth NGM plate, coating uniformly, culturing and storing for later use.
Resuscitation and synchronization of caenorhabditis elegans: freezing and thawing the nematode freezing and storing tube, centrifuging, discarding the supernatant, and pouring the nematodes into an NGM flat plate with Escherichia coli OP50 for resuscitation; after the nematodes grow into adults, sucking sterile water by using a pipette gun to repeatedly wash the flat plate, transferring the liquid containing the nematodes into a centrifuge tube, sucking sterile aqueous suspension containing the nematodes, adding the sterile aqueous suspension into a new centrifuge tube, adding a sodium hydroxide solution and a sodium hypochlorite solution, and fully and uniformly mixing; placing the centrifuge tube under a microscope for observation after 2-3 minutes of oscillation until no large nematode fragments can be seen; centrifuging and washing with sterile water; and (3) resuspending the strain by using an S culture medium liquid, culturing the S culture medium liquid containing the ova, centrifugally collecting L1-stage nematodes, transferring the L1-stage nematodes to an NGM culture plate with escherichia coli OP50, and culturing for 72 hours at 20 ℃ to obtain the L4-stage nematodes.
TABLE 5 statistical analysis of longevity of Campylobacter jejuni nematode infected after lactic acid bacteria intervention
a: calculating the survival rate of the nematodes on day 13 by using a Kaplan-Meier survival model; b: DT50, time required for 50% of nematode deaths.
Table 5 shows the way of handling nematodes by picking L4 nematodes with sterile picker and transferring them to modified NGM plates, each plate containing about 80-100 nematodes, and adding 10 final concentration to each of control group (E. coli + C. jejuni) and pretreatment group (CCFM1012+ C. jejuni, H29M-8M + C. jejuni and LGG + C. jejuni)8CFU/mL Escherichia coli OP50 and lactobacillus suspension 200 μ L, transferring living nematodes to new improved NGM plate every day, adding corresponding bacterial suspension to different groups, respectively, continuing for three days, stopping feeding lactobacillus to intervention group, and adding 10% to control group and intervention group8CFU/mL of Campylobacter jejuni bacterial suspension 200. mu.L, after which live nematodes were transferred to new NGM plates every day, 10 added to each group8CFU/mL of Campylobacter jejuni bacterial suspension 200. mu.L, in blank group (E. coli), was transferred to new NGM plates daily and added to final concentration of 108CFU/mL E.coli OP50, and the number of nematode deaths per group was recorded. From the results in the figure, the life of the nematodes after the infection of the Campylobacter jejuni can be remarkably prolonged by the Pediococcus pentosaceus CCFM1012, the survival rate of the nematodes can still reach 45.81% at day 13 in the CCFM1012+ C.jejuni group, and moreover, the number of days in which the nematodes die by 50% is prolonged to 14.46 days, and the prolonging effect is remarkably stronger than that of the Pediococcus pentosaceus H29M-8M and Lactobacillus rhamnosus LGG.
Example 5: antagonistic action of Pediococcus pentosaceus CCFM1012 on Campylobacter jejuni in mice infected with Campylobacter jejuni
In the embodiment, a C57BL/6 mouse infected by a mixture of toxoplasma gondii and campylobacter jejuni is used as an experimental mouse, and a large amount of campylobacter jejuni can be planted in the toxoplasma gondii infected mouse.
Preparation of mouse gavage agent:
campylobacter jejuni gavage agent: culturing activated 2 generation Campylobacter jejuni at 37 deg.C under three-atmosphere condition for 24 hr, centrifuging at 4 deg.C and 2800r/min for 6min to collect thallus, discarding supernatant, and resuspending thallus with sterile phosphate buffer solution to make Campylobacter jejuni concentration reach 3 × 109CFU/mL。
Lactobacillus gastric lavage agent: taking 2 generation activated lactobacillus and heating at 37 deg.C (5% O)2，10％CO2，85％N2) Culturing for 24h, centrifuging at 4 deg.C and 8000r/min for 3min to collect thallus, discarding supernatant, and resuspending thallus with sterile phosphate buffer solution to reach lactobacillus concentration of 5 × 109CFU/mL。
Toxoplasma gavage agent: after the mice infected with Toxoplasma gondii Me49 were sacrificed, brain tissue was collected, added with sterile phosphate buffer and ground thoroughly. 10 μ L of brain homogenate was dropped onto the slide, counted under a light microscope, and repeated 3 times. The concentration of the brain homogenate was adjusted according to the counting results, and 200. mu.L of the brain homogenate was perfused into each mouse, so that the intragastric dose of Toxoplasma gondii reached 20 cysts/mouse.
