CN105779366B - A kind of movement Shandong outstanding person Salmonella bacterial strain and its application - Google Patents

A kind of movement Shandong outstanding person Salmonella bacterial strain and its application Download PDF

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CN105779366B
CN105779366B CN201610341231.8A CN201610341231A CN105779366B CN 105779366 B CN105779366 B CN 105779366B CN 201610341231 A CN201610341231 A CN 201610341231A CN 105779366 B CN105779366 B CN 105779366B
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于明超
张晓华
郑艳芬
李志涛
汤开浩
李文涛
蔡秀磊
徐涛
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Ocean University of China
Tongwei Co Ltd
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Abstract

The present invention relates to a kind of movement Shandong outstanding person's Salmonella YJ3 bacterial strain and its application, the deposit number of bacterial strain YJ3 is CGMCC No.12381.The modes prevention and control aquiculture disease such as enzyme can be quenched by generating Antagonistic protein and density induction in the bacterial strain, and is able to suppress a variety of encountered pathogenic bacterias.More importantly bacterial strain YJ3 has significant inhibiting effect to the luminous intensity of Vibrio luminous, and it can reduce infection of the Vibrio luminous to artemia.Bacterial strain YJ3, without toxic side effect, can be used as feed addictive or breeding water body additive application in aquaculture to Penaeus Vannmei shrimp seedling and artemia.

