WO2019174002A1 - 戊糖片球菌ccfm1012、其发酵食品及其在制备拮抗空肠弯曲杆菌感染药物中的应用 - Google Patents

戊糖片球菌ccfm1012、其发酵食品及其在制备拮抗空肠弯曲杆菌感染药物中的应用 Download PDF

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WO2019174002A1
WO2019174002A1 PCT/CN2018/079153 CN2018079153W WO2019174002A1 WO 2019174002 A1 WO2019174002 A1 WO 2019174002A1 CN 2018079153 W CN2018079153 W CN 2018079153W WO 2019174002 A1 WO2019174002 A1 WO 2019174002A1
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campylobacter jejuni
ccfm1012
pediococcus pentosaceus
infection
preparation
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WO2019174002A8 (zh
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王刚
陈卫
金星
赵建新
张灏
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江南大学
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Publication of WO2019174002A8 publication Critical patent/WO2019174002A8/zh
Priority to US17/019,867 priority patent/US11160839B2/en

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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A—HUMAN NECESSITIES
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    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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Definitions

  • the invention belongs to the technical field of microorganisms, and particularly relates to Pediococcus pentosaceus CCFM1012, fermented food thereof and application thereof in preparing medicaments for antagonizing infection of Campylobacter jejuni.
  • Campylobacter jejuni is a Gram-negative bacterium. Campylobacter jejuni is widely distributed in nature and can be spread by animals, food, water, milk, etc., and can be colonized in the intestines of various wild animals and poultry and livestock. Exposure to poultry, ingestion of uncooked or contaminated chicken, beef, unsterilized milk, and contaminated water can cause human infection. In recent years, the incidence of Campylobacter jejuni has been increasing worldwide. In some developed countries, the number of cases of diarrhea caused by Campylobacter jejuni infection even exceeds that of Salmonella and Shigella, becoming the most common diarrhea pathogen. In developing countries, Campylobacter jejuni is the most common pathogen of infectious diarrhea in infants and young children.
  • Campylobacter jejuni After infection with Campylobacter jejuni, the most common cause gastroenteritis, diarrhea, fever and abdominal cramps, and people with low immunity will further cause a series of complications, such as cholecystitis, peritonitis, meningitis, sepsis and osteomyelitis. .
  • GBS Gullian-Barré Syndrome
  • Campylobacter jejuni infection the common method for clinical treatment of Campylobacter jejuni infection is antibiotics, but the use of antibiotics will bring resistance to antibiotics by Campylobacter jejuni and intestinal bacteria. Excessive use of antibiotics may lead to the retention of antibiotics in the body. Screening a strain that is edible and antagonistic to Campylobacter jejuni from nature can help to enrich the response to the inhibition of Campylobacter jejuni infection and improve the efficiency of treating diseases caused by Campylobacter jejuni infection.
  • the present invention has been made in view of the above technical deficiencies.
  • the present invention overcomes the deficiencies in the prior art and provides a Pepococcus pentosaceus CCFM1012 (Pediococcus pentosaceus) deposited on February 11, 2018 in the Guangdong Provincial Collection of Microorganisms and Cultures.
  • the deposit address is the Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, with the preservation number GDMCC No.60331.
  • the present invention overcomes the deficiencies in the prior art and provides a fermented food.
  • the present invention provides the following technical solution, wherein the fermented food is produced by fermentation production using Pediococcus pentosaceus CCFM1012, and the fermented food includes solid food, liquid food, and semi-solid food.
  • a preferred method of the fermented food of the present invention wherein the fermented food comprises dairy products, soy products, fruit and vegetable products, the dairy products include milk, sour cream, cheese; the fruit and vegetable products include cucumbers, carrots, Beets, celery and cabbage products.
  • the present invention overcomes the deficiencies in the prior art and provides the use of Pediococcus pentosaceus CCFM1012 for the preparation of in vivo colonization of probiotics.
  • the present invention overcomes the deficiencies in the prior art and provides the use of Pediococcus pentosaceus CCFM1012 in the preparation of a medicament for antagonizing infection with Campylobacter jejuni.
  • a preferred embodiment of the use of the Pediococcus pentosaceus CCFM1012 in the preparation of probiotics in vivo wherein: the Pediococcus pentosaceus CCFM1012 is resistant to gastric acid, bile salts, inhibiting the growth of Campylobacter jejuni, Reduce the in vivo colonization of Campylobacter jejuni, reduce the expression levels of Campylobacter jejuni virulence genes flaA, cadF, cdtB, cdtC and dnaJ, and alleviate the physiological damage caused by Campylobacter jejuni infection.
  • the present invention overcomes the deficiencies in the prior art and provides the use of the fermented food of claim 2 or 3 in the preparation of a functional food antagonizing infection with Campylobacter jejuni.
  • a preferred embodiment of the fermented food of the present invention for preparing a functional food for antagonizing the sensation of Campylobacter jejuni wherein the fermented food can inhibit the growth of Campylobacter jejuni and reduce the in vivo colonization rate of Campylobacter jejuni
  • the expression levels of the virulence genes flaA, cadF, cdtB, cdtC and dnaJ of Campylobacter jejuni were reduced, and the physiological damage caused by Campylobacter jejuni infection was alleviated.
  • the present invention overcomes the deficiencies in the prior art and provides a feed additive wherein the feed additive is a liquid, powder or granule containing Pediococcus pentosaceus CCFM1012, the feed comprising poultry, Feed for livestock.
  • the feed additive is a liquid, powder or granule containing Pediococcus pentosaceus CCFM1012, the feed comprising poultry, Feed for livestock.
  • the present invention provides the following technical solutions:
  • the P. stipitis CCFM1012 of the invention has good resistance to gastric acid and bile salts, and has strong inhibitory effect on the growth of Campylobacter jejuni, and the size of the inhibition zone can reach 15.65 in the Oxford Cup experiment. ⁇ 0.47 (mm), and has good adhesion to intestinal epithelial cells, its adhesion index can reach 15.7 ⁇ 2.1, single bacteria can prolong the lifespan of C.
  • the Pediococcus pentosaceus CCFM1012 of the present invention can be used to prepare dairy products, soy products and fruit and vegetable products having prevention of Campylobacter jejuni infection. It can also be used to prepare additives which can be added to poultry and livestock feed to reduce the infection and carrying of Campylobacter jejuni in poultry and livestock, and has a wide application prospect.
  • Figure 1 is a schematic diagram showing the effect of the present strain on occult blood in feces of mice infected with Campylobacter jejuni;
  • FIG. 2 is a schematic diagram showing changes in colonization of Campylobacter jejuni in three days and five days after intervention of mice infected with Campylobacter jejuni;
  • Figure 3 is a schematic view showing the pathological section of colon tissue after intervention of mice infected with Campylobacter jejuni;
  • Figure 4 is a schematic diagram showing the pathological score of colon tissue after intervention of mice infected with Campylobacter jejuni;
  • Fig. 5 is a schematic diagram showing the changes in the transcriptional activities of the virulence genes flaA, cadF, cdtB, cdtC and dnaJ of C. jejuni in the intestinal tract of mice infected with Campylobacter jejuni.
  • an embodiment or “an embodiment” as used herein refers to a particular feature, structure, or characteristic that can be included in at least one implementation of the invention.
  • the Pseudomonas pentosaceus CCFM1012 (Pediococcus pentosaceus) of the present invention was deposited at the Guangdong Provincial Microbial Culture Collection on February 11, 2018, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong province.
  • the deposit number is GDMCC No.60331.
