CN103525719A - Probiotic separated from intestinal tracts and application thereof - Google Patents
Probiotic separated from intestinal tracts and application thereof Download PDFInfo
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- CN103525719A CN103525719A CN201310403662.9A CN201310403662A CN103525719A CN 103525719 A CN103525719 A CN 103525719A CN 201310403662 A CN201310403662 A CN 201310403662A CN 103525719 A CN103525719 A CN 103525719A
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Abstract
The invention relates to the field of microbial disease control and in particular relates to a probiotic separated from intestinal tracts of turbots and an application thereof. The probiotic is Leclercia adecarboxylata P115, is collected in China General Microbiological Culture Collection Center (CGMCC) on June 24, 2013, and has a collection number of CGMCCNo.7813. The probiotic is separated from the intestinal tracts of healthy turbots. The probiotic and the pure culture thereof have obvious bacteriostatic effects on tritirachium album, klebsiella pneumoniae, staphylococcus aureus, vibrio parahaemolyticus, vibrio harveyi and vibrio anguillarum. The pure culture or composition of the probiotic can be used for control of aquaculture diseases and development of novel antibacterial materials, and has good application prospects.
Description
Technical field
The present invention relates to belong to microorganism disease control field, probiotic bacterium and the application thereof of a kind of separation specifically in enteron aisle.
Background technology
Probiotic bacterium (Probiotics) is the living microorganism that a class is useful to host, be to be colonizated in human body or animal intestinal, reproductive system, thereby can produce the active beneficial microorganism general name that definite health efficacy improves host's microecological balance, performance beneficial effect.It mainly plays a role by improving the balance of host enteric microorganism flora.It has the intestinal microflora of improvement, suppresses pathogenic bacteria, generates nutritive substance, improves immunity of organisms, eliminates carcinogen, reduces cholesterol and blood pressure, improves the functions such as lactose digestibility.Fuller gave a definition to probiotic bacterium first in 1989: " probiotic bacterium is by improving intestinal microflora balance, host health to be produced the viable bacteria additive of beneficial effect ".
In human lives, the probiotic bacterium of widespread use already comprises clostridium aceticum, milk-acid bacteria, bifidus bacillus, Lactobacterium acidophilum, actinomycetes, yeast etc.Along with the development, particularly probiotic bacterium of the method for the anti-evil of microorganism is in the prospect that replaces traditional antibiotic use, make probiotic bacterium and preparation thereof more and more be applied to culture fishery.At present probiotic bacterium and the preparation thereof of aquaculture class are mainly divided into totally two classes: 1. unitary agent, and as yeast, photosynthetic bacterium, milk-acid bacteria, lactobacillus, Lactococcus lactis, Pseudomonas fluorescens, genus bacillus etc.2. compound formulation, as U.S. bacterium side, compound active bio water purification agent etc., mainly contains the bacterial strains such as nitrobacteria and denitrifying bacterium, Bacillus subtilus, lactobacillus, bifidus bacillus, pseudomonas, streptococcus faecium, yeast and forms.Such beneficial flora is formed by the useful bacterial strain cooperation of directed screening, can improve the microecological balance in aquatic animal body, stimulates the immunity system of body, antagonism pathogenic microorganism, decomposing organic waste, occurs thereby reduce disease, promote aquatic animal to grow to grow, improve breeding ecological environment.Large quantity research with facts have proved, probiotic bacterium has played good effect in the application of aquaculture, and as the separated many probiotics that obtain in from animal bodies such as shrimp, lefteye flounders, thereby the microbe composite bacterial liquid of making can be improved substrate, stabilizing water quality is to guarantee the successful important technology of cultivation.Owing to not using chemical agent, thereby the fishery products of producing are more complete and free of contamination green foods again, and can avoid fishery products to produce so-called resistance.Use continuously for many years the water body of bacterium liquid must form the ecological dominance of efficacious microbial colony, reach the benign cycle of water surrounding, guarantee the sustainable development to aquaculture.
