CN109536418A - A kind of microbial bacterial agent and its application in aquaculture - Google Patents
A kind of microbial bacterial agent and its application in aquaculture Download PDFInfo
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Abstract
The invention discloses a kind of microbial bacterial agents, specifically disclose by one plant of pig source bacillus licheniformisBacillus licheniformis YHSH‑02The microbial bacterial agent of preparation and the application in aquaculture.The strain of the invention is in the preservation of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.15454, and the deposit date is on March 15th, 2018.Bacillus licheniformis of the inventionYHSH‑02It is to separate to obtain from wild boar enteron aisle, small peptide abundant can be generated, lipopeptid class bacillin, antibacterial protein etc. can inhibit vibrio parahaemolytious, Edwardsiella tarda, the growth of a variety of pathogenic bacterias such as Aeromonas hydrophila, Pseudoalteromonas, Vibrio vulnificus, it can be used for preventing and treating by vibrio parahaemolytious, Edwardsiella tarda, aquiculture disease caused by the various bacterias such as Aeromonas hydrophila, Pseudoalteromonas, Vibrio vulnificus.Microbial bacterial agent of the invention finds that prawn " black leg " can be effectively prevented for the first time, provides a kind of effective scheme for prevention and treatment prawn " black leg ", be of great significance.The bacillus licheniformis that the present invention is obtained from wild boar enteron aisleYHSH‑02It is the significant somatotrophic biological control potentiality bacterial strain that one plant of prevention and treatment is high-efficient, prevention and treatment range is wide, there is good application and development prospect.
Description
Technical field
The invention belongs to microorganisms technical fields, disclose a kind of microbial bacterial agent and its application in aquaculture,
One kind is specifically disclosed by bacillus licheniformisBacillus licheniformis YHSH-02Micro- life is prepared in fermentation
Object microbial inoculum and the application in aquaculture.
Background technique
In recent decades, the feed medicated premix based on antibiotic, hormone, preservative is to culture fisheries such as fishes and shrimps
Production brings very big benefit.However, also there is some drawbacks, such as the production of antibody-resistant bacterium with its extensive use
Life, the residual and suprainfection of antibiotic in livestock and poultry, fishes and shrimps aquatic products etc., common concern of the these problems by international community
With worry, more and more countries have been prevented or restricted from Multiple Classes of Antibiotics, hormone, preservative as feed addictive one after another.It is anti-
The application of the medicated feed additives such as raw element, hormone, preservative is by serious challenge.Therefore, developing and promote one kind can
Effective substitute antibiotics, hormone, preservative can effectively health care, epidemic prevention, somatotrophic novel green feed addictive have become again
For the task of top priority.
The disease problem of fishes and shrimps bacteriosis (such as vibrios) is the bottleneck system for hindering the sustainable development of raising fish and shrimp industry always
About, it in particular with the fast development of raising fish and shrimp and the raising of intensive culture degree, and is fed and is commonly used using man-made feeds
Disease-resistant chemical combination medicine such as antibiotic, hormone, preservative have the shortcomings that again compared with being also easy to produce drug resistance and side effect is big.Especially
Nearly 2 years, the south of Fujian Province Zhaoan, Zhangpu, East Guangdong front three, Fujian east was permanently happy, Xiapu one is broken out with the Penaeus Vannmei of high-density breeding
Property it is dead.There is apparent nigrescence symptom in crust near shrimp swimming foot of dying of illness, and raiser is known as " black leg ".National Bureau of Oceanography
Xu Chang'an, third institute of oceanography et al. has carried out the Pathogen test of various prawn, testing result by fluorescence quantifying PCR method
Show: the generation of " black leg " and a kind of novel shrimps irido virus (CQIV) are infected with relationship；But it is carried out in laboratory
Seldom there is " black foot " symptom in the artificial oral challenge experiment of CQIV, the dead prawn that falls ill.It is inferred that " black leg " shrimp goes out
Now in addition to infecting CQIV, it is likely that there is also environment or the factors of other cause of diseases.Other than novel iridescent virus, some researchs
Detect dead nodavirus steathily；Also have and be considered since bottom of pond pathogenetic bacteria is excessive, substrate is too poor caused.In fact, its
Concrete reason is come to a conclusion not yet, and therefore, black leg especially when dead shrimp quantity increases, has no effective once occurring at present
Treating method, it is proposed that go out shrimp in time, not delay, reduce loss as far as possible.Therefore, a kind of prevention and treatment " black leg " how is found
Active drug is an important topic of prawn culturing.