  • the Pediococcus pentosaceus CCFM1012 has the following biological properties:
  • Colony characteristics the colony is milky white, the edges are neat, spherical, convex, opaque, and the surface is moist and smooth;
  • the minimum growth temperature of the strain is 15 ° C, the highest growth temperature is 45 ° C, the best growth is carried out at a temperature of 35-37 ° C, the optimum growth pH is 6.5, and the stable phase is entered after 18 h of culture;
  • Pediococcus pentosaceus can significantly increase the lifespan of nematodes infected with Campylobacter jejuni in the Caenorhabditis elegans infection model
  • Pediococcus pentosaceus can effectively reduce the colonization of Campylobacter jejuni in mice infected with Campylobacter jejuni;
  • Pediococcus pentosaceus significantly inhibited the transcriptional activities of virulence factors such as Campylobacter jejuni flaA, cadF, cdtB, cdtC and dnaJ in mice infected with Campylobacter jejuni.
  • the extraction method of the Pediococcus pentosaceus CCFM1012 is:
  • the lactic acid bacteria obtained by the step (2) were cultured overnight, and 1 mL of the culture suspension was taken in a 1.5 mL centrifuge tube, centrifuged at 10,000 rpm for 2 min, and the supernatant was discarded;
  • Example 1 Tolerance of Pediococcus pentosaceus CCFM1012 to simulated gastrointestinal fluid
  • the cryopreserved Pediococcus pentosaceus CCFM1012 was streaked into MRS solid medium, and cultured in aerobic culture at a temperature of 37 ° C for 48 h, and then subcultured for 2 to 3 times with MRS medium, and then the culture medium of Pediococcus pentosaceus CCFM1012 was taken.
  • the survival rate is the ratio of the number of viable cells in the culture solution to the number of viable cells at the 0th hour, expressed in %.
  • samples were taken at 0 h, 1 h, 2 h, 3 h, and 4 h, and plate colonies were counted by pouring culture on MRS agar medium, and the viable count was determined and the survival rate was calculated.
  • the survival rate is a ratio of the number of viable cells at the time of sampling in the culture solution to the number of viable cells at the 0th hour, expressed in %.
  • the experimental results are shown in Table 1 and Table 2. It can be seen that Pediococcus pentosaceus CCFM1012 has good tolerance to artificial simulated gastric juice and intestinal fluid.
  • Example 2 Inhibition of Campylobacter jejuni growth in vitro by Pediococcus pentosaceus CCFM1012
  • Pediococcus pentosaceus CCFM1012, Pediococcus pentosus H29M-8M and L. rhamnosus LGG strains were taken out in a -80 ° C refrigerator, streaked into MRS plates, cultured at 37 ° C for 48 h, and single colonies were picked for MRS liquid.
  • Example 3 Adhesion ability of Pediococcus pentosaceus CCFM1012 to intestinal epithelial cells HT-29
  • Intestinal epithelial cell line HT-29 cells (purchased from the cell culture bank of the Chinese Academy of Sciences' Type Culture Collection Committee) were cultured using RPMI1640 medium (Gibco) (10% fetal bovine serum and 1% streptomycin added). The HT-29 cells were cultured in a cell incubator containing 5% CO 2 (37 ° C), and the culture solution was changed once every 48 hours, and culture was continued.
  • the growth was fused to 70%-80% of HT-29 cells for digestion, adjusted to a concentration of 2 ⁇ 10 5 /mL, and the sterile coverslips were placed in a 6-well cell culture plate, and 2 mL of cell culture suspension was added to each well.
  • the solution was incubated at 37 ° C in a 5% CO 2 incubator.
  • the cells were washed three times with PBS, and 1 mL of serum-free and antibiotic-containing RPMI-1640 cells containing 2 ⁇ 10 8 CFU/mL of lactic acid bacteria was added to each well.
  • the bacterial suspension was cultured, and RPMI-1640 cell culture medium (free of serum and antibiotics) was added to 2 mL, and incubated for 2 hours. After the incubation, the PBS was washed three times to remove the unadhered lactic acid bacteria, then fixed with methanol for 20 min, washed three times with PBS, and then subjected to Gram staining, and microscopically examined under a 100-fold oil microscope. The number of bacteria adhering per 100 cells was calculated by randomly selecting 20 fields of view as the adhesion index. The results of the adhesion test are shown in Table 4.
  • Pediococcus pentosaceus CCFM1012 has strong adhesion ability to intestinal epithelial cells HT-29, and the adhesion index can reach 15.7 ⁇ 2.1, and its adhesion ability is stronger than that of the control group, Pediococcus pentosus H29M-8M.
  • Lactobacillus rhamnosus LGG a strong adhesion ability of Pediococcus pentosus CCFM1012
  • Example 4 Effect of Pediococcus pentosaceus CCFM1012 on the lifespan of C. elegans infected with Campylobacter jejuni
  • Escherichia coli OP50 was inoculated in liquid medium and cultured with shaking. When the OD600 was 1.0-1.2, the bacteria were added to the NGM plate of C. elegans after shaking and mixing. The coating is uniform, cultured and stored for later use.
  • Resuscitation and synchronization of Caenorhabditis elegans freeze and thaw the nematode cryotubes, centrifuge, discard the supernatant, and pour the nematodes into the NGM plate with E. coli OP50 to resuscitate; after the nematode grows into adults, use pipetting Rinse the plate repeatedly with sterile water, transfer the liquid containing the nematode to the centrifuge tube, pipette the sterile aqueous suspension containing nematodes into a new centrifuge tube, add sodium hydroxide solution and sodium hypochlorite solution, mix thoroughly.
  • the tube was placed under a microscope until no large pieces of nematode were visible; centrifuged, washed with sterile water; resuspended in S medium, S medium containing eggs
  • the L1 stage nematode was collected by centrifugation, transferred to an NGM culture plate containing E. coli OP50, and cultured at 20 ° C for 72 hours to obtain L4 stage nematodes.
  • a The survival rate of the 13th antenna worm was calculated using the Kaplan-Meier survival model; b: DT50, the time required for nematode death to be 50%.
  • the treatment of nematodes in Table 5 was to use the sterile picker to pick up the L4 stage nematodes and transfer them to the modified NGM plate.
  • the number of nematodes contained in each plate was about 80-100, and the control group (E.coli+C.jejuni) And the intervention group (CCFM1012+C.jejuni, H29M-8M+C.jejuni and LGG+C.jejuni) were added with a final concentration of 10 8 CFU/mL E. coli OP50 and 200 ⁇ L of lactic acid bacteria suspension, respectively.
  • the nematodes were transferred to new modified NGM plates, and the corresponding bacterial suspensions were added to different groups. After three days, the intervention group stopped feeding the lactic acid bacteria.
  • the survival rate of nematodes can still reach 45.81% on the 13th day, not only that, nematode death
  • the 50% days were also extended to 14.46 days, and the prolongation effect was significantly stronger than that of Pediococcus pentrea H29M-8M and Lactobacillus rhamnosus LGG.
  • Example 5 Antagonistic effect of Pediococcus pentosaceus CCFM1012 on Campylobacter jejuni in mice infected with Campylobacter jejuni
  • C57BL/6 mice infected with Toxoplasma gondii and Campylobacter jejuni were used as experimental mice, and Campylobacter jejuni can be colonized in a large number of mice infected with Toxoplasma gondii.
  • Campylobacter jejuni gavage Take 2 generations of Campylobacter jejuni and incubate at 37 ° C for three hours, centrifuge at 6 ° C, 2800 r / min for 6 min, discard the supernatant and buffer with sterile phosphate. The cells were resuspended in a liquid to bring the concentration of Campylobacter jejuni to 3 ⁇ 10 9 CFU/mL.
  • Lactic acid bacteria gavage agent Take 2 generations of lactic acid bacteria and incubate at 37 ° C (5% O 2 , 10% CO 2 , 85% N 2 ) for 24 h, centrifuge at 3 ° C, 8000 r / min for 3 min to collect the cells, discard The supernatant was removed and the cells were resuspended in sterile phosphate buffer to a concentration of 5 x 10 9 CFU/mL.