Grouping and processing of mice:
each experimental group had 8 mice, 4 mice were divided into 1 cage, and the gavage dose was 300 μ L/mouse. On the 1st day of the animal experiment, mice except the control group were gavaged with toxoplasma gondii-encapsulating pathogens; normal rearing on days 2, 3 and 4 so that the pathogen begins to destroy the immune system of the mouse; sequentially irrigating the mice with lactobacillus and campylobacter jejuni every 5 th and 6 th day, wherein the interval between the two times of lavage is at least 1h, so as to avoid the mutual influence between the lavage agents; day 7, 8, 9, 10 mice exhibited symptoms of campylobacter jejuni infection; mice were sacrificed on day 11 and the animal experiment was terminated. The detailed grouping and processing method is shown in table 6.
TABLE 6 grouping and treatment of laboratory mice
And (3) detecting fecal occult blood of the mice: 1 freshly collected mouse feces was taken and fecal occult blood detection was performed using a fecal occult blood kit. The experimental method is as follows: uniformly coating the excrement on white filter paper, dropwise adding 3 drops of o-tolidine solution, continuously dropwise adding 2 drops of hydrogen peroxide, and observing a color development result:
after 3min, no blue-green color appeared, the result was negative, and the score was 0;
blue color appears within 30s-60s, the result is weak positive, and the score is 1;
blue-green color is immediately developed, the result is positive, and the score is 2;
a dark blue color immediately appeared, the result was a strong positive, and the score was 3.
Detecting the number of the live bacteria of the mouse fecal campylobacter jejuni: fresh mouse feces were taken, precisely weighed, and then soaked in sterile physiological saline for 30min to soften the feces. And (3) fully and uniformly mixing the fecal suspension, performing gradient dilution, selecting 100 mu L of diluent with proper gradient, uniformly coating the diluent on a Columbia blood plate added with campylobacter selective antibiotic, placing the Columbia blood plate in a three-gas incubator at 37 ℃ for culturing for 48h, and counting campylobacter jejuni colonies growing on the plate.
Preparation of mouse colon paraffin section, HE staining and pathological scoring: taking out the colon tissue soaked in paraformaldehyde with the mass fraction of 4%, making paraffin sections and performing HE staining.
The paraffin section preparation steps are as follows: the colon tissue is put into ethanol with volume fraction of 70%, 80%, 90%, 95%, 100% for 20min for dehydration. The dehydrated colon tissue is placed in a solution of ethanol to xylene in a ratio of 1:1 for 5min, after which the colon is immersed twice in xylene for 15 min. Wax dipping followed by slicing to a thickness of about 5 μm.
HE staining procedure was as follows: dewaxing with xylene for 2 times, each time lasting 5min, immersing the slices in 100%, 95%, 80%, 70% ethanol for 5min, washing with flowing water for 5min, staining with hematoxylin for 5min, and washing with ultrapure water for 2 times. Adding hydrochloric acid ethanol (70% ethanol and 0.5% hydrochloric acid), separating for 10s, washing with flowing water for 10min, and sequentially adding slices into 70%, 80%, and 95% ethanol for 5 min. Staining with eosin ethanol (95% ethanol and 0.5% eosin), sequentially adding into 95% and 100% ethanol for 5min, respectively, clearing with xylene for 2 times, and sealing with neutral gum.
Under microscope, blind scoring and pathological scoring are carried out, and the scoring standard is shown in the following table:
TABLE 7 Colon histopathology scoring criteria
Mouse feces were collected and their occult blood was assessed using a fecal occult blood kit, the results are shown in FIG. 1. The results show that the fecal occult blood of the mice infected by the toxoplasma gondii and the campylobacter jejuni is serious, the fecal occult blood symptoms of the mice are obviously relieved after the intervention of the pediococcus pentosaceus CCFM1012, the fecal occult blood score is reduced to 2.15, and the pediococcus pentosaceus CCFM1012 plays a more positive relieving role compared with the pediococcus pentosaceus H29M-8M and the lactobacillus rhamnosus LGG.
