CN106190894B - One plant of Pediococcus pentosaceus G11 and its screening and application - Google Patents
One plant of Pediococcus pentosaceus G11 and its screening and application Download PDFInfo
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Abstract
The present invention relates to one plant of Pediococcus pentosaceus G11 and its screenings and application, are preserved in China typical culture collection center, and deposit number is CCTCC NO:2016248.The Pediococcus pentosaceus G11 has tolerance to Compound New Nomin and cefotaxime;The Pediococcus pentosaceus G11 all has bacteriostatic activity to vibrio parahaemolytious, Aeromonas hydrophila, vibrio alginolyticus, staphylococcus aureus and beta streptococcus.Has certain resistance, enzymatic productivity, achroacyte dissolubility has no drug resistance to most antibiotic, harmless to host through dipping bath verification experimental verification, but also it can be improved the immunity of body, its application prospect is extensive, can be applied to probiotics preparation, can also be made into bacterium powder, it is added in feed as feed addictive, for inhibiting pathogenic bacteria in animal body to grow, mortality of animals is reduced, is one plant of probiotic strain for being worth exploitation.
Description
Technical field
The present invention relates to one plant of Pediococcus pentosaceus more particularly to one plant to have prebiotic active pentose piece ball to Scylla paramamosain
Bacterium G11 and its screening and application.
Background technique
It is dynamic to belong to the economic crust of the eurythermal seawater of wide salt for Scylla paramamosain (Scylla paramamosain), abbreviation mud crab
Object is distributed mainly on China southeastern coastal areas, is the main sea-farming kind in especially Shantou, East Guangdong, has growth
Fastly, individual is big, adaptability and disease resistance are strong, resistance to dry dew, easily transport, the features such as meat flavour is delicious unique, full of nutrition, quite by
The favor of domestic and international producers and consumers.(such as mud crab baculoviral disease, exhales intestines to mud crab disease especially viral in recent years
Lonely sample virosis), phytosis and parasitic disease etc. cause serious economic loss to our province or even national blue crab cultivation industry.
Such as since the autumn and winter in 2004, unprecedented sudden disease is had occurred in Guangdong blue crab cultivation, during especially fattening, cream
The death rate of crab is in 70 % or more.The Guangdong Nan Shui mud crab fattening death rate is in 90 % or more within 2005~2006 years.2006,2007
Disease it is still serious, incidence and disease time and first 2 years it is essentially identical.Over 4 years, only Shantou Niu Tianyang encloses tideland for cultivation the area masses
Blue crab cultivation estimates annual economic loss up to 50,000,000 yuan or more with regard to the underproduction 60%.Shantou City's blue crab cultivation area more than 100,000
Mu;Wherein Niu Tianyang encloses tideland for cultivation more than 30,000 mu of area.The morbidity of mud crab disease is rapid, range is wide, and main outburst is in annual 9-11 month.
It is generally believed that mud crab category shell-fish invertebrate, does not have specific immune system, it is immunized with non-specificity
Based on immune, therefore the reliability of vaccine control mud crab disease and popularity is used to still have arguement at present.So far, both at home and abroad
Many scholars have carried out a large amount of research and exploration from various aspects such as mud crab cause of disease, mud crab immune defence mechanism and breeding environments,
Many progress are achieved in terms of mud crab disease treatment, but these researchs are difficult fundamentally to solve the problems, such as the disease of mud crab.
Therefore, the ecological control method of " relying mainly on prevention " is found, it is considered to be solve current blue crab cultivation disease, improve mud crab quality
An effective way.
It is cultivated using the environmental health that probiotics carries out mud crab, is to improve mud crab immunity, prevention mud crab disease is especially
One of effective way of bacterial disease.Currently reported aquatic products probiotics has genus lactubacillus, Bifidobacterium, vibrios
Category, pseudomonas, bacillus, nitrifier, photosynthetic bacteria etc..Lactic acid bacteria, bacillus, saccharomycete etc. have been used as bait to add
Agent is applied to aquatic livestock, plays the role of good disease preventing and treating and salubrious.The Tiny ecosystem system designed for terrestrial organism
Agent is applied in aquaculture, different surely to become dominant bacteria as early as possible and carry out Tiny ecosystem adjusting, moreover, these microorganisms are held very much
Easily gradually wither away with the time.Probiotics is had been supplied at present on prawn, shellfish and other marine aquaculture animals, still
It might not work blue crab cultivation suitable for the probiotics of shrimp shellfish or function and effect are unobvious.Exploitation is directed to the benefit of mud crab
Raw strain is significant to mud crab high density and healthy ecology aquaculture model.
