CN106434411B - A kind of giant panda source lactobacillus plantarum and application - Google Patents

A kind of giant panda source lactobacillus plantarum and application Download PDF

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CN106434411B
CN106434411B CN201610430331.8A CN201610430331A CN106434411B CN 106434411 B CN106434411 B CN 106434411B CN 201610430331 A CN201610430331 A CN 201610430331A CN 106434411 B CN106434411 B CN 106434411B
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lactobacillus plantarum
group
giant panda
infection
etec
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CN106434411A (en
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曾东
王强
彭芝榕
倪学勤
牛李丽
潘康成
景波
刘倩
赖婧
周毅
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CHENGDU ZOO
Sichuan Agricultural University
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CHENGDU ZOO
Sichuan Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Abstract

This application discloses a kind of giant panda source lactobacillus plantarum and application, which was preserved in China typical culture collection center on May 04th, 2016, and deposit number is CCTCC NO:M2016245.Cent lymphocytes reduction, the neutrophil leucocyte percentage raising that serum IgA, IgM, IgG immune globulin antibody are horizontal, and Experimental Enterotoxigenic Escherichia coli Infection is inhibited to induce can be improved in giant panda source provided by the invention lactobacillus plantarum；It is significant to lower inflammation-related factor IL-1 β, IL-8, IL-6, TLR4 and MyD88mRNA expression, alleviate enteron aisle acute inflammatory reaction caused by enterotoxigenic escherichia coli.

Description

A kind of giant panda source lactobacillus plantarum and application
Technical field
The application belongs to field of biotechnology, specifically, being related to a kind of giant panda source lactobacillus plantarum and application.
Background technique
In in the past few decades, people are dedicated to giant panda nutrition and the improvement of reproduction technique, have ignored infectiousness stomach The prevention and control of intestines problem.Enterotoxigenic escherichia coli (Enterotoxigenic Escherichia coli, ETEC) It is one of Captive Giant Panda enterogastric diseases encountered pathogenic bacteria, the microbial traveller, livestock and poultry and developing country baby point Secreting property bacterial diarrhea morbidity and mortality with higher.Currently, ETEC infection and its control of induced diarrhea are very big Antibiotic is depended in degree, however the appearance of antibody-resistant bacterium and multi-drug resistant has potential threaten to public health.It seeks Ask economically practical antibiotics substitute such as organic acid, probiotics or prebiotics for controlling alimentary infection disease Disease is current research hotspot.
Probiotics is microorganism living (usually bacterium or yeast), is played when ingested in sufficient quantity host health beneficial Effect.Lactic acid bacteria is one of the prebiotic strain being most widely used, because of its long fermentation starter status and its fermented product It is extensive consumption and be typically considered safe.From Mei Qinikefu (Elie Metchnikoff) proposition in 1908, fermentation Yoghourt helps to maintain intestinal flora stable state, since the hypothesis for inhibiting corruption.More and more researches show that probiotic lactobacillus can To improve gut barrier function, maintain commensal gut microbial flora balance, enhance immunity of organism by reducing pathogen field planting The effects of power, mechanism resisted the infection of part gastrointestinal tract pathogen, including salmonella, C.perfringens, Listeria and cause Rushing down property Escherichia coli.However, probiotics adherency has host species specificity, may only be played using heterologous probiotics of short duration Immanoprotection action.Giant panda is confined to external strain as the distinctive rareness species in imminent danger of China, the exploitation of dedicated probiotics Biological feature study.
Summary of the invention
In view of this, the application is directed to above-mentioned problem, a kind of giant panda source lactobacillus plantarum and application are provided.
In order to solve the above-mentioned technical problem, this application discloses a kind of giant panda source lactobacillus plantarum, which is big Panda source lactobacillus plantarum BSGP201683, Lactobacillus plantarum, the bacterial strain were protected on May 04th, 2016 It is hidden in China typical culture collection center, deposit number is CCTCC NO:M2016245.