The separation of probiotic bacterium is generally by gathering a large amount of different bacterial strains, carries out in vitro selectivity cultivation or antibacterial experiment and obtains.Principle and the method for milk-acid bacteria selective medium in the summary of Gatesoupe, have at length been listed.The people such as Sugita isolate 10055 strain bacterial strains and carry out antibacterial experiment in 7 kinds of near sea fishes enteron aisles, and select good probiotic strain genus bacillus.Also occurred afterwards the new technology of isolation identification, for example, the people such as Vandenberghe have carried out the AFLP fingerprint map analyzing of complete sequence to common bacterial strain Vibro harveyi in Environment of Litopenaeus vannamei Low cultivation, thereby its hereditary feature is analyzed.The people such as Spanggard have adopted traditional dull and stereotyped culture of isolated and 16s rDNA sequencing method to differentiate rainbow trout intestinal microflora simultaneously.
Development along with China's culture fishery.The disease problem that batch production, high-density breeding cause becomes increasingly conspicuous.The much tens billion of units of loss that cause because of aquatic products disease every year.And treatment aquatic animal disease, routine still extensively adopts Chemo-Therapy therapy, antibiotic abuse develops immunity to drugs and drug residue water body bacterium, indirect hazard the mankind's health.Therefore, increasing expert starts to pay close attention to and utilizes the biological restoration of beneficial microorganism to culture environment of aquatic products in physical environment, with this, replace antibiotic use, so not only saved expense, also can not produce secondary pollution, and can also make water quality keep good state residual bait, movement and other objectionable impurities decomposition and inversion, make aquaculture organism in health level.Therefore, beneficial microorganism has extremely wide prospect to the research and development of the bioremediation technology of culture environment of aquatic products, also can be more and more extensive in the application of aquaculture field, bring larger economic benefit and social benefit simultaneously.
Summary of the invention
The object of the present invention is to provide probiotic bacterium and the application thereof of a kind of separation in enteron aisle.
For achieving the above object, the technical solution used in the present invention is:
The probiotic bacterium of a kind of separation in enteron aisle, probiotic bacterium is that bacterium (Leclercia adecarboxylata) P115 is strangled in non-decarboxylation Rec, on June 24th, 2013, in Chinese microorganism, preserve center preservation, deposit number is CGMCC No:7813, and depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Described probiotic bacterium can be used for preparing antiseptic feed or the antibacterials of aquatic products or mankind pathogenic bacteria.
Described aquatic pathogenic bacterium comprises Vibrio splindidus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa or Vibrio anguillarum; Mankind pathogenic bacteria comprises Candida albicans, streptococcus aureus or Klebsiella pneumonia.
Harmful bacterial growth composition of curing the disease, suppressing harmful bacterial growth composition of curing the disease is probiotic bacterium claimed in claim 1 or the culture of probiotic bacterium or the mixed culture that contains profitable probliotics.
Described composition can be used for preparing antibacterials or the antiseptic feed of aquatic pathogenic bacterium or people's bacterioid.
Bacterium (Leclercia adecarboxylata) P115 is strangled in non-decarboxylation Rec, and on 2216E solid plate, single colony diameter is 1.5mm left and right, and circle is light yellow, translucent, protrudes media surface, glossy.16sDNA sequential analysis shows that it is 99.9% that the similarity of the 16sDNA of bacterium EG88 is strangled in itself and non-decarboxylation Rec.
The present invention has advantages of: probiotic bacterium of the present invention is all from separated acquisition in the enteron aisle of healthy turbot.Probiotic bacterium and pure growth to causing Vibrio parahaemolyticus, Vibrio harveyi, the Vibrio anguillarum of aquiculture disease, have obvious fungistatic effect, the clinical cause of disease of the mankind is had to obvious fungistatic effect equally as Candida albicans, Klebsiella Pneumoniae, streptococcus aureus.Its pure growth or composition can be used for the prevention and control of aquaculture class disease and the exploitation of clinical novel antibacterial material, have a good application prospect.Probiotic bacterium of the present invention is the living microorganism that a class is useful to host, can improve survival rate and the resistance against diseases of industrialized culture turbot.
Embodiment
Around bacterial strain of the present invention and embodiment, be described in detail, but embodiments of the present invention are not limited to this.