The means of prevention of China directed toward bacteria property aquatic products disease still relies primarily on the chemicals such as antibiotic, antibiosis at present
The measures such as extract for treating can achieve the purpose that part control disease occurs, promotes cultivated animals growth, but thus bring cause of disease
The problems such as microorganism drug resistance, medicament residue, water environment pollution and fish quality, is increasingly severe;Simultaneously as this
The long-time service of a little drugs, also damaged while killing causal organism cultivated animals skin, enteron aisle etc. possessed it is normal
Microbial barrier, the endogenous pathogen or exogenous pathogen for so that those was inhibited originally by normal flora barrier are a large amount of
Breeding destroys the microecological balance of cultivated animals epidermis and enteron aisle normal microflora, and causing cultivated animals disease, a situation arises
It makes the matter worse.Inhibit pathogen using the antagonistic microbe in natural environment, prevent and treat the pathogen invasion of cultivated animals, controls disease
Occur, i.e. biological control is always a research hotspot.It is manually set up from nature separation, identification or by bioengineering beneficial
Microorganism, exploitation through the special process such as culture, fermentation, drying, processing be made containing viable bacteria and for animal biological agent or
Active bacteria formulation has important theory significance and practical application value.
Summary of the invention
The purpose of the present invention is to provide a kind of microbial bacterial agents, and being specifically to provide one kind can a variety of aquatic products of antagonism by one plant
Pathogen, a kind of microbial bacterial agent made of the significant somatotrophic pig source the lichen bacillus ferments of energy；Through overtesting, the bacterium
In the salinity of 0-7% can well-grown, can be in seawater and freshwater all well-growns, and to a variety of pathogenic bacterias
Such as trowel vibrios, vibrio parahaemolytious, Vibrio vulnificus, vibrio alginolyticus, Vibrio harveyi, streptococcus, tarda, vibrio fluvialis,
Vibrio mimicus, Acinetobacter bauamnnii, Pseudoalteromonas, Aeromonas hydrophila, which has, significantly inhibits effect, can preferably prevent and treat
Prawn " black leg ".The strain is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese microorganism strain preservation conservator
Meeting common micro-organisms center preservation, deposit number are CGMCC NO.15454, and the deposit date is on March 15th, 2018.
On the other hand, it is an object of the present invention to provide a kind of microbial bacterial agents (bacillus licheniformis preparation) to prepare antagonism water
Produce the application in the biological products of cultivation pathogen.
Wherein, the application, which is characterized in that the biological products are zymocyte liquid or bacterium powder.Its zymocyte liquid
Or bacterium powder can be prepared by existing fermentation technique, the present invention provide the preparation method comprises the following steps:
One, bacillus licheniformis is taken outBacillus licheniformis YHSH-02Culture presevation pipe, with LB solid culture
Base draws plate and recovers, and 37 DEG C are cultivated 48 hours.Picking single bacterium colony is inoculated in 50 milliliters of LB culture mediums under plate, is being trained
Support 37 DEG C shake culture 24 hours in case.Seed uses 5% inoculum concentration, and it is big by three to be seeded to the 5L that liquid amount is 2LLB culture medium
In the bottle of angle, 37 DEG C shake culture 20-28 hour, detect its bacterial concentration, viable bacteria content can be used as ground more than 2,000,000,000/milliliter
Clothing bacillus liquid strain；
Two, using the bacillus licheniformis liquid spawn of step 1 culture as seed, 600L fermentation medium, inoculum concentration are seeded to
Be 10%, open stirring 120r/min, minute ventilation volume liquid-gas ratio 1:0.8,37 DEG C culture 24-36 hours, it is not low to spore content
In 15,000,000,000 CFU/ml, can terminate to ferment, sterile filling obtains finished product bacillus licheniformis agent；Or add after obtaining fermentation liquid
Upper dextrin is spray-dried, and bacillus licheniformis pulvis is obtained, and pulvis spore content is not less than 10,000,000,000 CFU/g；
The wherein LB culture medium: peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, in solid medium plus
Enter agar 2%；
The wherein fermentation medium: 50 g/L of bean cake powder, 10 g/L of fish meal, 10 g/L of corn starch, glucose 5g/L are solvable
Property 10 g/L of starch, 5 g/L of peptone, 0.3 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, carbonic acid
Calcium 5 g/L, pH7.2；The condition of each medium sterilization are as follows: 0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
Effect of the invention: the bacillus licheniformis in microbial bacterial agent of the inventionYHSH-02It is from wild boar enteron aisle
What separation obtained, small peptide abundant, lipopeptid class bacillin can be generated, antibacterial protein etc. can inhibit vibrio parahaemolytious, slow love moral
Fahrenheit bacterium, the growth of a variety of pathogenic bacterias such as Aeromonas hydrophila, Pseudoalteromonas, Vibrio vulnificus, can be used for preventing
It controls by vibrio parahaemolytious, Edwardsiella tarda, caused by the bacteriums such as Aeromonas hydrophila, Pseudoalteromonas, Vibrio vulnificus
Aquiculture disease.
Bacillus licheniformis liquid of the present invention has immunological enhancement, and the true nontoxic substance in border, degree of safety is big.
Bacillus licheniformis preparation of the present invention can purify water, reduce pathogen in water body and shrimp body, improve cultivation density.
Bacillus licheniformis preparation of the invention finds that prawn " black leg " can be effectively prevented for the first time, for mentioning for prevention and treatment prawn " black leg "
For a kind of effective scheme, it is of great significance.
The bacillus licheniformis that the present invention is obtained from wild boar enteron aisleYHSH-02Being one plant, prevention and treatment is high-efficient, prevention and treatment range is wide
Biological control potentiality bacterial strain, have good application and development prospect.