Description

A kind of movement Shandong outstanding person Salmonella bacterial strain and its application
Technical field
The invention belongs to beneficial bacterium screening technique fields, and in particular to a kind of movement Shandong outstanding person Salmonella bacterial strain and its application.
Background technique
Prawn culturing is important one of the sea-farming industry in China, brings huge economic benefit and society for China Benefit.But with the intensive of prawn culturing, scale and high density production, disease problems become increasingly conspicuous.Vibriosis (vibriosis) it is most common, the maximum bacterial disease of harmfulness in prawn culturing, has made multiple national by tight in the world The economic loss of weight.The pathogen of vibriosis penaeus can be caused mainly to have Vibrio vulnificus (Vibrio vulnificus), molten algae Glue vibrios (V.alginolyticus), Vibrio anguillarum (V.anguillarum), Kan Beishi vibrios (V.campbellii), magnificent arc Bacterium (V.splendidus), kills prawn vibrios (V.penaeicida), vibrio parahaemolytious at beauty's vibrio piscium (V.damsela) (V.parahaemolyticus) and Vibrio harveyi (V.harveyi) etc..Illness prawn shows as poor vigor, anorexia, growth With metamorphosis process slowly, jejunum and the symptoms such as muscle is opaque, infect serious person's exhibit tissue and appendage necrosis, Er Qiechang It is often accompanied by septicemia.In recent years, a kind of catastrophic emerging disease " Deaths syndrome " (early mortality Syndrome, EMS) it was first appeared in 2009 in SOUTHERN CHINA, spread to Vietnam (2010), Malaysian (2011) again later With the ground such as Thailand (2012).The disease occurs mainly in the young shrimp for putting seedling 20-30 days, can usually cause 100% after infected The death rate.Estimate according to global aquaculture alliance (GAA), Asia prawn industry loses nearly 1,000,000,000 dollars because breaking out EMS.
Disease frequently significantly reduces the yield of prawn, forces people using antibiotic to treat disease.But It is that antibiotic is to prevent it from growing or incite somebody to action by interfering the synthesis (such as cell wall, DNA and RNA) of pathogen essential elements It is directly killed, and this mode will certainly cause strong selection pressure that bacterium is made resistance mechanisms occur, promotes antibody-resistant bacterium Generate, thus caused by vicious circle so that problem is growed in intensity, the serious Green Sustainable for hindering culture fishery.It is logical In normal situation, disease is since external environment deterioration causes prawn anti-adversity ability to decline, so as to cause pathogenic bacterial infection. Therefore, the disease resistance for improving prawn is the key that solve the problems, such as.
In recent years, it is gradually accepted using the theory that beneficial bacterium carrys out prevention and control the farming disease harms.Fuller (1989) by its It is defined as " a kind of viable bacteria additive of beneficial effect is played by improving host intestine colony balance ".This definition is to be directed to For the mankind and livestock animals.Since aquiculture animal is lived in water body throughout one's life, surrounding water environment and autodigestion Road is to maintenance body health no less important.Therefore, beneficial bacterium is defined as " by improving colony balance by Gram et al. (1999) (in vivo or/and external) and a kind of viable bacteria additive for playing beneficial effect ".The mode that different beneficial bacteriums plays a role is not Together, mainly include the following types: (1) generates digestive ferment, promote the digestion and absorption of food.Many beneficial bacteriums can generate amylase, The enzymes such as protease enable the food for being difficult to digest in host gastrointestinal tract to digest.(2) attachment site and battalion are competed with pathogen Support substance.Beneficial bacterium is colonized in enteron aisle and being adsorbed on intestinal mucosa, can prevent the attachment of pathogen, make it that intestines be discharged as early as possible Road, referred to as competitive exclusion effect.(3) antibacterial substance is generated.Beneficial bacterium can generate some substances to inhibit other thin around The growth of bacterium, such as bacteriocin, lysozyme and siderophore.(4) immunostimulation.Beneficial bacterium can stimulation of host generate it is thin Born of the same parents are immune, the activity including improving lysozyme, respiratory burst, superoxide dismutase, antiprotease and peroxidase etc., Increase the quantity of leucocyte and lymphocyte;It can also promote the generation of antibody in host body fluids and cell.(5) nutrition is provided.Have A little beneficial bacteriums can generate fatty acid, biotin and vitamin B12 etc., and supply host growth needs.(6) improve water quality.Some Bacterium can convert organic matter to the gases such as carbon dioxide, the content of ammonia nitrogen and cultured water in water body be reduced, to mitigate Water body burden.
The Antagonistic Fungi of screening pathogen is a kind of quickly and effectively means for obtaining beneficial bacterium, is especially come to marine bacteria It says, antagonism is even more generally existing.The beneficial bacterium majority used in the past is from land or other water environments, due to life Environment possibly can not incorporate purpose environment with locating microbiota difference, some beneficial bacteriums, to lose Tiny ecosystem adjusting The effect of.Therefore, the drawbacks of can not playing a role because of environment discomfort can be reduced by screening indigenous beneficial bacterium, i.e., from purpose environment Middle searching beneficial bacterium, reapplies in the environment, is conducive to that its microbial ecological is kept to balance.In addition, the use of beneficial bacterium is also Can effective substitute antibiotics, improve food safety, promote the sustainable development of culture fishery.
Summary of the invention
