CN109022313A - One lactobacillus plantarum - Google Patents
One lactobacillus plantarum Download PDFInfo
- Publication number
- CN109022313A CN109022313A CN201810870915.6A CN201810870915A CN109022313A CN 109022313 A CN109022313 A CN 109022313A CN 201810870915 A CN201810870915 A CN 201810870915A CN 109022313 A CN109022313 A CN 109022313A
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- CN
- China
- Prior art keywords
- fbl
- lactobacillus plantarum
- bacterium
- herbivore
- strain
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- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 43
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 43
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- 244000005700 microbiome Species 0.000 claims abstract description 5
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- 229960001231 choline Drugs 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 235000013406 prebiotics Nutrition 0.000 description 1
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- 238000011160 research Methods 0.000 description 1
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- 235000021391 short chain fatty acids Nutrition 0.000 description 1
- 150000004666 short chain fatty acids Chemical class 0.000 description 1
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- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 238000012549 training Methods 0.000 description 1
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Animal Husbandry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Fodder In General (AREA)
Abstract
The present invention discloses one plant of bacterial strain that can prevent and treat cattle and sheep diarrhoeal diseases.Lactobacillus plantarum of the invention, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on April 2nd, 2018, and deposit number is CGMCC NO:15546.Lactobacillus plantarum of the invention can be in the application during herbivore is raised, can also be in the application in the feed addictive of preparation herbivore, can also be in the application in the feed of preparation herbivore.Lactobacillus plantarum strain of the invention, have it is very strong stick and bacteriostasis, have good acidproof, resistance to gastrointestinal tract and bile tolerance ability, can effectively preventing cattle and sheep diarrhoeal diseases.
Description
Technical field
The present invention relates to the bacterial strains that one plant can prevent and treat cattle and sheep diarrhoeal diseases.
Background technique
Probiotics is defined as microorganism living, when taking in enough quantity, can give host health effect.These energy
The probiotics Pseudomonas of the specified genus at place beneficial to health has: lactobacillus, Bifidobacterium, saccharomyces, enterococcus spp, micrococcus
Belong to, Leuconostoc, bacillus etc..The probiotics studied and be most widely used at present is lactobacillus
(Lactobacillus) and Bifidobacterium (Bifidobacterium).Wherein it is present in body enteron aisle and wide in food
The lactobacillus species of general addition mainly have: Lactobacillus crispatus (Lactobacillus crispatus), lactobacillus gasseri
(Lactobacillus gassed) and lactobacillus plantarum (Lactobacillus plantarum).According to national " food safety
Method " and its implementing regulations pertinent regulations, health ministry, which is defended, to be done supervision (2010) No. 65 files of hair and defines and " can be used for food
Strain list ", wherein lactobacillus has 14 kinds, and lactobacillus plantarum is arranging.Lactic acid bacteria during the fermentation, can not only make body
In the substances such as lactose, protein be converted to lactic acid, galactolipin, amino acid and small molecule peptide chain etc. easily by human consumption and absorption
Small-molecule substance, it is also metabolizable to generate the substances such as a variety of B family vitamins that body needs and provide nutrition for body.Therefore, exist
Lactic acid bacteria is added in feed can meet the needs of body is to all kinds of nutriments well, greatly improve body to nutriment example
Such as the absorption and use efficiency of calcium substance, the lactic acid bacteria taken in feed may act on body inner wall of intestine formed profitable strain and
Biological barrier, and by generating the substances such as a large amount of short chain fatty acids (lactic acid, acetic acid etc.), hydrogen peroxide, bacteriocin or polypeptide,
To play biological containment effect, the invasion of pathogenic microorganism are prevented, good body intestinal microecology balance system is formed.
In addition, lactic acid bacteria somatic antigen and metabolin activate immunocompetent cell by stimulation intestinal mucosa lymph node, to generate special
Property antibody and sensitized lymphocyte, thus the reaction of controlling inflammation property enteropathy, Reduce allergy, the enhancing innate immunity.Edible addition benefit
The feed of raw bacterium also to abdominal distension, the diarrhea that can solve beef cattle, yak and sheep the problems such as.