  • Toxoplasma gondii gavage Brain tissue was collected from mice chronically infected with Toxoplasma gondii Me49, added to sterile phosphate buffer and ground thoroughly. 10 ⁇ L of brain homogenate was added dropwise to the glass slide, and counted under a light microscope, and repeated 3 times. According to the counting results, the brain homogenate concentration was adjusted, and each mouse was intragastrically administered with 200 ⁇ L of brain homogenate to make the dose of Toxoplasma gondii reached 20 capsules/only.
  • mice There were 8 mice in each experimental group, 4 mice were divided into 1 cage, and all mice were given a gastric perfusion dose of 300 ⁇ L/head.
  • the mice other than the control group were intragastrically administered with Toxoplasma gondii cysts; on days 2, 3 and 4, the animals were normally reared so that the pathogens began to destroy the mouse immune system; on the 5th and 6th day, the mice were sequentially The lactic acid bacteria and Campylobacter jejuni were intragastrically administered at a time interval of at least 1 h to avoid interaction between the gavage agents; the mice showed symptoms of Campylobacter jejuni infection on days 7, 8, 9, and 10; The mice were sacrificed and the animal experiment was over.
  • Table 6 The detailed grouping and processing methods are shown in Table 6.
  • Mouse fecal occult blood test Take a freshly collected mouse feces and use fecal occult blood test kit for fecal occult blood test.
  • the experimental method is as follows: uniformly apply the feces on the white filter paper, add 3 drops of o-toluidine solution, continue to drop 2 drops of hydrogen peroxide, and observe the color development result:
  • Detection of Viable Count of Campylobacter jejuni in Mice Take fresh feces from the mice, and accurately weigh the feces in sterile saline for 30 min to soften the feces. The fecal suspension was thoroughly mixed and diluted by gradient. 100 ⁇ L of the appropriate gradient was selected and uniformly applied to the Columbia blood plate supplemented with Campylobacter selective antibiotics. The cells were cultured in a three-gas incubator at 37 ° C for 48 h. The Campylobacter jejuni colonies were counted.
  • the paraffin section was prepared by the following steps: colon tissue was sequentially placed in 70%, 80%, 90%, 95%, 100% ethanol for 20 min for dehydration.
  • the dehydrated colon tissue was placed in a solution of ethanol to xylene ratio of 1:1 for 5 min, after which the colon was immersed twice in xylene for 15 min.
  • the wax was dipped and then sliced to a thickness of about 5 ⁇ m.
  • the HE staining procedure was as follows: dewaxing with xylene twice for 5 min each, then immersing the sections in ethanol with a volume fraction of 100%, 95%, 80%, 70% for 5 min, rinsing with running water for 5 min and staining with hematoxylin for 5 min. , ultra-pure water cleaning 2 times. Add hydrochloric acid (70% by volume of ethanol to a volume fraction of 0.5% hydrochloric acid) for 10 s, rinse with running water for 10 min, then place the sections in volume fractions of 70%, 80%, 95%. In ethanol for 5 min each.
  • Pediococcus pentosaceus CCFM1012 can effectively reduce the colonization of Campylobacter jejuni in mice.
  • the detection of Campylobacter jejuni in the feces on the third day and the fifth sky was able to reach 10 6 and 10 8 CFU/g feces, and the amount of colonization gradually increased with the passage of time;
  • the colonization of Campylobacter jejuni in the CCFM1012 intervention group was reduced to 10 4 -10 5 CFU/g feces, which was reduced by about 1.5 orders of magnitude compared with the control group; by the fifth day, due to the mice
  • the infection of Toxoplasma gondii was gradually serious, and the colonization of Campylobacter jejuni in mice was also improved accordingly.
  • FIG. 3 The results of HE staining were observed under a microscope, as shown in Fig. 3.
  • A represents a control group
  • B represents a C.j group
  • C represents a CCFM1012 group
  • D represents H29M-8M
  • E represents an LGG group. From the results, it can be seen that the intestinal epithelium and intestinal wall of the control group were intact, the cell morphology was normal, and no adverse changes were observed. Colonic lesions in mice infected with Toxoplasma gondii + Campylobacter jejuni are more serious. Typical features include disappearance of goblet cells, obvious inflammatory cell infiltration, crypt destruction, and mucosal damage. Combined with the pathological score analysis (Fig.
  • Pediococcus pentosaceus CCFM1012 decreased to 4, which greatly attenuated colonic lesions in mice.
  • the above results indicate that Pediococcus pentosaceus CCFM1012 can alleviate the damage caused by Campylobacter jejuni to the colon of mice by antagonizing Campylobacter jejuni.
  • Example 6 Effect of Pediococcus pentosaceus CCFM1012 on the transcriptional levels of virulence factors flaA, cadF, cdtB, cdtC and dnaJ of Campylobacter jejuni in mice infected with Campylobacter jejuni
  • 0.2 g of fresh colon tissue taken out after dissection of the mouse was repeatedly ground in a liquid nitrogen-added mortar (180 ° C, 4 h high temperature enzyme), and then 1 mL of Trizol reagent was added to the mortar, and the grinding was continued until the liquid was substantially clarified. After that, collect it into a 1.5 mL enzyme-free centrifuge tube, let stand for 15 min at room temperature, add 200 ⁇ L of chloroform solution to the centrifuge tube, gently shake for 15 s, let stand at room temperature for 10 min, centrifuge at 15 °C for 1 min, and centrifuge for 15 min at 12000 r/min.
  • the aqueous phase was transferred to another enzyme-free centrifuge tube and 500 ⁇ L of isopropanol was added. Mix upside down and let stand for 10 min at room temperature. After standing at the end, centrifuge at 10 °C and 12000 r/min for 10 min, discard the supernatant, leave a white precipitate formed by RNA at the bottom of the centrifuge tube, and add 1 mL of 75 prepared with DEPC water. % ethanol solution, vortex and resuspend, centrifuge at 4 ° C, 7500 r / min for 5 min, discard the supernatant, and naturally evaporate at room temperature. 30 ⁇ L of RNase free water was added to the dried RNA.
  • RNA concentration and purity were measured by Nanodrop, and the quality of the RNA was detected by agarose gel electrophoresis.
  • the extracted total RNA was used as a template, and the DNA was synthesized by reverse transcription according to the procedure of TaKaRa's PrimeScript 1st Strand cDNA Synthesis Kit kit, and stored at -20 °C.
  • the CFX96TM real-time PCR instrument performs PCR amplification and reads fluorescent signals.
  • the C.jejuni virulence gene qRT-PCR reaction system is:
  • the qRT-PCR reaction conditions of the C. jejuni virulence gene are:
  • Pediococcus pentosaceus CCFM1012 can effectively inhibit the transcriptional levels of Campylobacter jejuni flaA, cadF, cdtB, cdtC and dnaJ virulence factors in mice, and the corresponding virulence factor fold can be reduced to 0.17. 0.13, 0.51, 0.14, and 0.41 indicate that Pediococcus pentosaceus CCFM can antagonize Campylobacter jejuni infection by reducing the level of jejunal virulence factor.
  • Example 7 Producing fermented food containing the fungus using the present invention, Pediococcus pentosaceus CCFM1012
  • the juice After washing with fresh vegetables, the juice is juiced, and then sterilized by high temperature, and then heat-sterilized at a temperature of 140 ° C for 2 seconds, then immediately cooled to 37 ° C, and then connected to the fungus of Pseudomonas pentosaceus CCFM1012 prepared by the present invention.
  • the concentration was 10 6 CFU/mL or more, and it was stored at a temperature of 4 ° C in a refrigerated state, thereby obtaining a fruit and vegetable beverage containing the live bacteria of the P. pentosaceus CCFM1012 of the present invention.
  • fermented foods can be prepared by fermentation production using Pediococcus pentosaceus CCFM1012, which includes solid foods, liquid foods, and semi-solid foods.