Disclosed herein as well is above-mentioned giant panda source lactobacillus plantarums in the pre- of giant panda transmissible gastroenteric tract disease Application in anti-and control.
Further, transmissible gastroenteric tract disease is enteron aisle acute inflammation.
Disclosed herein as well is above-mentioned giant panda source lactobacillus plantarums to alleviate in enteron aisle acute inflammation drug in preparation Application.
Further, enteron aisle acute inflammation is as caused by enterotoxigenic escherichia coli.
Further, giant panda source lactobacillus plantarum be resistant to pH2.0 3 hours, 2% Pig cholate 4 hours.
Further, giant panda source lactobacillus plantarum is by improving serum IgA, IgM, IgG immune globulin antibody water It is flat, inhibit the cent lymphocytes of Experimental Enterotoxigenic Escherichia coli Infection induction to reduce, neutrophil leucocyte percentage raising；Significantly Inflammation-related factor IL-1 β, IL-8, IL-6, TLR4 and MyD88mRNA expression are lowered, it is big further to alleviate production enterotoxin Enteron aisle acute inflammatory reaction caused by enterobacteria.
Compared with prior art, the application can be obtained including following technical effect:
1) present invention provides 1 plant of isolated lactobacillus plantarum from healthy giant panda excrement sample, deposit number: CCTCC No.2016245。
2) giant panda source lactobacillus plantarum BSGP201683 provided by the invention can tolerate pH2.0 3 hours, 2% pig gall Salt 4 hours, pathogen enterotoxigenic escherichia coli, salmonella and the bacteriostatic diameter of staphylococcus aureus are reached respectively 22.48mm, 20.10mm and 17.90mm.
3) giant panda source provided by the invention lactobacillus plantarum can improve intestinal tight connection correlative protein expression, protection The intestinal tract injury of Experimental Enterotoxigenic Escherichia coli Infection induction.
4) serum IgA, IgM, IgG immune globulin antibody can be improved in giant panda source provided by the invention lactobacillus plantarum Level inhibits the cent lymphocytes of Experimental Enterotoxigenic Escherichia coli Infection induction to reduce, neutrophil leucocyte percentage raising；It is aobvious It writes and lowers inflammation-related factor IL-1 β, IL-8, IL-6, TLR4 and MyD88mRNA expression, alleviate and produce enterotoxin large intestine bar Microbial enteron aisle acute inflammatory reaction.
5) giant panda source provided by the invention lactobacillus plantarum can increase colon Bacillus acidi lactici, bifidobacteria, drop Low bacteroid, enterobacteria abundance, and significantly reduce Experimental Enterotoxigenic Escherichia coli Infection model colon enterobacteria and intolerant to warmheartedness poison Plain gene content.
Certainly, implement any product of the application it is not absolutely required to and meanwhile reach all the above technical effect.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present application, constitutes part of this application, this Shen Illustrative embodiments and their description please are not constituted an undue limitation on the present application for explaining the application.In the accompanying drawings:
Fig. 1 is influence of the application various dose lactobacillus plantarum to weight before and after mouse infection；
Fig. 2 is the application various dose lactobacillus plantarum to blood leucocyte sum before and after mouse infection and the shadow of classification It rings；
Fig. 3 is the application various dose lactobacillus plantarum to Serum DAO activity and D-ALPHA-Hydroxypropionic acid content before and after mouse infection Influence；
Fig. 4 be the application various dose lactobacillus plantarum to serum IgA before and after mouse infection, IgM, IgG content shadow It rings；
Fig. 5 is that the application various dose Bacillus acidi lactici closely connects GAP-associated protein GAP mRNA expression to jejunum before and after mouse infection Horizontal influence；
Fig. 6 is the application various dose lactobacillus plantarum to jejunum cell factor before and after mouse infection and Toll-like receptor The influence of mRNA expression；
Fig. 7 is influence of the application various dose lactobacillus plantarum to colonic microflora composition before and after mouse infection；
Fig. 8 is the application ETEC infection each test group mouse Colon heat-labile toxin gene content for 24 hours.