With residual of the antibiotic in livestock products to the harm of human health, excessive excretion to the pollution of environment, long-term
Using leading to problems such as the generation of antibody-resistant bacterium increasingly be valued by the people, many countries have been prevented or restricted from antibiosis at present
Use of the element in feed, nonreactive subtract anti-animal products and will become Developing mainstream, and current probiotics preparation is as safe, green
The feed addictive of color causes great concern.Exploitation has strong biocidal property, adhesion, strong acidproof, resistance to gastrointestinal tract and resistance to gallbladder
Salt ability, and the great application value of probiotics strain that animal immunizing power can be enhanced, and the lactic acid bacteria that prebiotic performance is fermented with it
Type is closely related, therefore it is most crucial as probiotic feed additive for obtaining the good lactic acid bacteria culturers for having exceptional function
Technology.
To solve the above problems, the feed addictive for finding a kind of safe green is the work side of staff in the industry
To.
Summary of the invention
The present invention provides a lactobacillus plantarum.
A lactobacillus plantarum of the invention is preserved in Chinese microorganism strain preservation conservator on April 2nd, 2018
Meeting common micro-organisms center, deposit number are CGMCC NO:15546.
Lactobacillus plantarum of the invention can be in the application during herbivore is raised, can also be in the feed of preparation herbivore
Application in additive, can also be in the application in the feed of preparation herbivore.
Lactobacillus plantarum strain of the invention, have it is very strong stick and bacteriostasis, have good acidproof, resistance to gastrointestinal tract
And bile tolerance ability, it can effectively preventing cattle and sheep diarrhoeal diseases.
Detailed description of the invention
The colonial morphology figure of Fig. 1 FBL-3a.
The Gram's staining figure of Fig. 2 FBL-3a.
The pcr amplified fragment map of Fig. 3 FBL-3a.
The growth and fermentation pH curve graph of Fig. 4 FBL-3a.
The influence that Fig. 5 temperature grows bacterium FBL-3a.
Fig. 6 bacterial strain FBL-3a adhesion.
Fig. 7 lactobacillus plantarum type strain CICC6009 adhesion.
Specific embodiment
It is to elaborate the solution of the present invention in conjunction with attached drawing below.
1. the separation of bacterial strain
Lactobacillus plantarum FBL-3a (Lactobacillus plantarum FBL-3a) of the present invention is from Xinjiang beef cattle excrement
Just separation obtains.Strain isolation purification process of the invention is: 1mL is drawn from the yak excrement dilution of Xinjiang, it is dilute respectively
Release 10-4, 10-5, 10-6Power respectively takes 0.1mL to be coated on the MRS plate of calcic, and after liquid to be diluted is absorbed by plate, 37 DEG C are detested
Oxygen culture for 24 hours, picks out single colonie 12 with the molten doubtful lactic acid bacteria of calcium circle, purifying of crossing on MRS solid plate respectively
Culture 3 times respectively chooses a single bacterium and falls in MRS fluid nutrient medium 37 DEG C of stationary cultures for 24 hours, take zymocyte liquid carry out resistance and
External probiotic properties test, finally picks out that 1 plant of adhesion is strong and biocidal property is good, there is good acidproof, resistance to gastrointestinal tract and resistance to gallbladder
The bacterial strain of salt ability saves and is named as FBL-3a.It is submitted on April 2nd, 2018 and is preserved in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, deposit number are CGMCC NO:15546.
The formula of the aforementioned culture medium used of the present invention is as follows:
(1) MRS culture medium (1L): peptone 10.0g；Beef extract powder 10.0g；Yeast extract 5.0g；Glucose 20.0g；
Tween 80 1.0mL；K2HPO4·3H2O 2.0g；Anhydrous sodium acetate 5.0g；Triammonium citrate 2.0g；MgSO4·7H2O
0.29g；MnSO4·H2O 0.058g.(the above ingredient is added in distilled water, constant volume to 1000mL, is dissolved by heating, adjust pH
It is 6.3,121 DEG C of high pressure sterilization 20min after packing.(note: solid medium adds 1.5% agar powder on this basis)
(2) LB culture medium (1L): tryptone 10g；Sodium chloride 10g；Yeast extract 5g, agar 20g, PH are adjusted to 7.5.