  • the fermented food product comprises a dairy product, a soy product, a fruit and vegetable product, the dairy product comprising milk, sour cream, cheese; the fruit and vegetable product comprises cucumber, carrot, beet, celery, cabbage product.
  • the fermented food can inhibit the growth of Campylobacter jejuni, reduce the in vivo colonization rate of Campylobacter jejuni, reduce the expression level of Campylobacter jejuni virulence genes flaA, cadF, cdtB, cdtC and dnaJ, and alleviate the infection caused by Campylobacter jejuni infection. Physiological damage.
  • Example 8 Production of a feed additive containing the fungus using the present invention, Pediococcus pentosaceus CCFM1012
  • the feed additive is a liquid or powder containing Pediococcus pentosaceus CCFM1012, which is prepared by transferring the cryopreserved Pediococcus pentosaceus CCFM1012 twice in MRS liquid medium at a ratio of 2% (v/v) (each time) After 18 hours), the secondary seed liquid was obtained after activation, and the secondary seed liquid was introduced into the fermenter containing MRS medium by 5% inoculum for fermentation culture, and aerobically cultured at 37 ° C for 48 hours, and the fermentation broth was centrifuged at 8000 rpm to obtain the bacteria.
  • the feed additive of Pediococcus pentosaceus CCFM1012 can be directly added to the finished feed for consumption by poultry and livestock according to 0.5-2% (mass ratio of culture solution to feed) to reduce the infection and carrying of Campylobacter jejuni in poultry and livestock.
  • the feed raw materials are mixed and fermented for several days to prepare feed.
  • the preparation process is as follows: the pulverized wheat bran, soybean meal and rice bran are selected, and the quality is uniformly mixed with water in a ratio of 8:1:1, so that the moisture of the material reaches 35%-40%.
  • the basic protein content is about 18%.
  • the feed additive of Pediococcus pentosaceus CCFM1012 is pre-melted according to the inoculation amount of 0.5% (mass ratio) and then added to the solid material. The mixture is evenly mixed, the temperature is controlled at 37 °C, and the anaerobic culture is 72 hours. The fermented material is dried at a low temperature of not higher than 45 ° C, and then stored at 4 ° C under refrigeration.
  • the live bacteria of Pediococcus pentosaceus CCFM1012 in the feed additive is not less than 5 ⁇ 10 8 CFU/g.
  • the feed additive can reduce the infection and carrying of Campylobacter jejuni in poultry and livestock.
  • the P. stipitis CCFM1012 of the invention has good resistance to gastric acid and bile salts, has strong inhibitory effect on the growth of Campylobacter jejuni, and has good adhesion ability to intestinal epithelial cells, single bacteria It can prolong the lifespan of C. elegans after infection with Campylobacter jejuni, and can significantly reduce the colonization of Campylobacter jejuni in mice infected with Campylobacter jejuni and the transcriptional activities of the virulence genes flaA, cadF, cdtB, cdtC and dnaJ of Campylobacter jejuni. Effectively relieves physiological damage caused by Campylobacter jejuni infection.
  • the Pediococcus pentosaceus CCFM1012 of the present invention can be used for the preparation of dairy products, soy products and fruit and vegetable products having prevention of Campylobacter jejuni infection. It can also be used to prepare additives which can be added to poultry and livestock feed to reduce the infection and carrying of Campylobacter jejuni in poultry and livestock, and has a wide application prospect.