Screening and the separation of embodiment 1 probiotic bacterium
1) sampling: get at random healthy turbot (the long 10-15cm of body) 5-10 bar, with the 1-2% vetanarcol anesthesia of 100-200ul, aseptic condition is dissected, and gets its enteron aisle.Rapidly with aseptic 2216E liquid nutrient medium (composition: peptone 5g, yeast extract paste 1g, high ferric phosphate 0.1g, Chen Haishui 1000mL, pH7.5,121 ℃ of sterilizing 20min) or aseptic PBS cleaning, the glass homogenizer homogenate of 5-10mL, homogenate with aseptic 2216E liquid nutrient medium or PBS according to 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6gradient dilution.Select 10
-3with 10
-6dilution homogenate, is uniformly coated on respectively on TSB, LB, 2216E solid plate.28-30 ℃, is inverted and cultivates 24-48h.
2) primary dcreening operation: single bacterium colony of having grown on above-mentioned flat board, according to colonial morphology constitutional features, provoke respectively, use the corresponding liquid nutrient medium of liquid used while cultivating with above-mentioned sampling to be cultured to OD
600=0.8-1.0, gets 50-100ul nutrient solution, and dibbling reaches or is greater than 10 at bacterial number
6on the 2216E agar plate of Vibrio anguillarum, 28-30 ℃, is inverted and cultivates 24-48h.
3) multiple sieve: be chosen in primary dcreening operation, by the bacterial strain that has produced obvious inhibition zone again dibbling on the above-mentioned 2216E agar plate that contains Vibrio anguillarum.
4) separation: screening is obtained to bacterial strain and rule in corresponding screening culture medium respectively, detect single colony morphology characteristic, single bacterium colony is cultured to OD with 2216E liquid nutrient medium
600=0.8-1.0, adds 30-50% glycerine ,-20-80 ℃ of prolonged preservation after mixing.
By screening, with separated, obtained and have fungistatic effect, Vibrio anguillarum is produced to the P115 bacterial strain of antagonistic action, it can grow on 2216E agarose plate.
The evaluation of embodiment 2:P115 and activation analysis
1.16S rDNA analyzes
A) extraction of P115 genomic dna: P115 is cultured to OD
600=1.0-1.5, utilizes the genomic dna of phenol chloroform method extracting P115 to producing without egg white layer, with a certain amount of TE solution, dissolves.
B) pcr amplification 16SrDNA:PCR reaction system is 0.5 μ l P115 genomic dna, 1 μ l 27F, 1 μ l 1492R, 4 μ l dNTP, 5 μ l Taqbuf, 0.4 μ l Taq enzyme, 36.1 μ l ddH
2o.Reaction conditions is 95 ℃ of denaturation 5min, 95 ℃ of sex change 1min, and 54 ℃ of renaturation 30s, 72 ℃ are extended 1min, move 30 circulations.72 ℃ are extended 10min.PCR product is wall scroll band by electrophoresis detection, and size is about 1.6kb, meets expection.Wherein, concrete primer 2 7F and 1492R, for conventional primer, belong to known field.
C) order-checking and analysis: after PCR product glue is reclaimed, send the order-checking of order-checking company.Result is analyzed through nBlast, determine that the 16SrDNA sequence of P115 and the 16SrDNA sequence similarity that bacterium (Leclercia adecarboxylata) EG88 is strangled in non-decarboxylation Rec are 99.9%, judge that it is that bacterium (Leclercia adecarboxylata) is strangled in the non-decarboxylation of strain Rec.
2. antibiotics resistance analysis:
A) preparation of microbiotic flat board: preparation is containing the antibiotic 2216E agarose plate of different concns respectively, and microbiotic final concentration is respectively kantlex 10,30,50,100 μ g/mL; Penbritin 50,100,150,200 μ g/mL; Spectinomycin 50,100 μ g/mL.
B) dibbling: get and be cultured to OD
600the P115 nutrient solution of=0.8-1.0, aseptic PBS, each 10-30ul of sterilized water, put on above-mentioned flat board respectively.Just putting 2-3h for 28-30 ℃, after dibbling liquid is absorbed by substratum, the incubator that is placed in 28-30 ℃ is inverted incubated overnight 24h.