2. the identification of strain
The morphologic observation of 2.1 strains
Colony morphological observation is carried out, referring to Fig. 1 to bacterium FBL-3a, Gram's staining, electron microscope are carried out to bacterial strain
Observe thalli morphology referring to fig. 2.The main morphological features of bacterium FBL-3a are as follows: bacterium colony circle, smooth, in the MRS culture medium of calcic
Upper is in milk yellow, and opaque, periphery of bacterial colonies has transparent molten calcium circle.Gram's staining display is positive, and thallus is rod-shaped, wide: 0.5-
It is 0.6 μm, long: 1.2-3.0 μm.
The identification of physiological and biochemical property:
Bacterium FBL-3a physiological and biochemical property is shown in Table 1, specifically: gelatin experiment and indoles experiment display are negative, how to test-manufacture ammonia
Experiment is the positive, and VP, MR, Starch Hydrolysis, hydrogen peroxide experiment display are negative, illustrates that the bacterium can decompose and utilizes the nitrogenous object of macromolecular
Breakdown of glucose cannot be generated pyruvic acid by matter, and cannot generate amylase for Starch Hydrolysis is glucide, cannot be utilized bright
Glue.Bacterium FBL-3a, which can ferment, simultaneously produces acid using alpha-lactose, D-Fructose, maltose, glucose, sucrose, does not produce gas.
The physiological and biochemical property of 1 bacterium FBL-3a of table
Note: physiologic character: ("+" indicates positive；"-" indicates negative)；Carbohydrate fermentation experimental result record sheet: ("+,+"
It indicates to produce acid, produces gas；"-,-" expression does not produce acid, does not produce gas.)
The molecular biology identification of 2.2 bacterium FBL-3a
1. the extraction of bacterial genomes DNA is extracted using the bacterial genomes DNA extraction kit of Takara company, extract
DNA EP pipe is sub-packed in afterwards to freeze in -20 DEG C.2. the PCR amplification of 16S rDNA sequence, it is about 1500bp that it is long, which to obtain target fragment,
(Fig. 3).Nanjing Genscript Biotechnology Co., Ltd. is entrusted to complete sequencing the product containing target fragment, what is obtained actually has
Effect nucleotides sequence be classified as 1425bp, in NCBI data to each 16S rRNA sequence carry out Blast compare analysis, obtain sequence with
The sequence homology of Lactobacillus plantarum is greater than 99%.FBL-3a gene order is submitted in ncbi database,
The application of the bacterium number of logining is carried out, the accession number applied is MH517566.
The 16SrDNA gene order of bacterium FBL-3a: the 16SrDNA gene order of lactobacillus plantarum FBL-3a is SEQ ID
NO.6, the homology analysis in morphologic observation and bio-chemical characteristics in conjunction with bacterium FBL-3a and phylogenetic tree, determines bacterium
FBL-3a is lactobacillus plantarum (Lactobacillus pLantarum) strain.
3. the Microbiological Characteristics research of lactobacillus plantarum FBL-3a
The preparation of 3.1 lactobacillus plantarum FBL-3a mother liquors
The lactobacillus plantarum FBL-3a for taking 100 μ L, 30% glycerol to save is inoculated in the MRS fluid nutrient medium of 10mL, and 37
For 24 hours, it is spare that strain mother liquor is made in DEG C stationary culture.