Specific embodiment
The separation of 1 bacillus licheniformis of embodiment
In December, 2014, bacillus licheniformis (Bacillus licheniformis) bacterial strain is in Hunan Province, beachhead town, Longhui County
It is isolated in the intestinal contents of the wild wild boar in field.Specific method is: the intestinal contents of wild wild boar being taken to be added
In the triangular flask for filling 10mL aqua sterilisa, shake culture 30min (200r/min) then takes suspension 1mL to be placed in 9mL sterile
In water, it is made 10-2Diluted suspension liquid, and so on, it is made 10-5, 10-6Dilute suspension；100 u L are respectively drawn wherein, are added to
Nutrient broth medium plate, MRS culture medium in PDA culture medium, are coated with uniformly, each processing is repeated 3 times with spreading rod.It will
Above-mentioned culture dish, be placed in 37 DEG C of constant incubators cultivate 1-2d(PDA culture medium set cultivated at 30 DEG C).Picking individual colonies switching
It is cultivated on to corresponding culture medium, after growing bacterium colony, carries out scribing line with oese and isolate and purify, purifying bacterial strain is saved in 4 DEG C.Always
It isolates to obtain 218 variant single colonies of form.
The YHSH-02 bacterial strain for separating, purifying acquisition monoclonal on nutrient broth medium therein grows to be formed
Bacterium colony be almond white, moisten glossy, slightly radial-like streak, after culture 48 hours, have on culture medium and obvious produce brown
Pigment；Cell shape be it is rod-shaped, cell size be 0.6 um -3.5 of um -0.8um × 1.5 um；Both ends blunt circle forms bud
Spore, gemma is closely oval, middle life, has peritrichous, can move.Do not form parasporal crystal；It is aerobic to be grown with anaerobism；It can
Liquefy gelatin, hydrolyzes starch, V-P reaction, contact enzyme reaction, casein decomposition reaction, milk, which peptonizes, to react for the positive；Yolk is anti-
It answers, indole reaction, phenylalanine deamination reaction, tyrosine catabolic reaction, milk Hirschfeld-Klinger reaction is feminine gender；Glucose can be utilized,
Xylose, L-arabinose, mannose etc..Optimal pH is 5.5-8.7, and optimum growth temperature is 25-38 DEG C, in the salinity of 0-7% all
Energy well-grown, 15-55 DEG C of growth temperature range is accredited as bacillus licheniformis by 16s rDNA.
In Vitro Bacteriostasis effect of 2 bacillus licheniformis of embodiment to common pathogen
Specific experimental method is as follows:
1、YHSH-02The preparation of bacteria suspension: taking inclined-plane seed, is seeded in 50m1 sterilizing TSB fluid nutrient medium, constant-temperature table shake
Swing culture 18h (30 DEG C of cultivation temperature, revolving speed 200rpm)；It is spare by bacteria suspension 8m1 in l0ml sterile centrifugation tube, it is existing
It does current.
2, the preparation of the bacteria suspension of the kinds of pathogenic vibrio such as Vibrio anguillarum
(1) lawn one ring in picking inclined-plane is seeded to 50m1 TSB fluid nutrient medium, shaking table shake culture 18h (cultivation temperature 30
DEG C, revolving speed 140rpm);
(2 take above-mentioned bacteria suspension 5m1 renewed vaccination newly to match TSB fluid nutrient medium, shaking table shake culture 18h (cultivation temperature to 50m1
30 DEG C, revolving speed 140rpm);
(3) supernatant is removed in centrifugation (5000rpm, l0min), then adjusts cell concentration about 1.0 × 10 with physiological saline8cfu/
Ml takes the uniform coated plate of 0.1 ml.
3, measuring method
(1) the above-mentioned Vibrio anguillarum bacteria suspension 10 prepared is taken8Other vibrios of cfu/ml(are tested in the same way) 0.1
Ml is spread evenly across TSA planar surface (each plate about 30m1 TSA solid medium);
(2) five holes, the diameter of punch about 7mm are uniformly made a call on the agar medium for be coated with pathogenic vibrio with punch.
(3) then that following liquid (about 0.2 ml/hole) bacteria suspension, physiological saline feminine gender are separately added into agar hole is right
According to;
(4) above-mentioned plate is set into constant incubator culture -48h for 24 hours, then observes outcome measurement antibacterial circle diameter.Experimental result
It is shown in Table 1.The result shows that bacillus licheniformis YHSH-02 is to the pathogen trowel vibrios in aquaculture, vibrio parahaemolytious, wound
Vibrios, vibrio alginolyticus, Vibrio harveyi, streptococcus, tarda, vibrio fluvialis, vibrio mimicus, Acinetobacter bauamnnii are false
Alteromonad, Aeromonas hydrophila etc. all have apparent inhibitory effect.