The object of the present invention is to provide one plant of movement Shandong outstanding person Salmonella bacterial strains and its application, i.e. one kind to be able to suppress Vibrio luminous Beneficial bacterium, to make up the deficiencies in the prior art.
Movement Shandong outstanding person Salmonella (Ruegeria mobilis) YJ3 bacterial strain of the invention, is preserved on April 21st, 2016 China Committee for Culture Collection of Microorganisms's common micro-organisms center's (address is the institute 3 of Chaoyang District Beijing North Star West Road 1), Deposit number are as follows: CGMCC No.12381.
The growth temperature range for moving Shandong outstanding person Salmonella YJ3 bacterial strain is 5-35 DEG C, and optimum culturing temperature is 25-30 DEG C;Growth PH value range is 5-11, and the most suitable growth pH is 7.
Bacterial strain of the invention can be used to prepare for inhibiting the bacterium of Vibrio luminous and other growth of pathogenic bacteria in aquaculture Preparation.
It include the viable bacteria for moving Shandong outstanding person Salmonella YJ3 bacterial strain in above-mentioned bacteria preparation.
Above-mentioned bacteria preparation is feed addictive or breeding water body additive.
Movement Shandong outstanding person Salmonella YJ3 bacterial strain of the invention can inhibit a variety of encountered pathogenic bacterias by generating Antagonistic protein, also can Enough the induction of secretion density is quenched enzyme and blocks the density induction access of pathogen, and then reduces the virulence of pathogen.It is demonstrated experimentally that bacterium Strain YJ3 has significant inhibiting effect to the luminous intensity of Vibrio luminous, and can reduce infection of the Vibrio luminous to artemia.Separately Outside, bacterial strain YJ3 can be used as feed addictive or breeding water body addition to Penaeus Vannmei shrimp seedling and artemia without toxic side effect Agent is applied in aquaculture.
Detailed description of the invention
Fig. 1: the bacterial strain YJ3 inhibiting effect to Vibrio luminous.
Fig. 2: the bacterial strain YJ3 protective effect to artemia.
Specific embodiment
Bacterial strain of the invention is described in detail below with reference to embodiment.
One, the screening of Shandong outstanding person Salmonella YJ3 bacterial strain is moved
In April, 2015, inventor was in the young shrimp shrimp seedlings of extra large one aquatic products seedling Co., Ltd selection, 10 health in Hainan, after grinding It is coated on marine bacteria culture medium 2216E plate, 28 DEG C of constant temperature incubation 48h.Random several single colonie dibblings of picking are in being coated with On the plate of Vibrio luminous, one faint yellow periphery of bacterial colonies of discovery has inhibition zone, and the picking single colonie carries out squamous subculture immediately, Until obtaining purifying bacterial strain, number YJ3.
The genomic DNA for extracting bacterial strain YJ3, expands its 16S rRNA gene order using bacterial universal primers, Determine the bacterial strain YJ3 that the present invention screens for movement Shandong outstanding person Salmonella (Ruegeria mobilis) after being sequenced.
Other two plants potentially beneficial bacterium (the Maxwell Alteromonads isolated from prawn seed-rearing environment AlteromonasmacleodiiS114 and Russia Pseudoalteromonas Pseudoalteromonasruthenica YJ1) with Movement Shandong outstanding person Salmonella YJ3 carries out following two experiment jointly, to inquire into a possibility that they are applied in production or commercial feed.
Two, movement Shandong outstanding person Salmonella YJ3 bacterial strain can reduce the luminous intensity of Vibrio luminous
By bacterial strain S114, YJ1 and YJ3 be added 2216E fluid nutrient medium (peptone 5g, yeast powder 1g, ferric phosphate 0.01g, Chen Haishui 1000ml, pH 7.6);Final concentration of 105Cfu/ml), it is (final concentration of that Vibrio luminous is added after shaken cultivation 12h 105Cfu/ml), continue to cultivate 12h, with the luminous intensity of Weak-luminescence instrument detection bacterium solution.Experimental setup three parallel, with one plant Common bacteria is co-cultured with Vibrio luminous as compareing, to eliminate the shadow caused by Vibrio luminous luminous intensity due to nutrient competition Ring (Vibrio luminous used is Kan Beishi vibrios, separates self-luminous son shrimp shrimp seedling).As a result, it has been found that the luminous intensity of control group is 323 Ten thousand luxs (Wlx), the luminous intensity that bacterial strain S114 is added is 325Wlx, illustrates bacterial strain S114 to the luminous intensity of Vibrio luminous There is no any inhibiting effect;The luminous intensity that bacterial strain YJ3 is added is 58.5Wlx, and the luminous intensity of Vibrio luminous can be made to weaken 82%;And the luminous intensity that bacterial strain YJ1 is added is essentially 0Wlx, can completely inhibit it and shine (Fig. 1).
Three, protective effect of the movement Shandong outstanding person Salmonella YJ3 bacterial strain to artemia
1. weighing 80mg artemia eggs, the centrifuge tube (rule being put in for several times equipped with 20ml antiseptic sea water are rinsed through antiseptic sea water Lattice: 50ml) in, in 170rpm, 28 DEG C of illumination (about 2000 lux) are incubated for for 24 hours.30 nauplius being incubated for out are taken to be put into In the vial for filling 20ml antiseptic sea water, environment 1h is adapted to.
2. picking fresh bacterial strain S114, YJ1 and YJ3 are inoculated in 2216E fluid nutrient medium, shaken cultivation is for 24 hours.Take 1ml Bacterium solution is centrifuged 10min in 4 DEG C, 4000rpm, abandons supernatant.Antiseptic sea water washing thalline is added, is centrifuged again with the same terms.It washes 1ml antiseptic sea water is added after washing 2-3 times, bacteria suspension is made.
3. being brought up in water to artemia and being separately added into S114, YJ1 and YJ3 bacteria suspension (final concentration of 105Cfu/ml), it is placed in 28 DEG C, it is cultivated in the shaking table of 80rpm, Vibrio luminous (final concentration of 10 is added after 22h3Cfu/ml), continue to bring up 18h, calculate halogen Worm survival rate.
