CN103525721B - There is probiotic bacterium and the application thereof of bacteriostasis - Google Patents
There is probiotic bacterium and the application thereof of bacteriostasis Download PDFInfo
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Abstract
The present invention relates to and belong to microorganism disease control field, specifically a kind of probiotic bacterium with bacteriostasis.Probiotic bacterium is Ludwig enterobacteria Enterobacter ludwigii-PC88, and preserve center preservation on June 24th, 2013 at China Microbiological, deposit number is CGMCC No:7814.Probiotic bacterium of the present invention is all separated and obtains in the enteron aisle of healthy Paralichthys olivaceus.The pure growth of probiotic bacterium to aquatic pathogenic bacterium as Vibrio splindidus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Vibrio anguillarum have stronger inhibition.Also can suppress to be known as the growth of the Candida albicans of human pathogen bacterium, streptococcus aureus, Klebsiella pneumonia.To penbritin, there is resistance.Its pure growth or composition can be used for the prevention and control of aquaculture class disease and the exploitation of novel antibacterial material, have a good application prospect.
Description
Technical field
The present invention relates to and belong to microorganism disease control field, specifically a kind of probiotic bacterium with bacteriostasis.
Background technology
Probiotic bacterium (Probiotics) is the living microorganism that a class is useful to host, be be colonizated in human body or animal intestinal, reproductive system, can definite health efficacy be produced thus improve host's microecological balance, play the active beneficial microorganism general name of beneficial effect.It plays a role mainly through improving the balance of host intestine microorganism species.It has improves intestinal microflora, suppresses pathogenic bacteria, generates nutritive substance, improves immunity of organisms, eliminates carcinogen, reduces cholesterol and blood pressure, improves the functions such as lactose digestion.Fuller gave a definition to probiotic bacterium first in 1989: " probiotic bacterium is the viable bacteria additive by improving intestinal microflora balance, host health being produced to 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 of the use of replacement conventional antibiotic, probiotic bacterium and preparation thereof is made more and more to be applied to culture fishery.The probiotic bacterium of current aquaculture class and preparation thereof are mainly divided into totally two classes: 1. unitary agent, 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 strain such as nitrobacteria and denitrifying bacterium, Bacillus subtilus, lactobacillus, bifidus bacillus, pseudomonas, streptococcus faecium, yeast composition.Such beneficial flora is formed by the useful bacterial strain cooperation of directed screening, can improve the microecological balance in aquatic animal body, stimulate the immunity system of body, antagonism pathogenic microorganism, decomposing organic waste, thus reduce disease generation, promote that aquatic animal to grow is grown, improve breeding ecological environment.A large amount of Researching and practicing proves, probiotic bacterium serves good effect in the application of aquaculture, and as being separated the many probiotics obtained in the animal body such as shrimp, lefteye flounder, thus the microbe composite bacterial liquid made can improve substrate, stabilizing water quality ensures the successful important technology of cultivation.Owing to not using chemical agent again, the fishery products thus produced are more complete and free of contamination green foods, and fishery products can be avoided to produce so-called resistance.Use the water body of bacterium liquid must form the ecological dominance of efficacious microbial colony for many years continuously, reach the benign cycle of water surrounding, ensure the sustainable development to aquaculture.
The separation of probiotic bacterium is generally by gathering bacterial strains different in a large number, carries out selectivity cultivation or antibacterial experiment acquisition in vitro.The Principles and ways of milk-acid bacteria selective medium is listed in detail in the summary of Gatesoupe.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 excellent probiotic strain genus bacillus.Also occurred the new technology of isolation identification afterwards, such as, the people such as Vandenberghe have carried out the AFLP fingerprint map analyzing of complete sequence to common bacterial strain Vibroharveyi in Environment of Litopenaeus vannamei Low cultivation, thus analyze its hereditary feature.The people such as Spanggard have employed traditional slat chain conveyor separation simultaneously and differentiate rainbow trout intestinal microflora with 16s rDNA sequencing method.
Along with the development of China's culture fishery.The disease problem that batch production, high-density breeding cause becomes increasingly conspicuous.Every year because of much tens billion of units of loss that aquatic products disease causes.And treat the disease of aquatic animal, routine still extensively adopts chemotherapy regimen, antibiotic abuse makes the bacterial resistance occurred and drug residue of water body, the indirect hazard health of the mankind.Therefore, increasing expert starts to pay close attention to and to utilize in physical environment beneficial microorganism to the biological restoration of culture environment of aquatic products, antibiotic use is replaced with this, so not only save expense, also secondary pollution can not be produced, and can also by residual bait, movement and other objectionable impurities decomposition and inversion, the state making water quality keep good, makes aquaculture organism be in health level.Therefore, the research and development of beneficial microorganism to the bioremediation technology of culture environment of aquatic products has extremely wide prospect, also can be more and more extensive in the application of aquaculture field, brings larger economic benefit and social benefit simultaneously.