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Abstract

戊糖片球菌CCFM1012、其发酵食品及其在制备拮抗空肠弯曲杆菌感染药物中的应用。所述戊糖片球菌CCFM1012能降低小鼠体内空肠弯曲杆菌的定植率以及空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的转录活性，缓解由空肠弯曲杆菌感染造成的生理损伤，可用于制备预防空肠弯曲杆菌感染的乳制品、豆制品与果蔬制品，还可用于制备可添加在家禽、家畜饲料中的添加剂，用以降低家禽、家畜中空肠弯曲杆菌的感染及携带。

Description

戊糖片球菌CCFM1012、其发酵食品及其在制备拮抗空肠弯曲杆菌感染药物中的应用 技术领域
本发明属于微生物技术领域，具体涉及戊糖片球菌CCFM1012、其发酵食品及其在制备拮抗空肠弯曲杆菌感染药物中的应用。
背景技术
空肠弯曲杆菌(Campylobacter jejuni)为革兰氏阴性菌。空肠弯曲杆菌广泛分布于自然界，可通过动物、食物、水、牛奶等传播，能定植于各种野生动物和家禽、家畜的肠道内。接触禽畜类，食入未煮熟或受污染的鸡肉、牛肉、未彻底杀菌的牛奶以及污染的水都可以引起人类感染。近些年来，空肠弯曲杆菌感染率在世界各地普遍呈上升趋势。在一些发达国家，空肠弯曲杆菌感染引起的腹泻病例数甚至超过了沙门氏菌和志贺氏菌，成为最常见的腹泻致病菌。在发展中国家，空肠弯曲杆菌是婴幼儿感染性腹泻最常见的病原菌。人感染空肠弯曲杆菌后，最常见的会引起肠胃炎、腹泻、发烧以及腹部绞痛，免疫力低下者会进一步引起一系列的并发症，例如胆囊炎、腹膜炎、脑膜炎、败血症和骨髓炎等。空肠弯曲杆菌引起的最严重的并发症是格林-巴利综合征(Guillian-Barré Syndrome,GBS)，它会引起轴突的损伤及不可逆的神经伤害，导致呼吸肌麻痹而死亡。
目前临床治疗空肠弯曲杆菌感染的常用手段为抗生素，但抗生素的使用会带来空肠弯曲杆菌及肠道菌对抗生素的耐药性，抗生素过度使用还会导致体内抗生素的残留。从自然界中筛选出一株可食用并对空肠弯曲杆菌具有拮抗作用的菌株，有助于丰富缓解空肠弯曲杆菌感染的对策，提高治疗由空肠弯曲杆菌感染导致的疾病的效率。
发明内容
本部分的目的在于概述本发明的实施例的一些方面以及简要介绍一些较佳实施例。在本部分以及本申请的说明书摘要和发明名称中可能会做些简化或省略以避免使本部分、说明书摘要和发明名称的目的模糊，而这种简化或省略不能用于限制本发明的范围。
鉴于上述的技术缺陷，提出了本发明。
因此，作为本发明其中一个方面，本发明克服现有技术中存在的不足，提 供一种戊糖片球菌CCFM1012(Pediococcus pentosaceus)，于2018年2月11日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所，保藏编号为GDMCC No.60331。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供一种发酵食品。
为解决上述技术问题，本发明提供如下技术方案，其中：所述发酵食品为使用戊糖片球菌CCFM1012发酵生产制得，所述发酵食品包括固态食品、液态食品、半固态食品。
作为本发明所述发酵食品的一种优选方法，其中：所述发酵食品包括乳制品、豆制品、果蔬制品，所述乳制品包括牛奶、酸奶油、干酪；所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供戊糖片球菌CCFM1012在制备体内定植益生菌中的应用。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供戊糖片球菌CCFM1012在制备拮抗空肠弯曲杆菌感染药物中的应用。
作为本发明所述戊糖片球菌CCFM1012在制备体内定植益生菌中的应用的一种优选方案，其中：所述戊糖片球菌CCFM1012能够耐胃酸、耐胆盐、抑制所述空肠弯曲杆菌生长、降低空肠弯曲杆菌的体内定植量、降低空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的表达水平、缓解由空肠弯曲杆菌感染造成的生理损伤。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供权利要求2或3所述的发酵食品在制备拮抗空肠弯曲杆菌感染的功能性食品中的应用。
作为本发明所述发酵食品在制备拮抗空肠弯曲杆菌感的功能性食品中的应用的一种优选方案，其中：所述发酵食品能够抑制所述空肠弯曲杆菌生长、降低空肠弯曲杆菌的体内定植率、降低空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的表达水平、缓解由空肠弯曲杆菌感染造成的生理损伤。
作为本发明另一个方面，本发明克服现有技术中存在的不足，提供一种饲料添加剂，其中：所述饲料添加剂为含有戊糖片球菌CCFM1012的液体、粉剂或颗粒，所述饲料包括家禽、家畜的饲料。
作为本发明所述饲料添加剂在制备拮抗空肠弯曲杆菌感染药物中的应用的一种优选方案，其中：所述饲料添加剂能够降低家禽、家畜的空肠弯曲杆菌感染及携带。
为解决上述技术问题，本发明提供了如下技术方案：
本发明的有益效果：本发明戊糖片球菌CCFM1012具有良好的耐胃酸、耐胆盐特性，对空肠弯曲杆菌的生长具有很强的抑制作用，在牛津杯实验中抑菌圈的大小可达15.65±0.47(mm)，同时对肠上皮细胞具有很好的黏附能力，其粘附指数可达15.7±2.1，单菌就能够延长空肠弯曲杆菌感染后线虫的寿命，且能显著的降低空肠弯曲杆菌感染小鼠体内空肠弯曲杆菌的定植量以及空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的转录活性，有效缓解由空肠弯曲杆菌感染造成的生理损伤。
本发明所述的戊糖片球菌CCFM1012可用于制备具有预防空肠弯曲杆菌感染的乳制品、豆制品与果蔬制品。还可用于制备可添加在家禽、家畜饲料中的添加剂，用以降低家禽、家畜中空肠弯曲杆菌的感染及携带，具有非常广泛的应用前景。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。其中：
图1为本菌株对空肠弯曲杆菌感染小鼠粪便隐血的影响示意图；
图2为本菌株干预空肠弯曲杆菌感染小鼠后三天及五天体内空肠弯曲杆菌的定植量变化示意图；
图3为本菌株干预空肠弯曲杆菌感染小鼠后结肠组织的病理切片示意图；
图4为本菌株干预空肠弯曲杆菌感染小鼠后结肠组织的病理评分示意图；
图5为本菌株干预空肠弯曲杆菌感染小鼠后小鼠肠道内空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ转录活性的变化示意图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂，下面结合具体实施例对本发明的具体实施方式做详细的说明。
在下面的描述中阐述了很多具体细节以便于充分理解本发明，但是本发明还可以采用其他不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本发明内涵的情况下做类似推广，因此本发明不受下面公开的具体实施例的限制。
其次，此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例，也不是单独的或选择性的与其他实施例互相排斥的实施例。
本发明的戊糖片球菌CCFM1012(Pediococcus pentosaceus)，于2018年2月11日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所，保藏编号为GDMCC No.60331。
所述戊糖片球菌CCFM1012具有以下生物学特性：
(1)菌体特征：革兰氏阳性，细胞球状，直径0.8～1.0μm，无鞭毛，无芽孢；
(2)菌落特征：菌落乳白色，边缘整齐，球状，凸起，不透明，表面湿润光滑；
(3)生长特性：该菌株的最低生长温度为15℃，最高生长温度为45℃，在温度35-37℃下生长最佳，最适生长pH为6.5，培养18h后进入稳定期；
(4)具有良好的耐酸耐胆盐特性；
(5)体外可显著抑制空肠弯曲杆菌生长；
(6)对肠上皮细胞具有较好的粘附能力；