In Vitro Bacteriostasis effect of 1. bacillus licheniformis of table to pathogen
| Pathogen | Bacteria suspension | Negative control (sterile saline |
| Trowel vibrios | 32±2 | 7 |
| Vibrio parahaemolytious | 29±2 | 7 |
| Vibrio vulnificus | 22±1 | 7 |
| Vibrio alginolyticus | 28±1 | 7 |
| Vibrio harveyi | 22±1 | 7 |
| Streptococcus | 28±1 | 7 |
| Tarda | 33±2 | 7 |
| Vibrio fluvialis | 27±1 | 7 |
| Vibrio mimicus | 24±1 | 7 |
| Acinetobacter bauamnnii | 20±1 | 7 |
| Pseudoalteromonas | 18±1 | 7 |
| Aeromonas hydrophila | 28±2 | 7 |
The preparation of the microbial bacterial agent of the present invention of embodiment 3 (bacillus licheniformis preparation)
One, culture presevation pipe is taken out, plate is drawn with LB solid medium and recovers, 37 DEG C are cultivated 48 hours.It is chosen under plate
Single bacterium colony is taken to be inoculated in 50 milliliters of LB culture mediums, in the incubator 37 DEG C shake culture 24 hours.Seed uses 5% inoculation
Amount, be seeded to liquid amount be 2LLB culture medium the big triangular flask of 5L in, 37 DEG C shake culture 20-28 hours, it is dense to detect its bacterium solution
Degree, viable bacteria content are more than 2,000,000,000/milliliter, can be used as bacillus licheniformis liquid spawn；
Two, using the bacillus licheniformis liquid spawn of step 1 culture as seed, 600L fermentation medium, inoculum concentration are seeded to
Be 10%, open stirring 120r/min, minute ventilation volume liquid-gas ratio 1:0.8,37 DEG C culture 24-36 hours, it is not low to spore content
In 15,000,000,000 CFU/ml, can terminate to ferment, sterile filling obtains finished product bacillus licheniformis agent；Or add after obtaining fermentation liquid
Upper dextrin is spray-dried, and bacillus licheniformis pulvis is obtained, and pulvis spore content is not less than 10,000,000,000 CFU/g；
The wherein LB culture medium: peptone 5g, beef extract 3g, sodium chloride 5g, water 1000mL, pH7.2, in solid medium plus
Enter agar 2%；
Wherein, the fermentation medium: 50 g/L of bean cake powder, 10 g/L of fish meal, corn starch 10 g/L, glucose 5g/L can
10 g/L of soluble starch, 5 g/L of peptone, 0.3 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 0.5 g/L of potassium dihydrogen phosphate, carbon
Sour 5 g/L of calcium, pH7.2；The condition of each medium sterilization are as follows: 0.10-0.15MPa, 121 DEG C sterilize 30 minutes.
The safety testing of the microbial bacterial agent of the present invention of embodiment 4 (bacillus licheniformis preparation)
The bacillus licheniformis liquid that the embodiment of the present invention 2 produces is in Shaoyang institute through rat acute toxicity test, conclusion The results table
Bright bacillus licheniformis liquid is to rat (feeding bacillus licheniformis liquid of the present invention 14 days by 0.6ml/10g weight) effect: rat
All survival, activity freely, hair smoothing, diet is normal, respiratory tract, eye and oral cavity etc. without secretion outside, do not find any
Poisoning symptom, weight have increase.With blank group control group, compare, the rat body weight for feeding this lichens bacillus liquid is obvious
Increase (P < 0.001).14 day observation period terminated, by rat dislocate put to death dissection, visually observe the heart, liver, spleen, lung, kidney, brain,
The internal organs such as intestines, small intestine are showed no obvious abnormalities performance.In addition, rat chest gland coefficient and Spleen coefficient the result shows that, with blank pair
Compare according to group, bacillus licheniformis liquid rats in test groups Spleen coefficient obviously increases (P < 0.01), prompts this bacillus licheniformis
Liquid has immunological enhancement, and the true nontoxic substance in border, degree of safety is big.
The analysis of microbial bacterial agent of the present invention (bacillus licheniformis preparation) antibacterial substance of embodiment 5
The measurement of 1 small peptide content
It accurately weighs and takes fermentation liquid 6g in 100ml beaker, it is accurate that 15% solution of trichloroacetic acid 50ml is added, it is uniformly mixed, stands
5min is discarded a little initial filtrate, filtrate is transferred to centrifuge tube, is centrifuged at 4000r/min with the filtering of middling speed qualitative filter paper
10min accurately pipettes its supernatant 10 and measures its crude protein content by crude protein determining method in digest tube, while doing sky
White test measures the crude protein content of non-inoculation fermentation liquid (culture medium).
The coarse extraction of 2 antibacterial proteins -- ammonium sulfate different saturation is saltoutd
It takes 100 ml fermentation liquids to be statically placed in ice bath respectively, is separately added into ammonium sulfate solids to 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90% saturation degree is stood overnight, and precipitating is collected by centrifugation in next day, is delayed precipitating is dissolved in l0ml phosphoric acid respectively
(in (PB) (0.01mol/L, pH6.8), after dialysis concentration, cylinder plate method measures its antibacterial (vibrio parahaemolytious) activity to fliud flushing.