Specific embodiment
Presently filed embodiment is described in detail below in conjunction with embodiment, whereby to the application how application technology hand Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
The separation and identification of 1 bacterium of embodiment
One, the separation of bacterium
Excrement sample 10g inside fresh giant panda excrement ball is weighed, is added equipped with 90mL sterile saline triangular flask (band glass Pearl), oscillation mixes 20min, and 10 times are serially diluted to 10-3Sample liquid.Take 100 μ L10-3Dilution sample liquid even spread MRS plate, After being placed in 37 DEG C of 24~48h of constant incubator Anaerobic culturel, the doubtful lactic acid bacteria single bacterium colony of oese picking is anti-on MRS plate Multiple scribing line purifying, until microscopy is without miscellaneous bacteria.
Two, it identifies
(1) (generation is insulted with " lactic acid bacteria separates identification and experimental method " according to " Berger bacterial identification manual " (the 8th edition) Text, east show pearl etc. are write, Beijing: China Light Industry Press, 1999.3) method described in, to bacterial strain BSGP201683 into Row morphological feature, cultural character and physio-biochemical characteristics identification, concrete outcome are as follows:
The form and physio-biochemical characteristics of thallus:
In MRS culture medium, 37 DEG C are cultivated 2 days, and thallus is rod-shaped, and diameter is about 3.0-8.0 μm of 0.9-1.2 μ m；Bacterium colony milk White, round, protrusion, neat in edge.
Physiological and biochemical property:
Catalase :-；Gelatin hydrolysis :-；Nitrate reduction :-；Aesculin hydrolysis :+；Tween80 hydrolysis :-；Indoles Experiment :-；Urase :-；H2S is generated :-；VP experiment :-；Anaerobic growth :+；6.5%NaCl:+；15 DEG C :+；45 DEG C :+；PH 4.5: +；Fructose :+；Glucose :+；Lactose :+；Cellobiose :+；Aesculin :+；Maltose :+；Mannitol :+；Gluconate :+； Sucrose :-；Sorbierite :-；Arabinose :-；Xylose :-.
(2) 16S rDNA is tested
The total DNA for extracting BSGP201683 utilizes bacterial 16 S rDNA universal primer (27F, 1492R) using it as template PCR amplification is carried out, the amplified production that length is about 1.5kb is obtained, amplified production is recycled and is sequenced, the sequence measured is such as Shown in SEQ ID No.1.Compared according to Gen-Bank sequence homology, bacterial strain BSGP201683 and Lactobacillus Plantarum KLAB10 (GenBank accession number KM497503.1) homology is 99%, determines the bacterium for lactobacillus plantarum (Lactobacillus plantarum)。
Based on features above, bacterial strain BSGP201683 is accredited as Lactobacillus plantarum.The bacterial strain in Being preserved in China typical culture collection center on May 4th, 2016, (abbreviation CCTCC NO:M2016245, address: Hubei Province is military The Wuchang District Han Shi Bayi Road 299, Wuhan University's collection, postcode 430072), deposit number is CCTCC NO:M2016245.
Three, acid resistance is tested
After bacterial strain BSGP201683 inoculation MRS fluid nutrient medium is incubated overnight, adjustment cell concentration to 108CFU/mL, It is seeded to the MRS fluid nutrient medium of pH 1.0, pH 2.0 and pH 3.0,37 DEG C of constant temperature incubation 3h under aseptic condition, and carries out dilute It releases plate count and calculates viable count, be reference with 0 hour viable count.
0 hour viable count of bacterial strain BSGP201683 is 6.67 × 108CFU/mL, 3 is small in pH3.0MRS fluid nutrient medium When after viable count be 2.33 × 108CFU/mL；After being cultivated 3 hours in pH2.0MRS fluid nutrient medium viable count be 3.33 × 107CFU/mL；Viable count is 0CFU/mL after cultivating 3 hours in pH1.0MRS fluid nutrient medium.