As shown in FIG. 2, Pediococcus pentosaceus CCFM1012 is effective in reducing the colonization amount of Campylobacter jejuni in mice. C.j, the amount of Campylobacter jejuni in feces in the body on the third day and the fifth day can reach 106And 108CFU/g faces, and the planting amount gradually increases along with the time; the field planting amount of Campylobacter jejuni in Pediococcus pentosaceus CCFM1012 stem group can be reduced to 10 at the third day4-105CFU/g faces decreased by about 1.5 orders of magnitude compared to the control; by the fifth day, the mice are gradually seriously infected by the toxoplasma, and the colonization of the campylobacter jejuni in the mice is correspondingly improvedHowever, the pediococcus pentosaceus CCFM1012 still shows good cleaning capability after intervention, and the colonization amount of the Campylobacter jejuni can be reduced to 106CFU/g faces decreased by about two orders of magnitude, and the clearance of Campylobacter jejuni in mice by Pediococcus pentosaceus CCFM1012 was much higher than that of Pediococcus pentosaceus H29M-8M and that of Lactobacillus rhamnosus LGG.
HE staining was observed under the microscope and is shown in fig. 3, where: a represents a control group, B represents C.j group, C represents CCFM1012 group, D represents H29M-8M, and E represents LGG group. From the results, it can be seen that the intestinal epithelium and intestinal wall of the control mice are intact, the cell morphology is normal, and no adverse change is observed. The colon lesions of mice infected by the toxoplasma gondii and the campylobacter jejuni are serious, and typical characteristics comprise goblet cell disappearance, obvious inflammatory cell infiltration, crypt destruction, mucosal injury and the like. In combination with pathology scoring analysis (fig. 4), the value of the pediococcus pentosaceus CCFM1012 component dropped to 4, greatly alleviating colonic lesions in mice. The above results show that the strain of Campylobacter jejuni on the colon of mice can be reduced by antagonizing Campylobacter jejuni by Pediococcus pentosaceus CCFM 1012.
Example 6: effect of Pediococcus pentosaceus CCFM1012 on the transcriptional levels of Campylobacter jejuni virulence factors flaA, cadF, cdtB, cdtC and dnaJ in mice infected with Campylobacter jejuni
The model of mouse campylobacter jejuni infection was established and processed as described in example 5.
Campylobacter jejuni virulence genes flaA, cadF, cdtB, cdtC and dnaJ q RT-PCR primer design and synthesis are shown in Table 8
TABLE 8Q PCR primers for Campylobacter jejuni virulence genes flaA, cadF, cdtB, cdtC, and dnaJ and reference genes
Extraction of colon tissue and campylobacter jejuni total RNA and synthesis of cDNA:
0.2g of fresh colon tissue taken out after the mouse is dissected is repeatedly ground in a mortar (180 ℃, 4h high temperature enzyme deactivation) added with liquid nitrogen, 1mL of Trizol reagent is added into the mortar, the grinding is continued, after the liquid is basically clarified, the fresh colon tissue is collected into a 1.5mL enzyme-free centrifuge tube, the centrifuge tube is kept still at room temperature for 15min, 200 mu L of trichloromethane solution is added into the centrifuge tube, the centrifuge tube is gently shaken for 15s, the centrifuge tube is kept still at room temperature for 10min, the centrifuge tube is centrifuged for 15min at 4 ℃ and 12000r/min, 600 mu L of upper colorless aqueous phase is taken out to another enzyme-free centrifuge tube, and 500 mu L of isopropanol is added. Mixing by reversing up and down, standing at room temperature for 10min, centrifuging at 4 deg.C and 12000r/min for 10min after standing, discarding the supernatant, leaving the white precipitate of RNA at the bottom of the centrifuge tube, adding 1m L75% ethanol solution prepared from DEPC water, vortex shaking for resuspension, centrifuging at 4 deg.C and 7500r/min for 5min, discarding the supernatant, and naturally volatilizing and drying at room temperature. To the dried RNA, 30. mu.L of RNase free water was added, and after the RNA was dissolved, the concentration and purity of the RNA were determined by Nanodrop, and the quality of the RNA was checked by agarose gel electrophoresis. The total RNA extracted was used as a template, and c DNA was synthesized by reverse transcription according to the procedure of the PrimeScript 1stStrand cDNA Synthesis Kit (TaKaRa), and stored at-20 ℃.
qRT-PCR reaction system and conditions:
by using
The CFX96TM real-time fluorescent quantitative PCR instrument performs PCR amplification and reads the fluorescent signal.