The coarse extraction of 3 other Substances
It takes 120m1 fermentation liquid tune pH value to 2-3, the extraction of equivalent ethyl acetate is added three times, and after extract liquor is filtered with filter paper,
Rotation is evaporated at 40 DEG C, obtains faint yellow crude extract, is dissolved with a small amount of ethyl acetate, organic system miillpore filter (0.45 μm of aperture)
Residue is filtered off, supernatant is settled to 5ml, i.e. Substance crude extract, 4 DEG C are sealed.
4 Substance chemical structure analysis
(1) thin-layer chromatography (TLC)
Capillary takes a small amount of crude extract point sample on silica G plate at (20 cm × 5cm × 0.25cm) edge 1.0- 1.5cm, room
Temperature is dried, chloroform: methanol: glacial acetic acid: two 6:3:1:1 of water is as solvent, the filter paper item that will impregnate in solvent first
It is pasted on four wall of chromatography cylinder, solvent is added into chromatography cylinder, volatilize 10-30min, and it is fully saturated to be unfolded agent to chromatography cylinder
Afterwards, the 60 DEG C of inclinations of excellent silica G plate will be put to be placed in chromatography cylinder, solvent front is lower than sampling point, and ascending method is opened up layer, is being unfolded
Solvent front has been remembered, iodine develops the color after drying away from thin plate is taken out when 1.0-2.0 cm at the top of silica G plate in agent forward position.By each spot
Corresponding component scrapes, and is extracted with lml methanol, cylinder plate method surveys its bacteriostatic activity, and calculates the Rf value of each component.Silica G is done simultaneously
Blank control.
(2) functional group's chromogenic reaction
Will be dry after antibacterial substance crude extract thin plate exhibition layer, ninhydrin reagent, phenolsulfuric acid reagent, ammonium molybdate-are used respectively
Perchloric acid colour reagent, 2,4-dinitrophenylhydrazine reagent, ferric trichloride reagent, Sonnenschein's reagent, the spray of p-aminobenzene sulfonic acid reagent
Mist colour developing, surveys its component.
3 results:
The fermentation liquid of bacillus licheniformis YHSH-02 bacterial strain after testing, the total initial albumen of small peptide content (30.89g/L) Zhan
The 84.5% of (36.5g/L) illustrates that this bacillus licheniformis generates protease abundant, can be substantially by the albumen base in dregs of beans
It is changed into small peptide material on this,
We test discovery, and ammonium sulfate saturation degree is lower than 30%, and when being higher than 80%, the crude protein saltoutd is seldom, slightly there is one
Point is also the foreign protein without biocidal property.From table 2 it can be seen that ammonium sulfate saturation degree has a large amount of antibacterial eggs within the scope of 30%-80%
White precipitation；Take the method for directly adding ammonium sulfate to reach terminal saturation degree, the suppression of thick leach protein when ammonium sulfate saturation degree reaches 60%
Bacterium activity is most strong, and antibacterial circle diameter is up to 35.8mm.When ammonium sulfate saturation degree 50%, biocidal property takes second place, and antibacterial circle diameter is
32.7mm。
The different ammonium sulfate saturation degree protein precipitation fungistatic effects of table 2
| Ammonium sulfate concentrations (%) | 20 | 30 | 40 | 50 | 60 | 70 | 80 | 90 |
| Antibacterial circle diameter (mm) | 0 | 24.5 | 18.6 | 32.7 | 35.8 | 28.2 | 13.4 | 0 |
For the fermentation liquid of bacillus licheniformis YHSH-02 bacterial strain after salt treatment, centrifuging and taking supernatant carries out fungistatic effect measurement, examination
Testing supernatant as the result is shown still has bacteriostatic activity.Fermentation liquid is after the ammonium sulfate precipitation of 80% saturation degree, while its fermentation liquid
Centrifuging and taking supernatant carries out fungistatic effect measurement, and test result shows that supernatant still has bacteriostatic activity.Fermentation liquid is by 80% saturation
After the ammonium sulfate precipitation of degree, supernatant is measured with bacteriostatic activity, illustrates the extracellular metabolin of bacillus licheniformis YHSH-02 bacterial strain
In there is also one or more of other Substances, such as bacteriocin, polypeptide other than antibacterial protein.This test
Primarily determine that there are also other bacteriostatic activity objects in addition to antibacterial protein in the extracellular metabolin of bacillus licheniformis YHSH-02 bacterial strain
Matter.
Measurement result (being shown in Table 3) shows that the antibacterial substance extracted by ethyl acetate, organic crude extract pass through paper chromatography and life
Object shows inhibition zone, and ninhydrin reagent reaction is positive, and occurs punctation at Rf0.84 in silica gel thin sheet, infers
The substance is polypeptide or amino acids substance;Phenolsulfuric acid reagent shows brown, occurs brown at Rf0.47 in silica gel thin sheet
Spot illustrates there is glycolipid substance, has bacteriostatic activity;One perchloric acid color developing agent of ammonium molybdate is positive, in silica gel thin sheet
Occur black-and-blue spot at Rf0.47, blue spot occur at Rf0.58, illustrates have antibacterial there are glycolipid, phospholipid substance
Activity;There is spot at thin plate Rf0.84 in Sonnenschein's reagent, illustrates that Substance is reducing substances;Ferric trichloride examination
Agent reaction is positive, and occurs green spot at silica gel thin sheet Rf0.84, shows that the substance contains phenol ring;2,4 1 dinitrobenzenes
Hydrazine reagent reaction is positive, and shows that the substance contains active carbonyl, but do not have bacteriostatic activity；The reaction of p-aminobenzene sulfonic acid reagent is in sun
Property, show the heterocyclic compound that the substance has amine and can be coupled.According to the chromatoplate chromogenic reaction preliminary analysis antibacterial work
Property substance may be peptide matters, glycolipid class, phospholipid substance.