Four, bile tolerance performance test
After bacterial strain BSGP201683 inoculation MRS fluid nutrient medium is incubated overnight, adjustment cell concentration to 108CFU/mL, 0.3%, 1.0%, 2.0% Pig cholate MRS fluid nutrient medium of addition is seeded under aseptic condition, and (% is quality volume hundred Divide than (g/100mL)), 37 DEG C of constant temperature incubation 4h, and carry out dilution plate and count calculating viable count, it is ginseng with 0 hour viable count It examines.
0 hour viable count of bacterial strain BSGP201683 is 4.67 × 108CFU/mL is trained containing 0.3% Pig cholate MRS liquid Supporting viable count after cultivating 4 hours in base is 4.33 × 107CFU/mL；Small containing culture 4 in 1% Pig cholate MRS fluid nutrient medium When after viable count be 1.20 × 107CFU/mL；It is being containing viable count after being cultivated 4 hours in 2% Pig cholate MRS fluid nutrient medium 1.23×106CFU/mL。
Five, pathogen antagonistic effect
Using Odontothrips loti detection bacterial strain BSGP201683 to the antagonistic activity of pathogen.Bacterial strain BSGP201683 is inoculated with MRS broth bouillon, for 24 hours in 37 DEG C of cultures.Culture bacterium solution is taken, 15,600 × g is centrifuged 10min and obtains cell-free supernatants.It will Enterotoxigenic escherichia coli, salmonella, staphylococcus aureus are inoculated in 10mL nutrient broth medium, 37 DEG C of cultures respectively After 18h, it is diluted to 107It is spare that CFU/mL makees indicator bacteria.100 μ L are taken to indicate the sterile LB solid medium of bacterium solution even spread, to After it is absorbed, it is disposed vertically diameter 8mm Oxford cup, pressing lightly on Oxford cup makes it combine closely with agar, draws 200 μ L separation Bacterial strain cell-free supernatants are into Oxford cup, 37 DEG C of culture 12h, measure antibacterial circle diameter.
Bacterial strain BSGP201683 is 22.48mm to the antibacterial circle diameter of enterotoxigenic escherichia coli, the suppression to salmonella Bacterium loop diameter is 20.10mm, and the antibacterial circle diameter to staphylococcus aureus is 17.90mm.
2 lactobacillus plantarum BSGP201683 of embodiment influences the response of coli-infection mouse immune and intestinal flora
1.1 animal packets and processing
90 4 week old Female ICR mices (16 ± 2g) of regular grade, adaptive feeding after a week, are randomly divided into 5 groups of (n= 18): blank group, Experimental Enterotoxigenic Escherichia coli Infection group, low dosage Bacillus acidi lactici group, middle dosage Bacillus acidi lactici group and high dose cream Acidfast bacilli group, experimental period 21d.1-14d is tested, blank control group and Experimental Enterotoxigenic Escherichia coli Infection group mouse feed daily The sterile PBS of 200 μ L, basic, normal, high dosage Bacillus acidi lactici group mouse then feed 200 μ L 5.0 × 10 for every respectively daily8CFU/ mL、5.0×109CFU/mL and 5.0 × 1010CFU/mL L.plantarum BSGP201683 bacteria suspension.Test 15d, blank Group still feeds sterile blank group and still feeds sterile PBS, remaining test group mouse every feeds the production intestines of 200 μ LPBS preparation Toxin Escherichia coli bacteria suspension (1.0 × 109CFU/mL).Mouse is with room sub-cage rearing, and 22 ± 2 DEG C of room temperature, natural lighting is free Feeding and drinking-water.
1.2 clinical observation
During test, mice clinical symptoms are observed and recorded daily, and every group randomly selects 6 mouse and weigh on an empty stomach, for uniting Meter analysis mouse weight situation of change.
1.3 blood leucocytes sum and its classification and Detection
In ETEC infect 0h, for 24 hours, 144h, every group randomly selects 6 mouse orbit veniplexes and takes a blood sample 200 μ L, using Pu Kang PE-6800VETThree points of groups automatic animal Blood cell analyzer detection blood middle leukocytes sum, lymphocyte, intermediate cell and in Property granulocyte percentage.