The qRT-PCR reaction system of the jejuni virulence gene is as follows:
the qRT-PCR reaction conditions of jejuni virulence gene are as follows:
pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, and annealing at 60 ℃ for 30s (40 cycles).
As shown in FIG. 5, the results show that Pediococcus pentosaceus CCFM1012 can effectively inhibit the transcription level of Campylobacter jejuni flaA, cadF, cdtB, cdtC and dnaJ virulence factors in mice, and the corresponding virulence factor multiples can be reduced to 0.17, 0.13, 0.51, 0.14 and 0.41, which indicates that Pediococcus pentosaceus CCFM can realize antagonism on Campylobacter jejuni infection by reducing the level of the Campylobacter jejuni virulence factors.
Example 7: production of fermented food containing Pediococcus pentosaceus CCFM1012 of the present invention
Selecting fresh vegetables, washing, juicing, carrying out high-temperature instant sterilization, carrying out high-temperature heat sterilization at 140 ℃ for 2 seconds, immediately cooling to 37 ℃, and inoculating the fermentation agent of the pediococcus pentosaceus CCFM1012 microbial inoculum prepared by the invention to ensure that the concentration of the fermentation agent reaches 106The fruit and vegetable beverage containing the pediococcus pentosaceus CCFM1012 live bacteria is obtained by storing the beverage at a temperature of more than 4 ℃ in a refrigeration mode.
The method can be used for producing other fermented foods by fermenting the pediococcus pentosaceus CCFM1012, wherein the fermented foods comprise solid foods, liquid foods and semi-solid foods. The fermented food comprises dairy products, bean products and fruit and vegetable products, wherein the dairy products comprise milk, sour cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
The fermented food can inhibit the growth of the campylobacter jejuni, reduce the in vivo colonization rate of the campylobacter jejuni, reduce the expression level of virulence genes flaA, cadF, cdtB, cdtC and dnaJ of the campylobacter jejuni, and relieve physiological damage caused by campylobacter jejuni infection.
Example 8: : production of feed additive containing pediococcus pentosaceus CCFM1012 of the present invention
The feed additive is liquid or powder containing pediococcus pentosaceus CCFM1012, and the preparation process comprises the following steps: inoculating the freeze-preserved pediococcus pentosaceus CCFM1012 in an MRS liquid culture medium according to the proportion of 2% (v/v) for two times (18 hours each time) to activate to obtain a secondary seed liquid, inoculating the secondary seed liquid into a fermentation tank containing the MRS culture medium according to the inoculation amount of 5% to perform fermentation culture, performing aerobic culture at 37 ℃ for 48 hours, centrifuging fermentation liquor at 8000rpm to obtain bacterial sludge, adding water accounting for 90% of the mass of the bacterial sludge, sucrose accounting for 40%, maltodextrin accounting for 50% and whey powder accounting for 50%, standing at normal temperature for 30 minutes, and then mixing with sodium carboxymethylcellulose accounting for 40% of the mass of the bacterial sludge, microcrystalline cellulose accounting for 8 times of the mass of the bacterial sludge and seaweed accounting for 10 times of the mass of the bacterial sludgeMixing sodium, calcium chloride with the mass of 10 times of the bacterial sludge and water with the mass of 20 times of the bacterial sludge, then carrying out wet granulation, and carrying out rotary flow drying at 37 ℃ to obtain the finished product of the feed additive containing pediococcus pentosaceus CCFM 1012. The viable count of the product is 1 × 1010CFU/g is higher than the standard. The Pediococcus pentosaceus CCFM1012 feed additive can be directly stirred and added into finished feed for poultry and livestock according to 0.5-2% (mass ratio of culture solution to feed) to reduce infection and carrying of campylobacter jejuni of the poultry and the livestock, and can also be uniformly mixed with feed raw materials and fermented for a plurality of days to prepare the feed, and the preparation process comprises the following steps: selecting crushed wheat bran, bean pulp and rice bran, wherein the mass ratio is 8: 1:1, adding water and uniformly mixing to ensure that the water content of the material reaches 35-40 percent, the content of basic protein is about 18 percent, pre-dissolving the feed additive of pediococcus pentosaceus CCFM1012 according to the inoculation amount of 0.5 percent (mass ratio), then inoculating the feed additive into a solid material, uniformly mixing, controlling the temperature to be about 37 ℃, carrying out low-temperature drying on the fermented material at the temperature of not higher than 45 ℃ after anaerobic culture for 72 hours, and carrying out cold storage and sealed storage at the temperature of 4 ℃ after the completion. The feed additive contains Pediococcus pentosaceus CCFM1012 with viable count not less than 5 × 108CFU/g. The feed additive can reduce Campylobacter jejuni infection and carrying of poultry and livestock.