4. three experimental groups in this experiment, are arranged, a control group.Three experimental groups are as follows: only add potentially beneficial bacterium, only Add Vibrio luminous, not only plus Vibrio luminous but add potentially beneficial bacterium.Control group is without any processing.Every group setting three parallel, real It tests and is repeated once to guarantee its repeatability.
As a result, it has been found that the survival rate of artemia is 83.3% in the vial of addition bacterial strain YJ3 and Vibrio luminous;And it only adds In the vial of Vibrio luminous, the survival rate of artemia only has 8.9%, illustrates that bacterial strain YJ3 can largely reduce luminous arc Infection of the bacterium to artemia.However, bacterial strain S114 and YJ1 does not have significant protective effect (Fig. 2) to artemia.Although bacterial strain YJ1 energy Enough growths for inhibiting Vibrio luminous in vitro, but it does not play a role in the living body.Lot of domestic and international research has also discovered Similar phenomenon, it may be possible to because of the action site of this bacterium in the living body and pathogen breeding and to infect site inconsistent. Therefore, the bacterial strain with antagonism is not meant to also play a role in vivo in vitro.
Four, movement Shandong outstanding person Salmonella YJ3 bacterial strain can degrade long-chain AHL molecule
Bacterial strain YJ3 is inoculated in 2216E fluid nutrient medium, for 24 hours in shaking table 170rpm, 28 DEG C of cultures.It is obtained in culture Bacterium solution in be added C12-HSL after 28 DEG C reaction for 24 hours.Centrifuging and taking supernatant reports that bacterium chromobacterium violaceum VIR24 detection is surplus with AHL Remaining AHL molecule, discovery bacterial strain YJ3 can degradable AHL molecules.And detect that density induction is quenched in its genome Enzyme illustrates that by the way that the signaling molecule that pathogen secretes is quenched may be a kind of mode that bacterial strain YJ3 plays beneficial effect.
Five, movement Shandong outstanding person Salmonella YJ3 bacterial strain is able to suppress encountered pathogenic bacteria
Show that there is movement Shandong outstanding person Salmonella YJ3 the potential quality as beneficial bacterium therefore to carry out it into one by testing above Step research.17 plants of encountered pathogenic bacterias are chosen from this laboratory strains library, are inoculated in 2216E fluid nutrient medium respectively, bacterium is made Liquid.With being coated on 2216E solid medium after normal saline dilution to a certain concentration, picking bacterial strain YJ3 dibbling is on plate. After cultivating 16-24h, whether have transparent circle, and measure diameter if observing around bacterial strain YJ3.As a result, it has been found that bacterial strain YJ3 is not only to luminous Vibrios has inhibiting effect, also has inhibiting effect to a lot of other pathogens, especially to Vibrio anguillarum and Edwardsiella tarda (Edwardsiellatarda) antagonism is most strong, to Aeromonas hydrophila (Aeromonashydrophila), mermaid The effect of vibrios, Vibrio vulnificus and vibrio mimicus (V.mimicus) is taken second place, and concrete outcome is as follows:
Six, movement Shandong outstanding person Salmonella YJ3 bacterial strain has safety
(1) Pnthogenic test of the bacterial strain YJ3 to shrimp seedling and artemia
YJ3 bacteria suspension (final concentration of 10 is added into the raising water of artemia6Cfu/ml), control group adds the sterile sea of equivalent Water, during which feeding, raising did not had no toxic side effect after 2 days.
YJ3 bacteria suspension (final concentration of 10 is added into young shrimp shrimp seedling cultivation water6Cfu/ml), control group addition equivalent is sterile Seawater, during which not feeding, continuous to be oxygenated, without death after 7 days.
(2) sensibility of the bacterial strain YJ3 to antibiotic
Can one plant of bacterium be measured be applied as beneficial bacterium, in addition to require its to experimental animal no pathogenicity other than, also The sensibility of the strains is evaluated, can effectively be controlled when being grown so that bacterial strain is explosive for some reason System.It is attached on the plate for being coated with bacterial strain YJ3 with sterile tweezers clamping antibiotic drug sensitive piece, each plate puts 6 kinds of susceptibility pieces, altogether 32 kinds.After culture for 24 hours, it is found that bacterial strain YJ3 to wherein 16 kinds of antibiotic sensitives, 7 kinds of medium sensitivities (such as following table), shows bacterial strain YJ3 meets the standard as beneficial bacterium application.
S is sensitivity, and R is resistance, and I is medium sensitivity
Therefore, movement Shandong outstanding person's Salmonella YJ3 bacterial strain of the invention can be used as effective inhibiting-bacteria preparation applied to aquaculture In, be conducive to keep microbial ecological balance in cultivated animals and breeding environment, realize the healthy sustainable development of culture fishery Exhibition.

Claims (5)

1. a kind of movement Shandong outstanding person Salmonella (Ruegeria mobilis) YJ3, which is characterized in that the guarantor of the movement Shandong outstanding person Salmonella Hiding number is CGMCC No.12381.
2. movement Shandong outstanding person Salmonella described in claim 1 inhibits the application in pathogenic bacteria growth product in preparation;Institute The pathogenic bacteria stated is Vibrio luminous.
3. application as claimed in claim 2, which is characterized in that the product is bacteria preparation.
4. a kind of bacteria preparation, it is characterised in that include movement Shandong described in claim 1 outstanding person Salmonella in the bacteria preparation Viable bacteria.
5. bacteria preparation as claimed in claim 4 is preparing the application in feed addictive or breeding water body additive.
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CN112322544B (en) * 2020-11-19 2022-03-15 中国海洋大学 Lujie's bacillus for prawn culture
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