Summary of the invention
Technical problem to be solved by the present invention lies in provide one plant of Pediococcus pentosaceus G11 and its screening and culturing and application
It solves the problems, such as of the existing technology.
In order to achieve the above purpose, it adopts the following technical scheme that:
One plant of Pediococcus pentosaceus G11 Pediococcus pentosaceus G11 is preserved in Chinese Typical Representative culture guarantor
Hiding center, depositary institution address are Wuhan, China university, and the deposit date is on May 5th, 2016, deposit number CCTCC
NO:M2016248, classification naming are Pediococcus pentosaceus G11 Pediococcus pentosaceus G11.
Further, the Pediococcus pentosaceus G11 has tolerance to Compound New Nomin and cefotaxime;The pentose piece
Coccus G11 all has suppression to vibrio parahaemolytious, Aeromonas hydrophila, vibrio alginolyticus, staphylococcus aureus and beta streptococcus
Bacterium activity.The Pediococcus pentosaceus G11 has tolerance to Compound New Nomin and cefotaxime;G11 pairs of the Pediococcus pentosaceus
Vibrio parahaemolytious, Aeromonas hydrophila, vibrio alginolyticus, staphylococcus aureus and beta streptococcus all have bacteriostatic activity.This
Outer Pediococcus pentosaceus G11 has certain resistance, enzymatic productivity, achroacyte dissolubility, to most antibiotic without resistance to
Pharmacological property, it is highly-safe, yolk lecithin enzyme and acidic materials (organic acid) are secreted, Gram-positive, pH tolerance is high, in pH=4
When still have 11.24% viable bacteria rate.It is harmless to host through dipping bath verification experimental verification, but also can be improved the immunity of body.
A kind of screening and culturing method of Pediococcus pentosaceus G11, mainly comprises the steps that
(1) strain isolation is in the water sample of cultivating pool;
(2) plate is applied using the dilution of MRS culture medium, 28 DEG C of constant temperature stationary cultures are for 24 hours;
(3) picking individual colonies shake bacterium solution overnight, are added into the Oxford cup being coated on vibrio parahaemolytious plate, choosing
Select the best bacterial strain of fungistatic effect, and glycerol adding, -80 DEG C of conservations.
Further, step (1) described strain isolation is in the method for the water sample of cultivating pool are as follows: sample sterilizing life
Reason salt water does gradient dilution coating MRS plate, 28 DEG C of culture 2-3 d, and the picking individual colonies on MRS culture medium shake bacterium solution, adopt
With Odontothrips loti, the bacterial strain best to vibrio parahaemolytious fungistatic effect is detected, it is drawn into plate again, isolates and purifies conservation.
A kind of Pediococcus pentosaceus G11 is inhibiting the purposes in vibrio parahaemolytious growth.
A kind of Pediococcus pentosaceus G11 is preparing the purposes in probiotics preparation.
A kind of probiotics preparation contains above-mentioned Pediococcus pentosaceus G11.
A kind of bacterium powder, using above-mentioned Pediococcus pentosaceus G11 preparation.
Purposes of the above-mentioned bacterium powder as feed addictive.
A kind of mud crab feed, containing above-mentioned Pediococcus pentosaceus G11, the Pediococcus pentosaceus G11 viable count is 1 × 108 - 1
×109cfu/g。
Compared with prior art, the present invention provides one plant of Pediococcus pentosaceus G11, the Pediococcus pentosaceus G11 is new to compound
Promise is bright and cefotaxime has tolerance;The Pediococcus pentosaceus G11 to vibrio parahaemolytious, Aeromonas hydrophila, vibrio alginolyticus,
Staphylococcus aureus and beta streptococcus all have bacteriostatic activity.But also can be improved the immunity of body, before application
Scape is extensive, can be applied to probiotics preparation, can also be made into bacterium powder, is added to feed as feed addictive
In, for inhibiting pathogenic bacteria in animal body to grow, mortality of animals is reduced, is one plant of probiotic strain for being worth exploitation.