1.4 serum diamine oxidase activity, antibody and D-ALPHA-Hydroxypropionic acid content detection
In ETEC infect 0h, for 24 hours, 144h, every group randomly selects the blood sampling of 6 eyeball of mouse, and 4 DEG C of all blood samples solidifications are high 4 DEG C of 3000 × g of refrigerated centrifuge are spent, 10min is centrifuged, careful separation serum is living for measuring diamine oxidase in serum (DAO) Property, D-ALPHA-Hydroxypropionic acid, IgA, IgM, IgG content, all detection methods are carried out according to kit specification.
1.5 jejunal tissue cell factors and Toll-like receptor detection
In ETEC infect 0h, for 24 hours, 144h, every group randomly selects 6 mouse, and dislocation is put to death, clip jejunum intestinal segment 6cm, in advance Cold PBS (PH ≈ 7.4) washes off intestinal tube content, is put into liquid nitrogen flash freezer rapidly after masking foil package, rear to take out in -80 DEG C of ice Case saves.Sample total serum IgE is extracted using RNAiso plus kit, concrete operation step is said referring to RNAiso plus kit Bright book.It usesUltramicron nucleic acid-protein analyzer and 1% agarose gel electrophoresis detect total serum IgE Purity and integrality.Satisfactory RNA sample is usedRT reagent Kit With GDNAEraser kit reverse transcription freezes at cDNA in -80 DEG C.
Using cDNA as template, using SYBR Premix Ex Taq II (Tli RNaseH Plus) dye method, in CFX ConnectTMEach gene (table 1) mRNA expression is detected on real-time PCR.25 μ L reaction systems include: 1 μ L upper and lower Primer, 2 μ L DNA profilings, ddH2O 8.5 μ L, 12.5 μ L SYBR Premix Ex TaqTMII.PCR response procedures are as follows: 95 DEG C, 5min；95 DEG C of 15s, Tm 30s, 72 DEG C of 30s, 35 circulations.Using β-actin as reference gene, using 2-ΔΔCTMethod calculates purpose Gene relative expression quantity.Destination gene expression amount (- Δ Δ CT)=(C i.e. to be measuredT, target geneOne CT, β-actin)Test groupOne (CT, target gene One CT, β-actin)Control group。
1 relative fluorescence quantitative detection primer of table
1.6 colon fecal like bacteria fluorogenic quantitative detections
In ETEC infect 0h, for 24 hours, 144h, collect colonic contents.It usesKit mentions Take bacteria total DNA.The total DNA extracted is usedUltramicron nucleic acid-protein analyzer and 1% agarose Its quality of detected through gel electrophoresis, sets -20 DEG C and saves backup.Using SYBR Premix Ex TaqTMII kit and Bio-Rad CFX96TMReal-time PCR Detection System carries out absolute fluorescence quantitative PCR detection to each DNA sample.25 μ L are anti- Answering system includes: the 1 upper and lower primer of μ L, 2 μ L DNA profilings, ddH2O 8.5 μ L, 12.5 μ L SYBR Premix Ex TaqTMII。 PCR response procedures are as follows: 95 DEG C, 5min；95 DEG C of 15s, Tm 30s, 72 DEG C of 30s, 35 circulations.It is absolute based on 16S rDNA gene Total bacterium in quantitative analysis sample, heavy wall bacillus, bacteroid, Bacillus acidi lactici, Bifidobacterium, streptococcus, enterococcus, enterobacteria and not The content (table 2) of heat-stable toxin gene LT.
2 bacterium absolute fluorescence quantitative detection primer of table
1.7 statistical procedures
Significance analysis is carried out using SPSS 19.0, different disposal comparison among groups use one-way analysis of variance, all numbers According toIt indicates, is as a result significant difference with P < 0.05.