Summary of the invention
The object of the invention is to provide a kind of probiotic bacterium and the application thereof with bacteriostasis.
For achieving the above object, the technical solution used in the present invention is:
Have a probiotic bacterium for bacteriostasis, probiotic bacterium is Ludwig enterobacteria Enterobacterludwigii-PC88, and preserve center preservation on June 24th, 2013 at China Microbiological, deposit number is CGMCC No:7814; Depositary institution address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Described probiotic bacterium can be used for the antibacterials preparing aquatic pathogenic bacterium or human bacterial.
Described aquatic pathogenic bacterium is Vibrio splindidus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa or Vibrio anguillarum; Human bacterial is that mankind pathogenic bacteria is as Candida albicans, streptococcus aureus or Klebsiella pneumonia.
A kind of suppression is harmful to bacterial growth composition of curing the disease, and suppresses harmful bacterial growth composition of curing the disease to be the culture of probiotic bacterium according to claim 1 or probiotic bacterium.
Described composition can be used for the antibacterials preparing aquatic pathogenic bacterium or human bacterial.
The advantage that the present invention has:
Probiotic bacterium of the present invention is all separated and obtains in the enteron aisle of healthy Paralichthys olivaceus.The pure growth of probiotic bacterium to aquatic pathogenic bacterium as Vibrio splindidus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Vibrio anguillarum have stronger inhibition.Also can suppress to be known as the growth of the Candida albicans of human pathogen bacterium, streptococcus aureus, Klebsiella pneumonia.To penbritin, there is resistance.Its pure growth or composition can be used for the prevention and control of aquaculture class disease and the exploitation of novel antibacterial material, have a good application prospect.
Ludwig enterobacteria (Enterobacter ludwigii) PC88, on TSB solid plate, single colony diameter is about 1.5mm, circular, light yellow, translucent, protrudes media surface, glossy.Its main bacteriostatic activity is see table 1.Antibiotics resistance is see table 2.
The analysis of table 1 bacteriostatic activity
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.
The analysis of table 2 antibiotics resistance
Embodiment
Be described in detail around bacterial strain of the present invention and embodiment, but embodiments of the present invention are not limited thereto.
The screening of embodiment 1 probiotic bacterium Ludwig enterobacteria (Enterobacter ludwigii) PC88 and separation
1) sample: get healthy Paralichthys olivaceus (the long 10-15cm of body) 5-10 bar at random, anaesthetize with the 1-2% vetanarcol of 100-200ul, aseptic condition is dissected, and gets its enteron aisle.Rapidly with aseptic TSB liquid nutrient medium (composition: Tryptones 17g, multivalence peptone 3g, yeast extract paste 6g, sodium-chlor 5g, dipotassium hydrogen phosphate 2.5g, glucose 2.5g, add water to 1L, pH=7.2-7.5) or aseptic PBS clean, the glass homogenizer homogenate of 5-10mL, homogenate with aseptic TSB 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 TSB, LB solid plate respectively.28-30 DEG C, is inverted and cultivates 24-48h.
2) primary dcreening operation: the single bacterium colony grown on above-mentioned flat board, according to colonial morphology constitutional features, provokes respectively, is cultured to OD with corresponding liquid nutrient medium
600=0.8-1.0, gets 50-100ul nutrient solution, and dibbling is on the flat board filling Vibrio anguillarum, and 28-30 DEG C, is inverted and cultivates 24-48h.
3) multiple sieve: be chosen in primary dcreening operation, create the bacterial strain of obvious inhibition zone.By its again dibbling on the flat board being coated with Vibrio anguillarum.
4) be separated: screening is obtained bacterial strain and rules in corresponding screening culture medium respectively, detect single colony morphology characteristic, the corresponding liquid nutrient medium of single bacterium colony is cultured to OD
600=0.8-1.0, adds 30-50% glycerine, preserves for a long time for-20-80 DEG C after mixing.
By screening and being separated, obtain alternative bacterium 320 strain with fungistatic effect.Feature all can produce antagonistic action to Vibrio anguillarum.PC88 is a wherein strain, and it can grow on TSB agarose plate.
The qualification of embodiment 2:PC88 and activation analysis
1) 16S rDNA analyzes
A) extraction of PC88 genomic dna: PC88 is cultured to OD
600=1.0-1.5, utilizes the genomic dna of phenol chloroform method extracting PC88 to produce, with a certain amount of TE solubilize to without egg white layer.
B) pcr amplification 16SrDNA:PCR reaction system is 0.5 μ l PC88 genomic dna, 1 μ l27F, 1 μ l1492R, 4 μ l dNTP, 5 μ l Taqbuf, 0.4 μ l Taq enzyme, 36.1 μ l ddH
2o.Reaction conditions is 95 DEG C of denaturation 5min, 95 DEG C of sex change 1min, 54 DEG C of renaturation 30s, and 72 DEG C extend 1min, run 30 circulations.72 DEG C extend 10min.PCR product is single slice by electrophoresis detection, and size is about 1.6kb, meets expection.