3.2 lactobacillus plantarum FBL-3a growth and fermentation PH curve determination, take lactobacillus plantarum FBL-3a mother liquor in 3.1
(similarly hereinafter) each 100 μ L is inoculated in 57 pipes equipped in the test tube of 10mL MRS fluid nutrient medium respectively, and 37 DEG C of standing for fermentation are cultivated, with
It does not connect the fluid nutrient medium of bacterium as control, takes 3 pipes to measure thallus OD in the different time sections of 0-96 (h) after fermentation respectively
(600nm) value and pH value, the results showed that (Fig. 4) enters logarithmic growth phase when bacterium FBL-3a fermentation is to 10h, when fermentation is to 18h
Into stationary phase, thallus OD (600nm) value is 3.09 ± 0.02 at this time.PH has been lowered to 4.17 when bacterium fermentation is to stationary phase, and
Stationary phase pH stablizes 3.66 or so.
The measurement of the most suitable fermentation temperature of 3.3 lactobacillus plantarum FBL-3a
Each 100 μ L of lactobacillus plantarum FBL-3a mother liquor is taken to be inoculated in the test tube equipped with 10mL MRS fluid nutrient medium respectively
In, 20,25,30,35,40,45,50 (DEG C) standing for fermentation cultures for 24 hours, measure fermentation liquid OD600nm) value, the results showed that (figure
5), bacterium FBL-3a optimum growth temperature is 35 DEG C, and thallus OD (600nm) value is 3.66 ± 0.02；Bacterium is at 40 DEG C, OD
(600nm) value still reachable 3.02 ± 0.02, OD (600nm) value of bacterium drops to 0.80 ± 0.01 in base when fermenting to 50 DEG C
This not growth conditions, illustrates that the bacterium is suitble to 20-40 DEG C of growth temperature.
The measurement of the most suitable fermentation pH value of 3.4 lactobacillus plantarum FBL-3a
Each 100 μ L of lactobacillus plantarum FBL-3a mother liquor is taken to be inoculated in the MRS liquid training of the different pH value equipped with 10mL respectively
37 DEG C of standing for fermentation cultures in the test tube of base are supported for 24 hours, to measure fermentation liquid OD600nm value, the results are shown in Table 2, lactobacillus plantarum FBL-
The most suitable fermentation pH value of 3a is 6.5, and it is 3.81 ± 0.02 that thallus OD (600nm) value for 24 hours is cultivated under this pH value, when pH value is less than 1.0
Or when being greater than 9.0, OD (600nm) value of bacterium is below 0.1, in not growth conditions, illustrates that bacterium is suitble to the pH value range of growth
Between 3.0-8.0.PH value range 4.0-7.5 MRS culture medium through lactobacillus plantarum FBL-3a fermentation after, under bacterium solution pH value
It drops between 3.2-3.8.Illustrate that lactobacillus plantarum FBL-3a has very strong production acid and acid-fast ability.
The measurement of the most suitable fermentation pH value of 2 bacterium FBL-3a of table
4. the probiotic properties of lactobacillus plantarum FBL-3a are studied
4.1FBL-3a studies gastrointestinal toleration
The thallus being incubated overnight is collected, is resuspended with sterile PBS, its concentration is adjusted to 1x109CFU/mL, and put
Enter 37 DEG C of cultures 1,2,3h in simulate the gastric juice (pH3.0), it is separately sampled.It after 3h, places into simulated intestinal fluid, 1mL culture
Add 9mL simulated intestinal fluid (pH8.0), after 37 DEG C of cultures 2,4,8h, separately sampled and use colony counting method measures total viable count, knot
Fruit is shown in Table 3, after handling 3h and simulated intestinal fluid processing 8h by simulate the gastric juice, the motility rate of bacterium FBL-3a still 90% or more,
Know that the bacterium can be resistant to gastrointestinal tract environment well.
The detection of 3 bacterium FBL-3a gastrointestinal toleration of table
The bile tolerance of 4.2 bacterium FBL-3a bacterial strains is tested
Take FBL-3a bacterium solution with 2% inoculum concentration be inoculated in containing different cholate (concentration gradients 0,0.1%, 0.2%,
0.3%, 0.6%) MRS fluid nutrient medium (PH6.3) is placed at 37 DEG C and cultivates 0,2,4h respectively, 3 repetitions of each processing.