2 results and analysis
2.1 clinical observations and changes of weight
It takes orally during feeding lactobacillus plantarum 14d, mouse does not show any abnormalities or the phenomena of mortality；After infecting ETEC, sense It is depressed to contaminate mouse spirit, feeding is reduced, and only infected group has 1 mouse mild diarrhea occur, remaining test group mouse has no diarrhea Symptom.Mouse weight during test is recorded and analyzed, the result is shown in Figure 1.Before infection, Bacillus acidi lactici group mouse weight is compared with blank Control group mice increased, but without significant difference (P > 0.05).After ETEC infection for 24 hours, infecting mouse weight is decreased obviously. During infection, infected group mouse is slow compared with mouse growth is uninfected by, but not significant (the P > of each test group changes of weight difference 0.05)。
2.2 blood leucocytes sum and Classification Change
Using the variation of total white blood cells and its distribution in automatic Hematometer monitoring peripheral blood in mice, Fig. 2 is as a result seen.Sense Contaminate ETEC 0h, each test group Mouse Peripheral Blood Lymphocyte percentage, intermediate cell percentage, neutrophil leucocyte percentage and Total white blood cells are without significant difference (P > 0.05).Infect ETEC for 24 hours, compared with blank group, infected group and Bacillus acidi lactici group Cent lymphocytes significantly reduce (P < 0.05), and neutrophil leucocyte percentage significantly increases (P < 0.05), and Bacillus acidi lactici Group and infected group are without significant difference (P > 0.05).Infect ETEC 144h, peripheral blood lymphocytes percentage between each test group, in Between cell percentages, neutrophil leucocyte percentage and total white blood cells without significant difference (P > 0.05).
2.3 Serum DAO activity and D-ALPHA-Hydroxypropionic acid changes of contents
ETEC 0h is infected, each test group Serum DAO activity and D-ALPHA-Hydroxypropionic acid content are without significant difference (P > 0.05).Infection For 24 hours, infected group DAO activity and D lactic acid content are all remarkably higher than blank group, Bacillus acidi lactici middle dose group (P < 0.05) to ETEC, But with Bacillus acidi lactici high dose group without significant difference (P > 0.05).Infect ETEC 144h, each test group Serum DAO activity and D- Lactic acid content without significant difference (P > 0.05), is shown in Fig. 3.
The variation of 2.4 serum IgAs, IgM, IgG content
Fig. 4 is shown in the variation of mice serum antibody level during test.ETEC 0h is infected, in Bacillus acidi lactici, low dosage serum IgA, middle and high dosage serum IgM and high dose serum IgG content dramatically increase (P < 0.05) compared with blank group.Infect ETEC For 24 hours, Bacillus acidi lactici group serum IgA content is significantly higher than blank group (P < 0.05), but with not significant (the P > of infected group difference 0.05).ETEC 144h is infected, serum IgA, IgM, IgG content are without significant difference (P > 0.05) between each test group.
2.5 jejunums closely connect the variation of GAP-associated protein GAP mRNA expression
Jejunum tight junction protein (Claudin-1, Occludin and ZO-1) mRNA expression is shown in Fig. 5.Infection ETEC0h, Bacillus acidi lactici middle dose group jejunum Occludin and ZO-1mRNA expression are significantly higher than blank group (P < 0.05). Infect ETEC for 24 hours, decreasing trend is presented in three kinds of tight junction protein expressions, wherein infected group Claudin-1 and The expression of Occludin mRNA is substantially less than blank group (P < 0.05), and Bacillus acidi lactici low dosage, middle dose group and sky Bai Zuwu significant difference (P > 0.05).Infect ETEC 144h, Claudin-1, Occludin and ZO-1mRNA between each test group Expression is without significant difference (P > 0.05).