Detailed description of the invention
Fig. 1 is the stereoscan photograph of bacterial strain G11 of the present invention;
Fig. 2 is the systematic evolution tree of bacterial strain G11 of the present invention;
Fig. 3 is fungistatic effect picture of the bacterial strain G11 of the present invention to mud crab pathogen vibrio parahaemolytious;
Fig. 4 is G11 to Aeromonas hydrophila, vibrio alginolyticus, staphylococcus aureus, the antibacterial reality of beta streptococcus Oxford cup
Test figure;
Fig. 5 is the expression of bacterial strain G11 of the present invention ALF gene in blood when mud crab vibrio parahaemolytious stimulates;
Fig. 6 is the expression of bacterial strain G11 of the present invention LYS gene in blood when mud crab vibrio parahaemolytious stimulates;
Fig. 7 is the expression of bacterial strain G11 of the present invention CAT gene in blood when mud crab vibrio parahaemolytious stimulates;
Fig. 8 is the accumulation survival rate after addition bacterial strain G11 of the present invention receives vibrio parahaemolytious and stimulates in feed in 3 days.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with attached drawing
Step ground detailed description.
Embodiment
1, the separation screening of bacterial strain and identification
Strain isolation is in the water sample (the mud crab well-grown of the cultivating pool, and disease-free generation) of cultivating pool.Gradient
Dilution spread MRS plate, the picking individual colonies on MRS culture medium shake bacterium solution, using Odontothrips loti, detect to vibrio parahaemolytious
The best bacterial strain of fungistatic effect, draws plate for it again, isolates and purifies conservation.
It is identification bacterial strain using scanning electric mirror observing cell form and the part biochemical reactions of test strain, scanning
Electronic Speculum is as shown in Figure 1, primarily determine that the bacterial strain is Pediococcus pentosaceus.
The primer further used by the analysis of 16SrDNA sequence is universal primer 27F (5'-
AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACCTTGTTACGACTT-3') carries out PCR, clones and is sequenced.
In the website NCBI Blast compare discovery, wherein one plant with 20336 homology of Pediococcus pentosaceus DSM most
High (99.74%), is determined as Pediococcus pentosaceus G11(hereinafter referred to as G11).Its systematic evolution tree such as Fig. 2 institute
Show.
2, the bacteriostatic test of bacterial strain
Bacteriostatic experiment: bacterial strain MRS culture medium culture is detected into the common arc cultivated to Scylla paramamosain using Odontothrips loti
The antibacterial effect of bacterium.Its fungistatic effect is assessed according to the size of inhibition zone.From figure 3, it can be seen that G11 has suppression to vibrio parahaemolytious
Bacterium effect.G11 is to Aeromonas hydrophila as can be seen from Figure 4, vibrio alginolyticus, staphylococcus aureus, and beta streptococcus has
There is significant bacteriostatic activity.
Drug susceptibility test: selected antibiosis is known as: Amoxicillin, penicillin, tetracycline, Augmentin, Enrofloxacin, ten thousand
Ancient mycin, cephazoline, meter Luo Huan element, polymyxin B, cephalo benzyl ammonia, neomycin, Compound New Nomin, chloramphenicol, cephalo he
Pyridine, amikacin, cefuroxime, medecamycin, ampicillin, gentamicin, erythromycin, oxacillin.G11 is only to multiple
Square radonil and cefotaxime tolerance are high, have very high safety.
Add G11 in feed and raise young crab experiment: blue crab cultivation selects young crab 160 of 25 ± 1g, and mud crab is divided into two
Class (feeds addition Pediococcus pentosaceus G11 about 1 × 108 - 1×109The feed of cfu/g and the feeding added without Pediococcus pentosaceus G11
Material), every class sets 4 groups, every group 20 young crabs.Respectively after feeding feed stuff 12 days, the feed (concentration for being added to vibrio parahaemolytious is fed
It is 1.99 × 1010cfu/g;Separated from Niu Tianyang blue crab cultivation area, this laboratory saves) 3 days.
1) by secondary molten after is added probiotics 15 days using the detection feeding of real-time fluorescence PCR (real time PCR) method
The immunological stress situation of blood vibrios stimulation, as can be seen that the discovery anti-grease polysaccharide of processing group immunogene from Fig. 5, Fig. 6 and Fig. 7
The factor (anti-lipopolysaccharide factor Precursor, ALF, FJ013272.1), catalase
The expression of (catalase, CAT, FJ774660.1), lysozyme (lysozyme, LYS, 304441008) is significantly higher than control
Group.
2) records the death condition in 3 days.As can be seen from Figure 8, it compared with control group (chow diet group), feeds
The amount for adding G11 is 1 × 108 - 1×109Cumulative survival rate in 3 days of the mud crab group of cfu/g feed is significantly higher than control group
(31.94%) higher than blank control group.
The above results show that Pediococcus pentosaceus G11 has significant prebiotic effect in the cultivation of Scylla paramamosain, to mud crab
It cultivates that common vibrio parahaemolytious preventive effect is significant, is one plant of probiotic strain for being worth exploitation.