Table 3 passes through different its antibacterial substance of reagent qualitative reaction and fungistatic effect
| Color developing agent | There are substances | As a result | Biocidal property |
| Ninhydrin reagent | Peptides, amino acids substance | + | ++ |
| Phenolsulfuric acid reagent | Glycolipid class | + | + |
| Ammonium molybdate-perchloric acid color developing agent | Phospholipid | + | + |
| Sonnenschein's reagent | Reducing substances, lipoid etc. | + | + |
| Ferric trichloride reagent | Phenolic substances | + | - |
| 2,4- dinitrobenzene hydrazine reagents | Aldehyde material | + | - |
| P-aminobenzene sulfonic acid reagent | Phenols, amine and the heterocyclic compound that can be coupled | + | + |
Embodiment 5 illustrates the effect of microbial bacterial agent of the present invention (also referred to as: bacillus licheniformis preparation) below with reference to test
1, test site: Panyu District of Guangzhou City sea-gull island；Breed variety: Penaeus Vannmei；Area: 12 mu；Culture style: Tu Tang
Mixed breeding；Put number of days in a suitable place to breed: 30 days；Water salinity is 3/1000ths.
2, problem describes: shrimp material feeding is slow, has steathily extremely, and liver pancreas color is not deep, and shrimp body vibrios is 5 × 105CFU/ml PH8-
8.5, dissolved oxygen 6.5mg/L, ammonia nitrogen 0.3mg/L, water body detect vibrios 3 × 104CFU/ml；
3, case study: prawn turns liver phase undergrowth, and the development of liver pancreas is not so good, and water vibrios are higher, seriously affect prawn
Existence, easily cause stress, but full pond aerator all has already turned on, and it is also unobvious to apply oxygenation agent effect.
4, solution: the addition of bacillus licheniformis preparation is stirred evenly in daily feed, and adding proportion control exists
0.1%；Simultaneously by the directly full pond water-sprinkling of bacillus licheniformis preparation, additive amount is 1L/ mus.
5, it result: after bacillus licheniformis preparation use of the present invention, after 24 hours, extremely significantly reduces steathily, water quality indicator are as follows:
PH8-8.5, dissolved oxygen 5.5mg/L, ammonia nitrogen 0mg/L, shrimp body vibrios are 8 × 103CFU/ml, water body detect vibrios 5 × 102CFU/ml；
After 48 hours, shrimp body colour improves, water quality indicator are as follows: PH8-8.4, dissolved oxygen 4.8mg/L, ammonia nitrogen 0mg/L；Shrimp body vibrios is
500CFU/ml, after water body detects vibrios 20CFU/ml third day, shrimp body colour is restored, and nothing is dead steathily, and water colour is in yellow green；From test
As a result as can be seen that this lichens bacillus preparation, which has, steals incruable disease caused by obvious treatment vibrios.It can purify water, reduce water
Vibrios in body and shrimp body improves cultivation density.
The microbial bacterial agent of the invention of embodiment 6 -- the controlling experiment knot of bacillus licheniformis preparation prawn vibrio infection
Fruit
1, test site: Zhanjiang City, the town Ji Jia, Leizhou City；Breed variety: Penaeus Vannmei；Area: ten mu；Culture style: Tu Tang
Mixed breeding；Put number of days in a suitable place to breed: 20 days；Water salinity is 10/1000ths.
2, problem describes: prawn, which is in, turns the liver phase, and dead serious steathily, hemolysis vibrion is more than 2 × 10 in shrimp body6CFU/g, water body
Vibrios is more than 5 × 104CFU/ml, PH8.0-8.5, dissolved oxygen 5-6.6mg/L, ammonia nitrogen 0.3mg/L, nitrite 0mg/L, hydrogen sulfide
0mg/L；
3, case study: should the early stage of prawn caused by prawn vibrios steal dead syndrome；
4, solution: the addition of bacillus licheniformis preparation is stirred evenly in daily feed, adding proportion is controlled 0.2%；
Simultaneously by the directly full pond water-sprinkling of bacillus licheniformis preparation, additive amount is 2L/ mus.
5, result: after bacillus licheniformis preparation use of the present invention, after 24 hours, extremely significantly reducing steathily, prawn body vibrios
Detection level has reduced two orders of magnitude and has reached 3 × 104CFU/g water vibrios are down to 2 × 102CFU/ml；After 48 hours,
Shrimp body colour improves, only a small amount of to steal extremely；Behind third day, shrimp body colour is restored, and nothing is dead steathily, and prawn body vibrios detection level is decreased
To 100CFU/g, water vibrios content is also reduced to 10 CFU/ml.