C) result: contrast the result of aseptic PBS and sterilized water, observe the production of P115 on each microbiotic flat board.Discovery is for the microbiotic of institute's detectable level, and P115 is all without significantly growth.And do not containing well-grown on any antibiotic 2216E agarose substratum.Illustrate that P115 is to tested antibiotic sensitive.
3. antimicrobial spectrum analysis
A) the dull and stereotyped preparation of pathogenic bacteria: Candida albicans, Klebsiella Pneumoniae, streptococcus aureus based on 37 ℃, are cultured to OD by 2216E liquid culture based on 28 ℃ by Vibrio parahaemolyticus, Harbin vibrios by LB liquid culture
600=0.8-1.0, gets 100-200ul nutrient solution, and the centrifugal 3-5min of 6000-8000rpm room temperature, abandons supernatant, the aseptic PBS Eddy diffusion of 100-200ul for precipitation.Candida albicans, Klebsiella Pneumoniae, streptococcus aureus suspension are coated on to LB solid medium flat board in 37 ℃, Vibrio parahaemolyticus, Harbin vibrios suspension are coated on to 2216E solid culture based on 28 ℃, just putting respectively fixedly 2-3h.
B) dibbling: get and be cultured to OD
600the P115 nutrient solution of=0.8-1.0, aseptic PBS, each 10-30ul of sterilized water, put respectively and filling on the above-mentioned flat board of pathogenic bacteria.Just putting 2-3h for 28-30 ℃, after dibbling liquid is absorbed by substratum, the incubator that is placed in 28 ℃ or 37 ℃ is inverted incubated overnight 24-48h.
C) result: contrast the result of aseptic PBS and sterilized water, observe the growth of P115 on each pathogenic bacteria flat board and the production of inhibition zone.Discovery is for detected bacterium, and P115 has produced obvious inhibition zone.Aseptic PBS and sterilized water have no obvious inhibition zone and produce.
4.P115 antipathogenic composition initial analysis
A) sample preparation: P115 is cultured to OD with 2216E liquid nutrient medium
600=0.8-1.0.Get 2-4ml nutrient solution, 6000-8000rpm, 4-8 ℃ of centrifugal 5-8min, supernatant filters and thoroughly removes the bacterium that the inside is contained with aseptic 0.22um strainer; After precipitation suspends with the aseptic PBS of 2-4ml, 6000-8000rpm, 4-8 ℃ of recentrifuge 5-8min, precipitation is standby with the aseptic PBS Eddy diffusion of 2ml.
B) prepare pathogenic bacteria flat board: utilize step in embodiment 2 steps 3 a) method of the dull and stereotyped preparation of pathogenic bacteria prepare pathogenic bacteria flat board.Selected aquatic pathogenic bacterium is respectively Vibrio splindidus 3 strains, vibrio fluvialis, solution bath vibrios, Vibrio vulnificus, intestinal bacteria, subtilis and two strain phytopathogen cucumber anthracnoses, grape anthracnose.
C) by the sample of preparation, with aseptic PBS and sterilized water in contrast.Dibbling is on the pathogenic bacteria flat board of preparation respectively.Just putting 2-3h for 28-30 ℃, after dibbling liquid is absorbed by substratum, the incubator that is placed in 28 ℃ or 37 ℃ is inverted incubated overnight 24-48h.
D) result: contrast aseptic PBS and sterilized water, in discovery, cleer and peaceful precipitation all has the inhibition having added for aquatic pathogenic bacterium, but for two detected strain phytopathogeic fungi effects.The fungistatic effect of simultaneously finding supernatant is slightly better than precipitation.Thereby infer that the composition that P115 bacterial strain plays bacteriostatic action should be the meta-bolites of bacterium, and can be a large amount of be secreted into born of the same parents' outer (table 1).