Above disclosed is only presently preferred embodiments of the present invention, cannot limit the right of the present invention with this certainly
Range, therefore equivalent changes made in accordance with the claims of the present invention, are still within the scope of the present invention.
SEQUENCE LISTING
<110>University Of Shantou
<120>one plants of Pediococcus pentosaceus G11 and its screening and application
<130> 2016
<160> 3
<170> PatentIn version 3.3
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gattgagatt ttaacacgaa gtgagtggcg aacgggtgag taacacgtgg gtaacctgcc 180
cagaagtagg ggataacacc tggaaacaga tgctaatacc gtataacaga gaaaaccgca 240
tggttttctt ttaaaagatg gctctgctat cacttctgga tggacccgcg gcgtattagc 300
tagttggtga ggtaaaggct caccaaggca gtgatacgta gccgacctga gagggtaatc 360
ggccacattg ggactgagac acggcccaga ctcctacggg aggcagcagt agggaatctt 420
ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggg tttcggctcg 480
taaagctctg ttgttaaaga agaacgtggg taagagtaac tgtttaccca gtgacggtat 540
ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 600
cgttatccgg atttattggg cgtaaagcga gcgcaggcgg tcttttaagt ctaatgtgaa 660
agccttcggc tcaaccgaag aagtgcattg gaaactggga gacttgagtg cagaagagga 720
cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca ccagtggcga 780
aagcggctgt ctggtctgca actgacgctg agctcgaaag catgggtagc gaacaggatt 840
agataccctg gtagtccatg ccgtaaacga tgattactaa gtgttggagg gtttccgccc 900
ttcagtgctg cagctaacgc attaagtaat ccgcctgggg agtacgaccg caaggttgaa 960
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Claims (6)
1. one plant of Pediococcus pentosaceus G11, which is characterized in that be preserved in China typical culture collection center, deposit number is
CCTCC NO:2016248.
2. Pediococcus pentosaceus G11 according to claim 1, which is characterized in that the Pediococcus pentosaceus G11 is to Compound New Nomin
There is tolerance with cefotaxime;To vibrio parahaemolytious, Aeromonas hydrophila, vibrio alginolyticus, staphylococcus aureus and B-mode
Streptococcus all has bacteriostatic activity.
3. Pediococcus pentosaceus G11 is preparing the purposes in probiotics preparation according to claim 1.
4. a kind of probiotics preparation, which is characterized in that the probiotics preparation contains Pediococcus pentosaceus as described in claim 1
G11。
5. a kind of bacterium powder, which is characterized in that the bacterium powder is prepared using Pediococcus pentosaceus G11 as described in claim 1.
6. a kind of mud crab feed, which is characterized in that the feed contains Pediococcus pentosaceus G11 as described in claim 1, and described penta
Sugar-tablet coccus G11 viable count is 1 × 108-1×109cfu/g。
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CN108060103B (en) * | 2018-01-29 | 2019-01-18 | 四川农业大学 | A kind of Pediococcus pentosaceus and its application, screening calibration method |
CN108373983B (en) * | 2018-03-15 | 2020-05-12 | 江南大学 | Pediococcus pentosaceus CCFM1012, fermented food thereof and application of pediococcus pentosaceus CCFM1012 in preparation of medicines for antagonizing campylobacter jejuni infection |
CN109136126B (en) * | 2018-08-21 | 2021-04-13 | 云南农业大学 | Pediococcus pentosaceus for preventing diarrhea of weaned piglets |
CN109439565A (en) * | 2018-09-14 | 2019-03-08 | 湖南师范大学 | One plant of Pediococcus pentosaceus bacterial strain, its probiotics and preparation method thereof |
CN109504637B (en) * | 2018-12-29 | 2021-10-08 | 福建省农业科学院农业工程技术研究所 | Pediococcus pentosaceus and application thereof |
CN110734874A (en) * | 2019-10-17 | 2020-01-31 | 广东海洋大学 | lactic acid bacteria zy-B with food preservation and fresh-keeping function and application thereof |
CN114085789B (en) * | 2021-11-03 | 2023-08-15 | 中国农业大学 | Pediococcus pentosaceus MA.WTPQJ01 and application thereof |
CN117264850B (en) * | 2023-11-09 | 2024-05-14 | 潍坊君薇生物科技有限责任公司 | Pediococcus pentosaceus SW006 with auxiliary treatment of colpitis and immunity enhancing functions and application thereof |
CN117757658A (en) * | 2023-11-13 | 2024-03-26 | 江苏科技大学 | Pediococcus pentosaceus from silkworm intestinal tract and application thereof |
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