2.6 jejunal tissue cell factors and the variation of Toll-like receptor mRNA expression
Fig. 6 is shown in infection front and back jejunal tissue cell factor and the variation of Toll-like receptor expression.ETEC 0h is infected, only Bacillus acidi lactici high dose group IL-1 β and IL-8mRNA expression is significantly higher than blank group (P < 0.05), remaining detecting factor exists Without significant difference (P > 0.05) between each test group.Infect ETEC for 24 hours, infected group IL-1 β, IL-8, IL-6, TLR4 and MyD88mRNA expression is significantly higher than in blank group and Bacillus acidi lactici, low dose group (P < 0.05), and the high agent of Bacillus acidi lactici Amount group IL-8 expression and infected group are without significant difference (P > 0.05).Infect ETEC 144h, infected group IL-1 β mRNA expression Level is significantly higher than blank group and each dosage group of Bacillus acidi lactici (P < 0.05).
The composition variation of 2.7 colonic microfloras
Each test group colonic microflora testing result in ETEC infection front and back is as shown in Figure 7.ETEC 0h is infected, in Bacillus acidi lactici Dosage group colon Bacillus acidi lactici and bifidobacteria dramatically increase (P < 0.05) compared with blank group, and bacteroid, enterobacteriaceae number Amount significantly reduces (P < 0.05).Infect ETEC for 24 hours, infected group enterobacteria quantity is significantly higher than each dosage group of Bacillus acidi lactici (P < 0.05).ETEC 144h is infected, institute's detection bacterium is without significant difference (P > 0.05) between each test group.
The variation of 2.8 colon heat-labile toxin gene contents
ETEC infect for 24 hours, infected group colonic contents heat-labile toxin LT gene copy number be significantly higher than blank group and Each dosage group of Bacillus acidi lactici (P < 0.05), and without significant difference (P > 0.05) (Fig. 8) between each dosage group.ETEC infect 0h and Heat-labile toxin LT gene is not detected in 144h, each test group mouse.
3 discuss
In this test, most Escherichia coli CVCC196 (LT+STa-STb+) infecting mouse do not occur apparent diarrhea Or it is dead, only show the symptoms such as weight loss and slow growth.Its reason may be experimental animal sensibility and infection strain Caused by Virulence Difference.So far, probiotics be used to promote domestic animal growth 30 years existing.The monitoring of this test mice changes of weight The results show that not observing the significant growth promoting function of lactobacillus plantarum either before infection or after infecting.Equally, Supplement lactobacillus plantarum has no significant effect mouse weight.These results indicate that different lactic acid bacillus mycopremnas are to animal Growth promoting function effect is different.
In this test, feed in, low dosage lactobacillus plantarum significantly suppress ETEC infect caused by Serum DAO activity It is increased with D-ALPHA-Hydroxypropionic acid content.In this test, jejunal tissue transmembrane protein Claudin-1 and Occludin mRNA expression is aobvious It writes and lowers, show that ETEC infection destroys intestinal tight connection.The display of this test result, it is significant to feed various dose Bacillus acidi lactici Infection preceding serum IgA, IgG and IgM content are improved, but is not made significant difference to serum immunoglobulin level after infection；This examination It tests and only total IgG, IgA and IgM in serum is detected, can lactobacillus plantarum BSGP201683 improve serum or enteron aisle The anti-ETEC specific antibody level of mucous membrane needs further to be studied.
This test also finds the great expression of ETEC infection induced jejunal tissue pro-inflammatory cytokine and Toll-like receptor, Including IL-1 β, IL-6, IL-8, MCP-1, MyD88 and TLR4.In this test, the downward of TLR4 and MyD88mRNA expression With the reduction of pro-inflammatory cytokine expression.Feeding low, middle dosage plant bacillus significantly reduces infection jejunal tissue for 24 hours IL-1 β, IL-6 and IL-8mRNA expression, and high dose group infection for 24 hours IL-8mRNA expression compared with infected group without significant Difference；ETEC infects 144h, and only infected group IL-1 β is significantly higher than blank group and Bacillus acidi lactici dosage group.These results indicate that raising In feeding, low dosage lactobacillus plantarum BSGP201683 can be relieved enteron aisle acute inflammatory reaction caused by ETEC infects.