(7)戊糖片球菌在秀丽隐杆线虫感染模型中能显著提高感染空肠弯曲杆菌后线虫的寿命；
(8)戊糖片球菌能有效降低空肠弯曲杆菌感染小鼠体内空肠弯曲杆菌的定植量；
(9)戊糖片球菌在空肠弯曲杆菌感染小鼠体内中能显著抑制空肠弯曲杆菌flaA、cadF、cdtB、cdtC和dnaJ等毒力因子的转录活性。
所述戊糖片球菌CCFM1012的提取方法为：
(一)乳酸菌的分离筛选：
(l)收集若干份健康家鸡的粪便样本，将样品在含有山梨醇GM17培养 基中35℃富集12h；
(2)将富集样品进行梯度稀释后涂布于添加了0.02％嗅甲酚紫的GM17固体平板上，培养24-48h；
(3)选取变色圈明显，并且符合乳酸菌目基本形态的单菌落进行平板划线纯化，筛选分离出乳酸菌；
(4)将上述单菌落培养于液体GM17培养液中培养24h后进行革兰氏染色，选取革兰氏阳性菌进行后续试验。
(二)乳酸菌的初步鉴定：溶钙圈测定法
(l)将步骤(一)所筛选得到的乳酸菌在液体山梨醇GM17培养液中培养24h，然后取l mL培养物8000rpm离心2min；
(2)用0.05M KH 2PO 4溶液洗涤两次；
(3)将所得菌泥重悬，划线在山梨醇GM17-0.75％CaCO 3的固体培养基上，培养24h；
(4)选取溶钙圈明显，且呈凸面圆形、细密色白、无菌丝体的菌落，革兰氏染色后显微镜观察菌体为球状即初步判定为球菌。
(三)乳酸菌的分子生物学鉴定：
(l)单菌基因组抽提：
A.将步骤(二)所筛选得到的乳酸菌培养过夜，取培养过夜的菌悬液1mL于1.5mL离心管，10000rpm离心2min，弃上清得菌体；
B.用1mL无菌水吹洗菌体后，10000rpm离心2min，弃上清得菌体；
C.加入200μLSDS裂解液，80℃水浴30min；
D.加入酚-氯仿溶液200μL于菌体裂解液中，其中酚-氯仿溶液的组成成分及体积比为Tris饱和酚:氯仿:异戊醇＝25:24:1，颠倒混匀后，12000rpm离心5-10min，取上清200μL；
E.加入400μL冰乙醇或冰异丙醇于200uL上清中，﹣20℃静置1h，12000rpm离心5-10min，弃上清；
F.加入500μL70％(体积百分数)冰乙醇重悬沉淀，12000rpm离心1-3min，弃上清；
G.60℃烘箱烘干，或者自然晾干；
H.50μLddH 2O重溶沉淀以备PCR；
(2)16S rDNA PCR
A.细菌16S rDNA 50μLPCR反应体系：
10×Taq buffer，5μL；dNTP，5μL；27F，0.5μL；1492R，0.5μL；Taq酶，0.5μL；模板，0.5μL；ddH 2O，38μL。
B.PCR条件：
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃2min；
(3)制备1％琼脂糖凝胶，之后将PCR产物与10000×loading buffer混合，上样量5μL，120V跑30min，然后进行凝胶成像；
(4)将16S rDNA的PCR产物进行测序分析，将得到的序列结果使用BLAST在GeneBank中进行搜索和相似性比对，选取测序结果鉴定为属于戊糖片球菌的一种新发现的菌株，-80℃保藏备用。
实施例1：戊糖片球菌CCFM1012对模拟胃肠液的耐受性
将冷冻保存的戊糖片球菌CCFM1012划线接种于MRS固体培养基中，在温度37℃有氧培养48h，再经MRS培养液传代培养2～3次后，取戊糖片球菌CCFM1012培养液，8000r/min离心5min收集菌体，重悬于(1:1)pH 2.5的人工模拟胃液(含1％胃蛋白酶、pH＝2.5的MRS培养基)混合，然后在37℃下厌氧培养，分别在开始(0h)、1h、2h和3h时取样，用MRS琼脂培养基浇注培养进行平板菌落计数，测定活菌数并计算其存活率。存活率是在该培养液中的活菌数与在第0h时活菌数之比，以％表示。
取戊糖片球菌CCFM1012的培养液，8000r/min离心5min收集菌体，重悬于(1:1)人工模拟肠液(含0.3％牛胆盐、1％胰蛋白酶、pH＝8.0的MRS培养基)中，在37℃下有氧培养，分别在0h、1h、2h、3h和4h时取样，用MRS琼脂培养基浇注培养进行平板菌落计数，测定活菌数并计算其存活率。存活率是在该培养液中取样时的活菌数与在第0h时活菌数之比，以％表示。实验结果如表1、表2所示，可以看出戊糖片球菌CCFM1012对人工模拟胃液及肠液均具有良好的耐受性。
表1戊糖片球菌CCFM1012在人工模拟胃液中的耐受性
  人工模拟胃液
处理时间(h) 1 2 3
存活率(％) 95.45±2.36 88.37±4.26 82.59±3.77
表2戊糖片球菌CCFM1012在人工模拟肠液中的耐受性
Figure PCTCN2018079153-appb-000001
实施例2：戊糖片球菌CCFM1012体外抑制空肠弯曲杆菌生长
将戊糖片球菌CCFM1012、戊糖片球菌H29M-8M以及鼠李糖乳杆菌LGG菌种于-80℃冰箱取出后，划线于MRS平板中，37℃培养48h，挑取单菌落于MRS液体管中，37℃培养18h，以2％的体积量接种于新的MRS液体培养基中，于37℃培养18h，按照同样的方式再次培养一代，然后将乳酸菌菌悬液在8000r/min、4℃条件下离心8min，吸取上清液，使用0.22μm水系滤膜进行过滤除菌，得到乳酸菌发酵上清液，用1mol/L的NaOH将发酵液调节至pH＝6.5。
空肠弯曲杆菌NCTC11168菌株(购自美国典型培养物保藏中心ATCC)在两项培养基上(即布氏琼脂(青岛海博生物技术有限公司产品)与脑心浸液肉汤培养基(Oxoid公司培养基))于温度37℃，5％O 2、10％CO 2和85％N 2的三气环境下培养，按照同样的方式传代培养两代后，在2800r/min条件下离心6min，使用磷酸盐缓冲液(PBS，pH＝7.2)洗涤后，重悬于PBS至菌浓度为10 8CFU/mL。
吸取空肠弯曲杆菌菌悬液(250μL，10 8CFU/mL)，均匀涂布于哥伦比亚血琼脂平板中，待菌液自然干燥后，放置牛津杯，在牛津杯中加入100μL乳酸菌发酵上清(pH＝6.5)，于4℃扩散2h后放入三气培养箱中经37℃培养48h后测量抑菌圈的直径。以加入pH为6.5的灭菌MRS作为阴性对照，0.30mg/mL诺氟沙星广谱抗生素为阳性对照，检测抑菌圈直径(mm)。
表3抑菌圈直径的测量结果
Figure PCTCN2018079153-appb-000002
Figure PCTCN2018079153-appb-000003
抑菌效果见表3，结果表明戊糖片球菌CCFM1012发酵上清液显著抑制了空肠弯曲杆菌的生长，其抑菌圈直径可达15.65±0.47(mm)，且效果显著强于对照组戊糖片球菌H29M-8M以及鼠李糖乳杆菌LGG。
实施例3：戊糖片球菌CCFM1012对肠道上皮细胞HT-29的粘附能力
使用RPMI1640培养液(Gibco公司)(添加10％胎牛血清和1％青链霉素)培养肠上皮细胞株HT-29细胞(购自中国科学院典型培养物保藏委员会细胞库)。HT-29细胞在含5％CO 2的细胞培养箱中进行培养(37℃)，每培养48h后更换培养液1次，连续培养。
将生长融合至70％-80％的HT-29细胞进行消化，调整浓度至2×10 5个/mL，将无菌盖玻片放置在6孔细胞培养板中，每孔加入2mL细胞培养悬液，于5％CO 2培养箱中37℃培养，待细胞长至单层时，用PBS清洗三次，每孔加入1mL含2×10 8CFU/mL乳酸菌的无血清及抗生素的RPMI-1640细胞培养菌悬液，补加RPMI-1640细胞培养液(不含血清及抗生素)至2mL，孵育2h。孵育结束后PBS用清洗三次，以除去未粘附的乳酸菌，然后用甲醇固定20min，PBS清洗三次后进行革兰氏染色，于100倍油镜下镜检。随机选取20个视野计算每100个细胞所粘附的细菌数，作为粘附指数。粘附实验结果列于表4。
表4乳酸菌在HT-29细胞表面的黏附情况
Figure PCTCN2018079153-appb-000004
由表4结果可知，戊糖片球菌CCFM1012对肠道上皮细胞HT-29有较强的黏附能力，粘附指数可达15.7±2.1，其黏附能力强于对照组戊糖片球菌H29M-8M以及鼠李糖乳杆菌LGG，具有较强黏附能力的戊糖片球菌CCFM1012在肠道定植后，可以有效地防止病原微生物接触并黏附肠粘膜细胞，从而可以 预防肠道致病菌引起肠道疾病。
实施例4：戊糖片球菌CCFM1012对感染空肠弯曲杆菌的秀丽隐杆线虫的寿命的影响