The microbial bacterial agent of the invention of embodiment 7 -- bacillus licheniformis preparation, the prevention and treatment effect in the quick-fried head disease of Pelteobagrus fulvidraco
Fruit
1, test site: Hunan Province Lixian County raiser, Pelteobagrus fulvidraco cultured area reach 80 mu, and morbidity pond is 18 mu, weight
In 2-4 two.
2, problem describes: July 5, begins with fish and quick-fried head disease i.e. " tassel flower " disease occurs, uses other desinsections, antibiotic
Drug effect is unobvious, July 8, and dead fish amount increases obviously, and water colour is more muddy, ammonia nitrogen 0.4mg/L, nitrite 0.2mg/L,
Hydrogen sulfide 0mg/L.
3, case study: the disease is mostly by the parasitogenic bacillary intercurrent disease of the helminths such as Trichodina.It is " Ai Dehuashi
Caused by bacterium " chronic infection.Its route of infection is via intranasal application infection olfactory cell, brain is entered back into, through infection of meninges skull.It should
Disease mostly occurs with other diseases, and most commonly septicemia easily occurs splitting head disease after occurring.Because of the infection of " tassel flower " disease
It is out of fish brain to outside brain, so once falling ill, external sterilizing, effects of antibiotics for oral administration are not very good.
4, solution: bacillus licheniformis preparation of the present invention addition is stirred evenly in daily feed, adding proportion
Control is 0.2%；Simultaneously by the directly full pond water-sprinkling of bacillus licheniformis preparation, additive amount is 1L/ mus, by increasing all in pond
Oxygen machine standard-sized sheet 48 hours.
5, result: after bacillus licheniformis preparation use of the present invention, after 24 hours, oneself loses and increases dead fish, and water colour also has
Marked improvement, after 48 hours, dead fish is significantly reduced；Behind third day, fish body color restores, and without dead fish, water colour is yellowish green, ammonia nitrogen
0.1mg/L, nitrite 0mg/L, hydrogen sulfide 0mg/L, effect highly significant.
The microbial bacterial agent of the invention of embodiment 8 -- bacillus licheniformis preparation, in the control efficiency of prawn " black leg "
1, test site: the Zhanjiang City town Jian Xin；Breed variety: Penaeus Vannmei；Area: every mouthful of three mu of pond, 8 mouthfuls in total, often
Put seedling 700,000 in mouth pond；Culture style: feed coefficient；Water salinity is 12/1000ths；Test group: Si Koutang has weekly three days
Continuously bacillus licheniformis preparation of the present invention addition is stirred evenly in daily feed, adding proportion is controlled 0.1%；Simultaneously
Weekly that the directly full pond water-sprinkling of bacillus licheniformis preparation is primary, additive amount is 1L/ mus, by aerator standard-sized sheet all in pond
48 hours.Control group:: Si Koutang has weekly conventional microbial inoculum (multienzyme benefit rhzomorph etc.) will continuously add in the market daily for three days
It is stirred evenly in feed, adding proportion is controlled 0.2%；Simultaneously weekly by effective microorganisms(EM) or photosynthetic bacteria (conventional microbial inoculum in the market)
Directly full pond water-sprinkling is primary, and additive amount was 5L/ mus, by aerator standard-sized sheet 48 hours all in pond；Other operations are identical.
Test result: cultivation in first 65 days is normal, and every mouthful of pool of control group is every 65 jin of meal material feeding or so average, and specification is averagely
60-70 head.Every mouthful of pool of test group is every 75 jin of meal material feeding or so average, and specification is averagely 50-55 head.After being come due to cold air, water
Temperature drop speed is very fast, there is Liang Koutang (control group 1 and control group 3) in control group, and when cultivation was to 70 days, it is dead steathily to begin with shell,
But it shows unobvious.Shell, which is also not just to slough off for 2-3 days, to be over, but slowly sloughs off, and has steal on a small quantity extremely before this, 2-3 jins daily, and the 74th
It starts mortality and starts more and more, and 50-100 jin daily, the shrimp cardinal symptom sucked out is exactly swimmeret nigrescence, and in pond
3-4 at shrimp also have the symptom of " black foot ".Sick shrimp living can swim over to water layer of the pond center from 20 centimeters of the water surface or so and float, vigor
It is very poor, get see all be jejunum sky stomach shrimp.Other two mouthfuls of pools of control group also start appearance one when cultivation was to 78 days
Sample have several steal extremely with black foot.