The invention has the beneficial effects that: the pediococcus pentosaceus CCFM1012 has good gastric acid resistance and cholate resistance, has a strong inhibiting effect on the growth of campylobacter jejuni, has good adhesion capacity on intestinal epithelial cells, can prolong the service life of nematodes after the campylobacter jejuni is infected by a single bacterium, can obviously reduce the planting amount of the campylobacter jejuni in a mouse infected by the campylobacter jejuni and the transcriptional activity of virulence genes flaA, cadF, cdtB, cdtC and dnaJ of the campylobacter jejuni, and effectively relieves physiological damage caused by the campylobacter jejuni infection.
The pediococcus pentosaceus CCFM1012 can be used for preparing dairy products, bean products and fruit and vegetable products for preventing campylobacter jejuni infection. Can also be used for preparing additives which can be added into the feed of poultry and livestock, is used for reducing the infection and carrying of campylobacter jejuni in the poultry and the livestock, and has very wide application prospect.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (7)
1. A Pediococcus pentosaceus (Pediococcus pentosaceus) CCFM1012 is preserved in Guangdong province microbial strain preservation center in 2018, 2 and 11 months, and is preserved by Guangzhou city, the China institute for microorganisms, Guangzhou province, No. 59 building, No. 5 building, No. 59 institute for Zhonglu-Jie, Middleya, and the preservation number is GDMCC number 60331.
2. A fermented food product characterized by: the fermented food is prepared by fermenting pediococcus pentosaceus CCFM 1012; wherein the fermented food comprises solid food, liquid food, and semi-solid food; the pediococcus pentosaceus CCFM1012 is preserved in Guangdong province microbial strain preservation center in 2018, 2 and 11 months, is preserved at Guangzhou city, Jielizhou No. 59 building, 5 building, Guangdong province microbial research institute, and has the preservation number of GDMCC number 60331.
3. The pediococcus pentosaceus CCFM1012 is stored in a Guangdong province microbial strain storage center in 2018, 2 and 11 days, and the storage address is the microbial research institute of Guangdong province, No. 59 building, No. 5 building, Guangzhou, Michelia elizae, No. 100 college, Michelia.
4. The pediococcus pentosaceus CCFM1012 is stored in Guangdong province microbial strain storage center 2 and 11 days 2018, the storage address is Guangzhou city Michelia intermedia No. 100 college No. 59 building No. 5 building Guangdong province microbial research institute, and the storage number is GDMCC number 60331.
5. Use of the fermented food product according to claim 2 for the preparation of a functional food product for antagonizing campylobacter jejuni infection.
6. A feed additive, characterized in that: the feed additive comprises liquid, powder or granules containing pediococcus pentosaceus CCFM1012, the feed comprises feed for poultry and livestock, the pediococcus pentosaceus CCFM1012 is stored in Guangdong province microorganism strain preservation center in 2018, 2 and 11 days, the preservation address is No. 59 building of No. 5 building of Guangdong province microorganism research institute of Middlexueli Middleway No. 100, Guangzhou, and the preservation number is GDMCC number 60331.
7. Use of a feed additive according to claim 6 for the preparation of a medicament for antagonizing campylobacter jejuni infection, wherein: the feed additive can reduce Campylobacter jejuni infection and carrying of poultry and livestock.
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