1mL culture is respectively taken to mix in 9mL physiological saline, preparation 10-4、10-5、10-6A dilution solution, take 0.1mL dilution in
It is coated on MRS solid plate, Anaerobic culturel records for 24 hours (each dilution does 3 in parallel) in 37 DEG C of carbon dioxide incubators
Clump count in calculate flat board.It the results are shown in Table 4, the increment of bacterium FBL-3a still reaches after gallbladder salinity is 0.3% processing 4h
To (3.13 ± 0.28) × 107(CFU/mL), it is known that bacterium FBL-3a has good resistance to choline ability.
The detection of 4 bacterium FBL-3a bile tolerance ability of table
The acid resistance test of 4.3 bacterium FBL-3a bacterial strains
Take bacterium FBL-3a mother liquor by 100 μ L inoculation strain in different pH value (pH gradient 1.0,1.5,2.0,2.5,3.0)
10mL MRS fluid nutrient medium, be placed at 37 DEG C and cultivate 0,2,4h respectively, 3 repetitions of each processing.Respectively take 1mL sample bacterium solution
It is mixed in 9mL PBS, preparation 10-4、10-5、10-6A dilution solution, takes 0.1mL dilution to apply on MRS solid plate
Cloth is inverted the bacterium colony cultivated in (each dilution does 3 in parallel) record calculate flat board for 24 hours in 37 DEG C of carbon dioxide incubators
Number.The results are shown in Table 5, bacterium FBL-3a is not grown substantially when pH value is 1.0 and 1.5, but at 2.0 or more, cultivate 2h and
There is growth after 4h, is 2 in pH, after cultivating 4h, bacterium colony can reach (3.77 ± 0.25) × 107(CFU/mL), well-grown is said
The bright bacterium has very strong acid-fast ability, between 2.0-3.0, cultivates the bacterium colony of 4h less than 2h, illustrates within the scope of this pH,
With the extension of processing time, the growth of bacterium is suppressed.
The detection of 5 bacterium FBL-3a acid-fast ability of table
The surface nature of 4.4 bacterium FBL-3a measures
It is incubated overnight lactobacillus plantarum FBL-3a (MRS fluid nutrient medium), (the LB liquid of staphylococcus aureus ATCC 6538
Body culture medium) 18h obtains bacterium solution, and it is respectively placed in 5000r/min, is centrifuged 10min at 4 DEG C, thallus is collected, respectively with pH's 7.2
Sterile phosphate buffer (PBS) washs 2 times and (PBS is added in thallus, piping and druming after mixing, sets 5000r/min, 4 DEG C
Lower centrifugation 10min collects thallus).From agglutination rate (%): lactobacillus plantarum FBL-3a being formed in wavelength with sterile PBS
The bacteria suspension that light absorption value at 600nm is 0.5 ± 0.02 (A0), stands and measures light absorption value A24 afterwards for 24 hours, from agglutination rate (%) public affairs
Formula is (A0-A24)/A0 × 100%；His agglutination rate (%): with sterile PBS by FBL-3a and staphylococcus aureus ATCC
6538 outstanding bacterium solution is adjusted to the mix suspending bacterium solution that the light absorption value at wavelength 600nm is 0.5 ± 0.02 (A0).It stands for 24 hours
Light absorption value A24 is measured afterwards, his agglutination rate (%) formula is (A0-A24)/A0 × 100%；Hydrophobicity (%): will with sterile PBS
Bacterium FBL-3a is formed in the bacteria suspension that the light absorption value at wavelength 600nm is 0.5 ± 0.02 (A0), and dimethylbenzene is added to bacteria suspension
In, at room temperature by two-phase system vortex oscillation 120s after preculture 10min, 37 DEG C of standings co-culture 1h, then small
The heart removes dimethylbenzene phase, and the light absorption value A1 of water phase is measured at 600nm.Hydrophobicity (%): formula be (A0-A1)/A0 ×
100%.Measurement result is shown in Table 6, it is known that FBL-3a is 77.43% from agglutination rate, his agglutination rate is 25.24%, and hydrophobicity is
48.28%, illustrate that lactobacillus plantarum FBL-3a can assemble with pathogenic bacteria (staphylococcus aureus), and hydrophobic performance is good, it can
Increase test basis to stick experiment.