C) order-checking and analysis: after being reclaimed by PCR primer glue, send order-checking company to check order.Result is analyzed through nBlast, determines that the 16SrDNA sequence of PC88 and Ludwig enterobacteria (Enterobacterludwigii) 16SrDNA sequence similarity are 100%, judges that it is a strain Ludwig enterobacteria (Enterobacter ludwigii).
2) antibiotics resistance analysis:
A) preparation of microbiotic flat board: prepare TSB agarose plate respectively, 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 PC88 nutrient solution of=0.8-1.0, aseptic PBS, each 10-30ul of sterilized water, put respectively on above-mentioned flat board.Just put 2-3h for 28-30 DEG C, after dibbling liquid is absorbed by substratum, incubated overnight 24h is inverted by the incubator being placed in 28-30 DEG C.
C) result: the result contrasting aseptic PBS and sterilized water, observes the production of PC88 on each microbiotic flat board.Find that PC88 is that the penbritin of 50 μ g/mL or the flat board of spectinomycin can grow at final concentration, the flat board of kantlex does not grow.Sterilized water and PBS do not grow simultaneously.Therefore PC88 can with these two kinds of microbiotic separately or be combined, thus easily for purifies and separates and the enlarged culturing of bacterial strain PC88.
3) antimicrobial spectrum analysis
A) pathogenic bacteria flat board preparation: by the vibrio fluvialis preserved, separate bath vibrios, Vibrio vulnificus, Vibrio harveyi, Vibrio anguillarum, Vibrio splindidus, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Pseudomonas fluorescens, subtilis, Candida albicans, Klebsiella Pneumoniae, streptococcus aureus and two strain phytopathogen cucumber anthracnoses, the corresponding substratum of grape anthracnose, 28 or 37 DEG C are cultured to OD
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 precipitation 100-200ul.By suspension on the flat board of suitable culture medium, just putting fixing 2-3h for 28 or 37 DEG C.
B) dibbling: get and be cultured to OD
600the PC88 nutrient solution of=0.8-1.0, aseptic PBS, each 10-30ul of sterilized water, put respectively on the above-mentioned flat board filling pathogenic bacteria.Just put 2-3h for 28-30 DEG C, after dibbling liquid is absorbed by substratum, incubated overnight 24-48h is inverted by the incubator being placed in 28 DEG C or 37 DEG C.
C) result is as shown in table 1.PC88 to aquatic pathogenic bacterium as Vibrio splindidus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Vibrio anguillarum have stronger inhibition.Also can suppress to be known as the growth of the Candida albicans of human pathogen bacterium, streptococcus aureus, Klebsiella pneumonia.Its pure growth or composition can be used for the prevention and control of aquaculture and bacterial diseases in humans and the exploitation of novel antibacterial material.There is good application prospect.
Claims (5)
1. have a probiotic bacterium for bacteriostasis, it is characterized in that: probiotic bacterium is Ludwig enterobacteria Enterobacter ludwigii-PC88, preserve center preservation on June 24th, 2013 at China Microbiological, deposit number is CGMCC No:7814.
2. by the probiotic bacterium with bacteriostasis according to claim 1, it is characterized in that: described probiotic bacterium can be used for preparing the antibacterials of aquatic pathogenic bacterium or human bacterial.
3. by the probiotic bacterium with bacteriostasis according to claim 2, it is characterized in that: described aquatic pathogenic bacterium is Vibrio splindidus, Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa or Vibrio anguillarum; Human bacterial is Candida albicans, streptococcus aureus or Klebsiella pneumonia.
4. suppress a detrimental bacterial growth composition, it is characterized in that: suppression detrimental bacterial growth composition is the culture of probiotic bacterium according to claim 1 or probiotic bacterium.
5., by the application of suppression detrimental bacterial growth composition according to claim 4, it is characterized in that: described composition can be used for preparing the antibacterials of aquatic pathogenic bacterium or human bacterial.
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| CN103966123B (en) * | 2014-04-18 | 2016-02-24 | 中国科学院南海海洋研究所 | One strain Huo Shi enterobacteria and the application in preparation antibacterials thereof |
| CN106520619B (en) * | 2016-11-15 | 2019-03-26 | 山东科技大学 | A kind of Ludwig enterobacteria and its in induction magnesium ion at the application in mine |
| CN108085275B (en) * | 2017-12-19 | 2020-11-17 | 山东省科学院生态研究所 | Enterobacter ludwigii for inhibiting pathogenic fungi of white muscardine silkworm and application thereof |
| CN111117910B (en) * | 2019-12-27 | 2021-08-31 | 江西农业大学 | Enterobacter ludwigii PN6 and application thereof |
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| Occurrence of Strains Producing Specific Antibacterial Inhibitory Agents in Five Genera of Enterobacteriaceae;Jan Smarda;《CURRENT MICROBIOLOGY》;20071231;第54卷;摘要部分 * |
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