大肠杆菌(Escherichia coli)OP50的制备：将大肠杆菌OP50接种于液体培养基中振荡培养，当OD600为1.0-1.2时，震荡混匀后吸取菌液滴加在秀丽隐杆线虫生长NGM平板上并涂布均匀，培养并贮存备用。
秀丽隐杆线虫的复苏及同步化：将线虫冻存管冻融，离心，弃上清，将线虫倒入长有大肠杆菌OP50的NGM平板使其复苏；待线虫长成成虫后，用移液枪吸取无菌水反复冲洗平板，并将含有线虫的液体转移至离心管中，吸取含有线虫的无菌水悬液加入新的离心管中，并加入氢氧化钠溶液和次氯酸钠溶液，充分混匀；每振荡2-3分钟后将离心管置于显微镜下观察，直到看不到大片的线虫碎片；离心，无菌水清洗；用S培养基液重悬，将含有虫卵的S培养基液培养，离心收集L1期线虫，转移到长有大肠杆菌OP50的NGM培养板中，20℃培养72h获得L4期线虫。
表5乳酸菌干预后感染空肠弯曲杆菌线虫的寿命统计分析
Figure PCTCN2018079153-appb-000005
a：用Kaplan-Meier生存模型计算第13天线虫的存活率；b：DT50，线虫死亡50％所需时间。
表5中线虫的处理方式为采用无菌挑虫器挑取L4期线虫转移至改良NGM平板中，每板中含有线虫条数约为80-100条，对照组(E.coli+C.jejuni)和干预组(CCFM1012+C.jejuni、H29M-8M+C.jejuni和LGG+C.jejuni)中分别加入终浓度为10 8CFU/mL大肠杆菌OP50和乳酸菌菌悬液200μL，此后每天将活体线虫转移至新的改良NGM平板中，不同组别分别加入对应菌悬液，持续三天后，干预组停止喂食乳酸菌，对照组和干预组中均加入10 8CFU/mL的空肠弯曲杆菌菌悬液200μL，此后每天将活体线虫转移至新的NGM平板中，各组中均加入10 8CFU/mL的空肠弯曲杆菌菌悬液200μL，空白组(E.coli)中则每天转移至新的NGM平板中并加入终浓度为10 8CFU/mL大肠杆菌OP50，记录每组线 虫死亡条数。从图中结果来看，戊糖片球菌CCFM1012能够显著延长感染空肠弯曲杆菌后线虫的寿命，CCFM1012+C.jejuni组在第13天时，线虫的存活率仍可达到45.81％，不仅如此，线虫死亡50％的天数也延长至14.46天，其延长效果显著强于戊糖片球菌H29M-8M以及鼠李糖乳杆菌LGG。
实施例5：戊糖片球菌CCFM1012在感染空肠弯曲杆菌小鼠体内对空肠弯曲杆菌的拮抗作用
本实施例采用弓形虫及空肠弯曲杆菌混合感染的C57BL/6小鼠作为实验小鼠，在弓形虫感染小鼠体内空肠弯曲杆菌可以大量定植。
小鼠灌胃剂的制备：
空肠弯曲杆菌灌胃剂：取活化2代的空肠弯曲杆菌并于37℃三气条件下培养至24h，于4℃、2800r/min离心6min收集菌体，弃去上清并用无菌磷酸盐缓冲液重悬菌体，使空肠弯曲杆菌浓度达到3×10 9CFU/mL。
乳酸菌灌胃剂：取活化2代的乳酸菌并于37℃(5％O 2，10％CO 2，85％N 2)下培养至24h，于4℃、8000r/min离心3min收集菌体，弃去上清并用无菌磷酸盐缓冲液重悬菌体，使乳酸菌浓度达到5×10 9CFU/mL。
弓形虫灌胃剂：将慢性感染弓形虫Me49的小鼠处死后取脑组织，加入无菌磷酸盐缓冲液并研磨充分。滴加10μL脑匀浆于载玻片上，于光镜下计数，重复3次。根据计数结果调整脑匀浆浓度，每只小鼠灌胃200μL脑匀浆液，使弓形虫灌胃剂量达到20个包囊/只。
小鼠分组及处理：
每个实验组有8只小鼠，4只小鼠分为1笼，所有小鼠灌胃剂剂量为300μL/只。动物实验第1天，对除对照组以外的小鼠灌胃弓形虫包囊病原；第2、3、4天正常饲养以便病原开始破坏小鼠免疫系统；第5、6天每天对小鼠依次灌胃乳酸菌和空肠弯曲杆菌，两次灌胃时间间隔至少1h，以避免灌胃剂之间相互影响；第7、8、9、10天小鼠表现出空肠弯曲杆菌感染症状；第11天将小鼠处死，动物实验结束。详细分组及处理方法见表6。
表6实验小鼠分组及处理方法
Figure PCTCN2018079153-appb-000006
Figure PCTCN2018079153-appb-000007
小鼠粪便隐血检测：取1颗新鲜收集的小鼠粪便，使用粪尿隐血试剂盒进行粪便隐血检测。实验方法如下：将粪便均匀涂布于白色滤纸上，滴加邻联甲苯胺溶液3滴，继续滴加过氧化氢2滴，观察显色结果：
3min后不显蓝绿色，结果为阴性，评分为0；
30s-60s内显蓝色，结果为弱阳性，评分为1；
立即显蓝绿色，结果为阳性，评分为2；
立即显深蓝色，结果为强阳性，评分为3。
小鼠粪便空肠弯曲杆菌活菌数检测：取小鼠新鲜粪便，精确称重后将粪便在无菌生理盐水中浸泡30min以软化粪便。将粪便悬液充分混匀并梯度稀释，选取合适梯度的稀释液100μL，均匀涂布于添加了弯曲杆菌选择性抗生素的哥伦比亚血平板，置于三气培养箱37℃培养48h，对平板上长出的空肠弯曲杆菌菌落进行计数。
小鼠结肠石蜡切片制作、HE染色及病理学评分：将浸泡于质量分数为4％的多聚甲醛中的结肠组织取出，制作石蜡切片并进行HE染色。
石蜡切片制作步骤如下：依次将结肠组织放入体积分数为70％、80％、90％、95％、100％的乙醇中各20min用以脱水。将脱水后的结肠组织放入乙醇与二甲苯比例为1:1的溶液中5min，之后将结肠两次浸入二甲苯中15min。浸蜡，之后进行切片，厚度约5μm。
HE染色步骤如下：使用二甲苯脱蜡2次，每次持续5min，然后将切片浸入体积分数为100％、95％、80％、70％的乙醇中各5min，使用流水冲洗5min并用苏木素染色5min，超纯水清洗2次。放入盐酸乙醇(体积分数为70％的乙醇中加入体积分数为0.5％的盐酸)中分色10s，使用流水冲洗10min，然后将切片依次放入体积分数为70％、80％、95％的乙醇中各5min。使用伊红乙醇(体积分数为95％的乙醇中加入体积分数为0.5％的伊红)染色5min，然后依次放入体积分数为95％、100％的乙醇中各5min，用二甲苯透明2次，最后使用中性树胶封固。
显微镜下盲法阅片并进行病理学评分，评分标准见下表：
表7结肠组织病理学评分标准
Figure PCTCN2018079153-appb-000008
Figure PCTCN2018079153-appb-000009
收集小鼠粪便，采用粪便隐血试剂盒评估其隐血情况，结果见图1。结果显示，弓形虫+空肠弯曲杆菌复合感染小鼠粪便隐血情况严重，而戊糖片球菌CCFM1012干预之后，显著减轻了小鼠粪便隐血症状，其粪便隐血评分分值下降至2.15，与戊糖片球菌H29M-8M和鼠李糖乳杆菌LGG相比，戊糖片球菌CCFM1012发挥了更加积极的缓解作用。
如图2所示，戊糖片球菌CCFM1012能有效降低空肠弯曲杆菌在小鼠体内的定植量。C.j组中，第三天以及第五天空肠弯曲杆菌在体内粪便中的检出量能够达到10 6以及10 8CFU/g feces，且随着时间的推移，定植量逐渐升高；戊糖片球菌CCFM1012干预组在第三天时，空肠弯曲杆菌定植量可以减少到10 4-10 5CFU/g feces，与对照组相比，下降了约有1.5个数量级；到第五天时，由于小鼠受弓形虫感染逐步严重，空肠弯曲杆菌在小鼠体内的定植也相应的得到了提高，但戊糖片球菌CCFM1012干预后依然表现出良好的清除能力，其空肠弯曲杆菌定植量可以减少到10 6CFU/g feces，下降了约有两个数量级，戊糖片球菌CCFM1012在小鼠体内对空肠弯曲杆菌的清除率远高于戊糖片球菌H29M-8M以及鼠李糖乳杆菌LGG的处理组别。
显微镜下观察HE染色结果，见图3，图中：A代表对照组，B代表C.j组，C代表CCFM1012组，D代表H29M-8M，E代表LGG组。从结果中可以看出对照组小鼠肠上皮以及肠壁结构完整，细胞形态正常，未见不良变化。弓形虫+空肠弯曲杆菌复合感染的小鼠结肠病变较为严重，典型特征包括杯状细胞消失、明显 的炎性细胞浸润、隐窝破坏、粘膜损伤等。结合病理学评分分析(图-4)，戊糖片球菌CCFM1012组分值下降到4，很大程度缓解了小鼠结肠病变。以上结果表示戊糖片球菌CCFM1012对空肠弯曲杆菌发挥拮抗作用后可以减轻空肠弯曲杆菌对小鼠结肠造成的损伤。
实施例6：戊糖片球菌CCFM1012对感染空肠弯曲杆菌小鼠体内空肠弯曲杆菌毒力因子flaA、cadF、cdtB、cdtC和dnaJ转录水平的影响
小鼠感染空肠弯曲杆菌模型的建立及处理方式见实施例5。