6 bacterial strain FBL-3a surface nature table of table
| Agglutination test | A0±s | A24±s | Agglutination rate (%) |
| Self-solidifying test | 0.506±0.027 | 0.115±0.042 | 77.43% |
| He is solidifying to test | 0.511±0.014 | 0.382±0.035 | 25.24% |
| Hydrophobicity tests | A0±s | A1±s | Hydrophobicity (%) |
| 0.495±0.028 | 0.256±0.036 | 48.28% |
The Adhering capacity of 4.5 bacterium FBL-3a measures
Activated lactobacillus plantarum FBL-3a is inoculated in MRS fluid nutrient medium with 2% inoculum concentration, 37 DEG C of cultures
18h, thalline were collected by centrifugation (8000r/min, 4 DEG C, 10min) is then washed 2 times with sterile PBS (pH7.2), and thallus is resuspended in
In Caco-2 cell culture fluid, cell concentration is adjusted to 1 × 10 with culture solution8CFU/mL.The Caco-2 of single layer will have been grown up to
Cell is rinsed 2 times with sterile PBS (p H 7.2), and every hole is added in the cell culture fluid of 200 μ L mycetomes.Set 37 DEG C, 5%
CO2, relative humidity 95% carbon dioxide incubator in be incubated for 2h, with sterile PBS (p H 7.2) rinse cell 3 times, with remove
The bacterium that do not stick.The fixed 10min of anhydrous methanol, the dyeing of Ji's nurse Sa, oil mirror microscopy are added into 24 orifice plates.Every hole random counter
30 visuals field, by counting the lactobacillus number being attached on individual cells, to judge the size of Adhering capacity, with lactobacillus plantarum
Type strain CICC6009 is control.Stick and the results are shown in Table 7, lactobacillus plantarum FBL-3a is reached with colon cancer cell Caco-2 adherence rate
To 2375 ± 112.4CFU/30cell, and check plant lactobacillus type strain CICC6009 and colon cancer cell Caco-2 sticks
Rate only reaches 359 ± 45.2CFU/30
Cell, then explanation has very strong Adhering capacity from separating plant lactobacillus FBL-3a.
7 bacterial strain FBL-3a Adhering capacity table of table
Application study of the 5 bacterium FBL-3a in cattle and sheep diarrhoeal diseases
Lactobacillus plantarum FBL-3a is mixed in a certain ratio with laboratory from the another three probiotics strain separated, is made micro-
Ecological agent.Feeding experiment is carried out to Zhangye, total 40 sheep, 80 oxen of the different degrees of diarrhea case in southern Jiangsu.The result shows that raising
Second day observation ill domestic animal is fed, discovery symptom of diarrhea takes an evident turn for the better, and after continuous feeding three days, the ill domestic animal that symptoms are mild is fullyed recover from an illness substantially
More, the more serious ill domestic animal of symptom is also almost recovered after a week in feeding.And have 1 ox of serious symptoms, more days before feeding
Hematochezia is not fed, and the state of an illness is excessively serious, fails to cure.But on the whole, curative effect is very significant, feeds by one week,
The symptom of diarrhea of 40 sheep improves, and cure rate reaches 97.5%, and phenomena such as Relapse rate does not occur in later observation, and 80
The symptom of diarrhea of head ox also improves, but has the seriously ill ox in both ends to fail to cure, but cure rate still reaches 98.75%.
Claims (4)
1. a lactobacillus plantarum is preserved in China Committee for Culture Collection of Microorganisms's commonly micro- life on April 2nd, 2018
Object center, deposit number are CGMCC NO:15546.
2. application of the lactobacillus plantarum described in claim 1 in herbivore is raised.
3. application of the lactobacillus plantarum described in claim 1 in the feed addictive of preparation herbivore.
4. application of the lactobacillus plantarum described in claim 1 in the feed of preparation herbivore.
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