From the above results, the microbiotic that P115 bacterium is used routine does not have obvious resistance.P115 is to being under the jurisdiction of Candida albicans, Klebsiella Pneumoniae, streptococcus aureus, the Pseudomonas aeruginosa of mankind pathogenic bacteria, to being under the jurisdiction of Vibrio harveyi, Vibrio vulnificus, Vibrio anguillarum, Vibrio splindidus, the vibrio fluvialis of aquatic pathogenic bacterium, for intestinal bacteria, subtilis, the Vibrio parahaemolyticus of infecting both domestic animals and human, all there is obvious bacteriostatic action.Its pure growth or composition can be used for the prevention and control of aquaculture class disease and the exploitation of novel antibacterial material.There is good application prospect.
Table 1 fungistatic effect cartogram
Note: the diameter of " ﹢ ﹢ ﹢ " inhibition zone is 0.8～1.3cm; The diameter of " ﹢ ﹢ " inhibition zone is 0.6～0.8cm; The diameter of " ﹢ " inhibition zone is 0.4～0.6cm; "-" produces without inhibition zone.
Claims (5)
1. the separation probiotic bacterium in enteron aisle, it is characterized in that: probiotic bacterium is that bacterium (Leclercia adecarboxylata) P115 is strangled in non-decarboxylation Rec, on June 24th, 2013, in Chinese microorganism, preserve center preservation, deposit number is CGMCC No:7813.
2. by the probiotic bacterium of separation claimed in claim 1 in enteron aisle, it is characterized in that: described probiotic bacterium can be used for preparing antiseptic feed or the antibacterials of aquatic products or mankind pathogenic bacteria.
3. by the probiotic bacterium of separation claimed in claim 2 in enteron aisle, it is characterized in that: described aquatic pathogenic bacterium comprises Vibrio splindidus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa or Vibrio anguillarum; Mankind pathogenic bacteria comprises Candida albicans, streptococcus aureus or Klebsiella pneumonia.
4. suppress harmful bacterial growth composition of curing the disease, it is characterized in that: suppressing harmful bacterial growth composition of curing the disease is probiotic bacterium claimed in claim 1 or the culture of probiotic bacterium or the mixed culture that contains profitable probliotics.
5. by the application of the harmful bacterial growth composition of curing the disease of inhibition claimed in claim 4, it is characterized in that: described composition can be used for preparing antibacterials or the antiseptic feed of aquatic pathogenic bacterium or people's bacterioid.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106085935A (en) * | 2016-06-14 | 2016-11-09 | 李倩 | A kind of processing method of aquaculture wastewater |
| WO2017066898A1 (en) * | 2015-10-23 | 2017-04-27 | 广州康泽医疗科技有限公司 | Optimized method for isolating human intestinal probiotics |
| CN106635932A (en) * | 2017-02-23 | 2017-05-10 | 遵义医学院 | Probiotic group and preparation method thereof |
| CN107779422A (en) * | 2017-11-29 | 2018-03-09 | 中国农业科学院油料作物研究所 | One plant height effect suppresses the Leclercia adecarboxylata biocontrol bacterial strain of aspergillus flavus synthesis aflatoxin |
| CN109971781A (en) * | 2019-03-29 | 2019-07-05 | 中国人民解放军军事科学院军事医学研究院 | A kind of new transposon Tn6505 and its application |
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2013
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017066898A1 (en) * | 2015-10-23 | 2017-04-27 | 广州康泽医疗科技有限公司 | Optimized method for isolating human intestinal probiotics |
| CN106085935A (en) * | 2016-06-14 | 2016-11-09 | 李倩 | A kind of processing method of aquaculture wastewater |
| CN106635932A (en) * | 2017-02-23 | 2017-05-10 | 遵义医学院 | Probiotic group and preparation method thereof |
| CN107779422A (en) * | 2017-11-29 | 2018-03-09 | 中国农业科学院油料作物研究所 | One plant height effect suppresses the Leclercia adecarboxylata biocontrol bacterial strain of aspergillus flavus synthesis aflatoxin |
| CN109971781A (en) * | 2019-03-29 | 2019-07-05 | 中国人民解放军军事科学院军事医学研究院 | A kind of new transposon Tn6505 and its application |
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