空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ q RT-PCR引物设计与合成按表8所示
表8空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ及内参基因的q PCR引物
Figure PCTCN2018079153-appb-000010
结肠组织及空肠弯曲杆菌总RNA的提取和cDNA的合成：
将小鼠解剖后取出的新鲜结肠组织0.2g在加有液氮的研钵(180℃，4h高温灭酶)中反复研磨，再向研钵中加入1mL Trizol试剂，继续研磨，待液体基本澄清后，收集至1.5mL无酶离心管中，室温静置15min，向离心管中加入200μL三氯甲烷溶液，轻摇15s，室温静置10min，4℃、12000r/min离心15min， 取600μL上层无色水相至另一只无酶离心管中，加入500μL异丙醇。上下颠倒混匀，室温下静置10min，静置结束后，4℃、12000r/min离心10min，弃去上清，留下RNA在离心管底部形成的白色沉淀，加入1mL用DEPC水配制的75％的乙醇溶液，漩涡震荡重悬，4℃、7500r/min离心5min，弃去上清，室温自然挥发干燥。向干燥的RNA中加入30μL RNase free water，待RNA溶解后，以Nanodrop测定RNA浓度及纯度，并通过琼脂糖凝胶电泳检测RNA的质量。以提取的总RNA为模板，根据TaKaRa公司的PrimeScript 1st Strand cDNA Synthesis Kit试剂盒说明书操作步骤逆转录合成c DNA，-20℃下保存。
qRT-PCR反应体系及条件：
用Bio-Rad
Figure PCTCN2018079153-appb-000011
CFX96TM实时荧光定量PCR仪进行PCR扩增，并读取荧光信号。
C.jejuni毒力基因qRT-PCR反应体系为：
Figure PCTCN2018079153-appb-000012
C.jejuni毒力基因qRT-PCR反应条件为：
95℃预变性30s，95℃变性5s，60℃退火30s(40个循环)。
结果如图5所示，戊糖片球菌CCFM1012能够有效抑制空肠弯曲杆菌flaA、cadF、cdtB、cdtC和dnaJ毒力因子在小鼠体内的转录水平，其相对应毒力因子倍数可下降至0.17、0.13、0.51、0.14、0.41，表明戊糖片球菌CCFM能够通过降低空肠弯曲杆菌毒力因子的水平实现对空肠弯曲杆菌感染的拮抗作用。
实施例7：利用本发明戊糖片球菌CCFM1012制造含该菌的发酵食品
选用新鲜蔬菜洗净后榨汁，接着进行高温瞬间灭菌，在温度140℃下高温热杀菌2秒后，立即降温至37℃，再接入本发明制备的戊糖片球菌CCFM1012菌剂发酵剂，使其浓度达到10 6CFU/mL以上，在温度4℃下冷藏保存，于是得到含有本发明戊糖片球菌CCFM1012活菌的果蔬饮料。
利用本发明能够使用戊糖片球菌CCFM1012发酵生产制备其他发酵食品，所述发酵食品包括固态食品、液态食品、半固态食品。所述发酵食品包括乳制品、豆制品、果蔬制品，所述乳制品包括牛奶、酸奶油、干酪；所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
所述发酵食品能够抑制所述空肠弯曲杆菌生长、降低空肠弯曲杆菌的体内定植率、降低空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的表达水平、缓解由空肠弯曲杆菌感染造成的生理损伤。
实施例8：：利用本发明戊糖片球菌CCFM1012制造含该菌的饲料添加剂
饲料添加剂为含有戊糖片球菌CCFM1012的液体或粉剂，其制备工艺为：将冷冻保存的戊糖片球菌CCFM1012按2％(v/v)比例在MRS液体培养基中转接两次(每次18小时)活化后得到二级种子液，将二级种子液按5％接种量接入含MRS培养基的发酵罐中进行发酵培养，在37℃有氧培养48h，将发酵液8000rpm离心得到菌泥，加入菌泥质量90％的水、40％的蔗糖、50％的麦芽糊精、50％的乳清粉，常温静置30分钟后，再与含有40％菌泥质量的羧甲基纤维素钠、8倍菌泥质量的微晶纤维素、10倍菌泥质量的海藻酸钠、10倍菌泥质量的氯化钙以及20倍菌泥质量水的溶液混合，然后进行湿法制粒、并在37℃条件下旋流干燥，得到含戊糖片球菌CCFM1012饲料添加剂成品。成品中活菌数在1×10 10CFU/g以上。戊糖片球菌CCFM1012饲料添加剂可按0.5-2％(培养液与饲料的质量比)直接搅拌加入成品饲料中供家禽、家畜食用，以降低家禽、家畜的空肠弯曲杆菌感染及携带，也可与饲料原始物料混匀发酵数天制备成饲料，制备工艺为：选用粉碎后的麦麸、豆粕和米糠，质量按8：1：1的比例加水混合均匀，使物料水分达到35％-40％，基础蛋白含量在18％左右，将戊糖片球菌CCFM1012的饲料添加剂按0.5％(质量比)接种量预溶化后接入固态物料中，混合均匀，温度控制在37℃左右，厌氧培养72h后，将发酵好的物料在不高于45℃的温度下进行低温烘干，完成后于4℃下冷藏密封保存。饲料添加剂中戊糖片球菌CCFM1012的活菌数不低于5×10 8CFU/g。所述饲料添加剂能够降低家禽、家畜的空肠弯曲杆菌感染及携带。
本发明的有益效果：本发明戊糖片球菌CCFM1012具有良好的耐胃酸耐胆盐特性，对空肠弯曲杆菌的生长具有很强的抑制作用，同时对肠上皮细胞具有很好的黏附能力，单菌就能够延长空肠弯曲杆菌感染后线虫的寿命，且能显著的降低空肠弯曲杆菌感染小鼠体内空肠弯曲杆菌的定植量以及空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的转录活性，有效缓解由空肠弯曲杆菌感染造成的生理损伤。
本发明所述的戊糖片球菌CCFM1012可用于制备具有预防空肠弯曲杆菌 感染的乳制品、豆制品与果蔬制品。还可用于制备可添加在家禽、家畜饲料中的添加剂，用以降低家禽、家畜中空肠弯曲杆菌的感染及携带，具有非常广泛的应用前景。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。

Claims (10)

  1. 一种戊糖片球菌CCFM1012(Pediococcus pentosaceus)，于2018年2月11日保藏于广东省微生物菌种保藏中心，保藏地址为广州市先烈中路100号大院59号楼5楼广东省微生物研究所，保藏编号为GDMCC No.60331。
  2. 一种发酵食品，其特征在于：所述发酵食品为使用戊糖片球菌CCFM1012发酵生产制得，所述发酵食品包括固态食品、液态食品、半固态食品。
  3. 如权利要求2所述的发酵食品，其特征在于：所述发酵食品包括乳制品、豆制品、果蔬制品，所述乳制品包括牛奶、酸奶油、干酪；所述果蔬制品包括黄瓜、胡萝卜、甜菜、芹菜、圆白菜制品。
  4. 戊糖片球菌CCFM1012在制备体内定植益生菌中的应用。
  5. 戊糖片球菌CCFM1012在制备拮抗空肠弯曲杆菌感染药物中的应用。
  6. 如权利要求5所述的应用，其特征在于：所述戊糖片球菌CCFM1012能够耐耐胃酸、耐胆盐、抑制所述空肠弯曲杆菌生长、降低空肠弯曲杆菌的体内定植量、降低空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的表达水平、缓解由空肠弯曲杆菌感染造成的生理损伤。
  7. 权利要求2或3所述的发酵食品在制备拮抗空肠弯曲杆菌感染的功能性食品中的应用。
  8. 如权利要求7所述的应用，其特征在于：所述发酵食品能够抑制所述空肠弯曲杆菌生长、降低空肠弯曲杆菌的体内定植量、降低空肠弯曲杆菌毒力基因flaA、cadF、cdtB、cdtC和dnaJ的表达水平、缓解由空肠弯曲杆菌感染造成的生理损伤。
  9. 一种饲料添加剂，其特征在于：所述饲料添加剂包括含有戊糖片球菌CCFM1012的液体、粉剂或颗粒，所述饲料包括家禽、家畜的饲料。
  10. 权利要求9所述的饲料添加剂在制备拮抗空肠弯曲杆菌感染药物中的应用，其特征在于：所述饲料添加剂能够降低家禽、家畜的空肠弯曲杆菌感染及携带。
PCT/CN2018/079153 2018-03-15 2018-03-15 戊糖片球菌ccfm1012、其发酵食品及其在制备拮抗空肠弯曲杆菌感染药物中的应用 WO2019174002A1 (zh)

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