Pass through detection, it has been found that the vibrios content of water body is higher, reaches 2 × 105CFU/ml；, due to it was reported that black
Pedopathy is that (but can not be led with iridescent virus disease or nodavirus challenge test caused by iridescent virus disease or nodavirus
Prawn is caused black leg occur, therefore black leg concrete reason is also not very clear), while sample presentation to Guangdong Ocean University examines
It surveys, it is aobvious positive from that can detect irido virus in the shrimp that dies of illness in some shrimp, but not all dead shrimp has；It takes measures:
Stop expecting, by reducing water level (dropping to 1-1.2 meter from original 1.8-2 rice) and periodically sterilizing (before seldom sterilize), simultaneously
The oxidation-reduction potential of water body is improved to be handled, but is produced little effect.The pond of preceding two mouthfuls of morbidities, dead shrimp is too many, 78
It when sell shrimp, clean up the pond, last 1 average size of control group is 62.4/jin, survival rate only 54.96%, the output value only 86312.8
Member；3 average size of control group is 60.6/jin, survival rate only 57.89%, only 96962.9 yuan of the output value.When cultivating 78 days,
Due to test group, prawn feeding, body colour, liver pancreas is all normal, and when starting morbidity with the 4th mouthful of pool for the 2nd mouthful of control group, it supports
Field technology factory director is grown, then is directly splashed 5L/ mus of bacillus licheniformis liquid of the present invention to two mouthfuls of pools of control group privately, aerator
Standard-sized sheet, due to stop over material, without spice.Second day, wherein a bite pool, dead shrimp reached more than 50 jin, in addition a bite pool,
15 jin of dead shrimp or so；But it draws a design, water body shrimp body colour obviously has improvement；Behind third day, dead shrimp is significantly reduced, only how many, water body
Vibrios is down to 200CFU/ml；4th day, only several dead shrimps start to mix to halve the lichens gemma bar for feeding intake, and mixing 0.2%
Bacteria liquid microbial inoculum, operation is identical as test group later.Final test result is shown in Table 4, from table 4, it can be seen that whole use lichens
The survival rate of the test group of bacillus bacterium solution is not below 74%, and the highest survival rate of control group also only have 64.05%(this
A or later period also uses Bacillus licheniformis liquid), from specification, it is 40.8 tails/jin that control group, which supports maximum specification, and
The minimum specification of test group can reach 28.4 tails/jin；Obvious test group is growed faster, and there are many shrimp high survival rate；Compare from the output value,
The output value is less than 670,000 yuan in total in control group four mouthfuls of ponds, and the output value on 3 a bite pool of test group pond has just reached 76.9 ten thousand yuan, and four
Cause for gossip tests the pool gross output value and has reached 2,580,000 yuan, is 3.85 times of the control group output value；Economic benefit is huge.More importantly by
Arranging pool rate in prawn black leg is more than 80%, huge to the destruction of prawn industry, and uses lichens gemma bar of the present invention no
When bacteria preparation, can not substantially it treat.Therefore, Bacillus licheniformis liquid of the invention is to prevent and treat mentioning for prawn black leg
For a kind of effective scheme, it is of great significance.
4 bacillus licheniformis of table is in prevention and treatment black leg test effect
| Group | Whether fall ill | Cultivate number of days | Shrimp specification (tail/jin) out | Yield (jin) | Survival rate | The pool mouthful valence (member/jin) | The output value (member) |
| Control group 1 | It is | 78 | 62.4 | 6165.2 | 54.96% | 14 | 86312.8 |
| Control group 2 | It is | 95 | 42.1 | 10506.5 | 63.19% | 22 | 231143 |
| Control group 3 | It is | 78 | 60.6 | 6687.1 | 57.89% | 14.5 | 96962.9 |
| Control group 4 | It is | 96 | 40.8 | 10988.4 | 64.05% | 22.5 | 247239 |
| Test group 1 | It is no | 100 | 28.4 | 18892.6 | 76.65% | 28 | 528992.8 |
| Test group 2 | It is no | 100 | 26.3 | 21564.3 | 81.02% | 28.5 | 614582.6 |
| Test group 3 | It is no | 102 | 22.1 | 25231.7 | 79.66% | 30.5 | 769566.9 |
| Test group 4 | It is no | 102 | 23.5 | 22289.8 | 74.83% | 30 | 668694 |
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although by referring to this
Invention has been described for the preferred embodiment of invention, it should be appreciated by those of ordinary skill in the art that can be
Various changes are made to it in form and in details, without departing from essence of the invention defined by the appended claims
Mind and range.
Claims (4)
1. a kind of microbial bacterial agent, which is characterized in that this microbial bacterial agent is by bacillus licheniformisBacillus licheniformis YHSH-02Fermentation is prepared, wherein the strain is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number are CGMCC NO.15454, preservation
Date is on March 15th, 2018.
2. a kind of microbial bacterial agent according to claim 1, which is characterized in that the microbial bacterial agent is preparing antagonism aquatic products
It cultivates pathogen and promotes the application in the biological products grown.
3. application according to claim 2, which is characterized in that the pathogenic bacteria is trowel vibrios, secondary haemolysis
Vibrios, Vibrio vulnificus, vibrio alginolyticus, Vibrio harveyi, streptococcus, tarda, vibrio fluvialis, vibrio mimicus, Bao Man is not
Lever bacterium, Pseudoalteromonas, Aeromonas hydrophila.
4. application according to claim 2, which is characterized in that the biological products are zymocyte liquid or bacterium powder.
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