It is right although feeding high dose Bacillus acidi lactici significantly improves healthy mice serum IgG antibody levels in this test The adjustment effect of enteron aisle acute inflammatory reaction caused by ETEC infects be inferior in, low dosage.Its reason may be high dose lactic acid The intake of bacillus is exempted from infection pre-induction the overexpression of proinflammatory factor (such as IL-8, L-1 β) to upset intestinal mucosa Epidemic disease function.
The display of this test result, mouse Colon Bacillus acidi lactici and bifid can be increased by feeding lactobacillus plantarum BSGP201683 The abundance of bacillus reduces bacteroid and enterobacteria quantity.The reduction and weight of enteron aisle gramnegative bacterium especially bacteroid Phenotype is closely related.The reduction of colon bacteroid quantity may accelerate organism metabolism, to promote the weight gain of mouse.This In test result, feeds lactobacillus plantarum BSGP201683 and significantly reduce colon enterobacteria and LT virulence factor content, it can Its protective effect to ETEC infection can inherently be explained.
4, brief summary
1) feed various dose giant panda source lactobacillus plantarum BSGP201683 can improve to some extent serum IgA, IgG, IgM antibody are horizontal, increase colon Bacillus acidi lactici, bifidobacteria, reduce enterobacteria, bacteroid quantity, but only high dose Amount significantly improves jejunal tissue IL-1 β and IL-8mRNA expression.
2) though mouse infection enterotoxigenic escherichia coli CVCC196 does not occur obvious diarrhea, show weight loss, The symptoms such as slow growth.Feeding low, middle dosage giant panda source Bacillus acidi lactici, to can inhibit periphery hemolymph caused by ETEC infects thin Born of the same parents' percentage reduces and neutrophil leucocyte percentage increases；Inhibit the decline of close connection GAP-associated protein GAP mRNA expression, drop Low blood plasma DOA activity and D-ALPHA-Hydroxypropionic acid content, so that intestinal mucosa caused by pathogenic bacterial infection be hindered to damage；Improve enterobacteriaceae group At reduction colon enterobacteria and heat-labile toxin gene content；Proinflammatory regulatory factor expression is lowered, alleviates ETEC infection and causes Acute intestinal inflammation reaction, and in, low dosage lactobacillus plantarum BSGP201683 is to Experimental Enterotoxigenic Escherichia coli Infection Intervention effect is better than high dose.
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention In the protection scope that benefit requires.

Claims (3)

1. a kind of giant panda source lactobacillus plantarum, which is characterized in that the bacterial strain was preserved on 05 04th, 2016 China typical culture collection center, deposit number are CCTCC NO:M2016245.
2. giant panda source lactobacillus plantarum described in claim 1 alleviates answering in enteron aisle acute inflammation drug in preparation With.
3. the application according to claim 2, which is characterized in that the enteron aisle acute inflammation is by production enterotoxin large intestine bar It is microbial.
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AR115757A1 (en) * 2018-07-13 2021-02-24 Cj Cheiljedang Corp COMPOSITION INCLUDING LACTOBACILLUS PLANTARUM STRAIN CJLP475 AND LACTOBACILLUS PLANTARUM STRAIN CJLP17 AND THE USE OF THE SAME
AR115758A1 (en) * 2018-07-13 2021-02-24 Cj Cheildang Corp COMPOSITION INCLUDING THE LACTOBACILLUS PLANTARUM STRAIN CJLP475 AND THE LACTOBACILLUS PLANTARUM STRAIN CJLP243 AND USE OF THE SAME
CN110317743A (en) * 2019-03-27 2019-10-11 四川农业大学 One plant of giant panda source fusion Wei Si Salmonella and its application
CN111484958B (en) * 2020-01-08 2021-12-10 博智农贸易(深圳)有限公司 Lactobacillus plantarum with ETEC (ethylene-tetra-ethyl-carbonate) inhibition effect, fermentation product and application thereof
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CN114196575B (en) * 2021-12-02 2022-07-22 四川农业大学 Panda source streptococcus paris and application thereof
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