CN103478509A - Production method of bacillus amyloliquefaciens antimicrobial lipopeptide and application thereof in prawn feed - Google Patents
Production method of bacillus amyloliquefaciens antimicrobial lipopeptide and application thereof in prawn feed Download PDFInfo
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Abstract
The invention relates to a production method of bacillus amyloliquefaciens antimicrobial lipopeptide and application thereof in prawn feeds and belongs to the technical fields of aquiculture and biology in the field of agriculture. The production method comprises the following steps that bacillus amyloliquefaciens fmb-50 with the collection number of CGMCC No.6249 is used as a strain and is sequentially subjected to seed culture, amplification culture and fermentation production to obtain fermentation liquor; the thalli of the fermentation liquor are removed through microfiltration or membrane filtration and nanofiltration; corn starch or porous starch is added for absorption and spray drying is carried out, thus obtaining an antibacterial substance feed additive with the antibacterial valence of 40000-50000 IU/g or 250000 IU/g. The antibacterial substance which is produced from the bacillus amyloliquefaciens fmb-50 through fermentation can be industrially produced in a large scale; the production period is short and production cost is greatly reduced; when used in the prawn feeds, the antibacterial substance can reduce the mortality rate, improve the utilization ratio of the prawn feeds, promote the nonspecific immunoreaction of prawns and enhance the energy metabolism level.
Description
Technical field
The invention belongs to technical field of aquaculture and biological technical field in agriculture field, relate to a kind of production method of bacillus amyloliquefaciens antibacterial lipopeptid and the application in prawn feed thereof.
Background technology
Culture fishery is being produced and process use antibiotic, is mainly prevention (prophylactic) and treatment (curative) bacterial disease.Adding antibiotic in feed can prevent and treat the aquatic products disease, promotes aquatic products output to increase and save feed.But, often use antibiotic to form drug resistance in the flora in breeding environment, once the virulent bacterium antibiotic shared to people and animals obtained drug resistance, after the human infection, likely original effectively antibiotic curative effect can reduce even invalid.In addition, the antibiotic remained in aquatic products enters human body, directly affects the mankind's health.
Food and feed belongs to same range of management in the U.S., is applicable to same law.Denmark government, in order to guarantee food security, has formulated in Feed Manufacturing and has banned use of antibiotic regulation; Within 2006, European Union has forbidden using antibiotic as feed promoter in animal feeding.Along with the raising of living standards of the people, increasing to the demand of animal product, more and more higher to the cry of pollution-free food.Some agricultural byproducts of China are because agricultural chemicals, antibiotic etc are residual, and the technical indicators such as content of beary metal surpass the quality standard of European Union and main body entrance state, rejected, detain and termination of the contract etc.In recent years, the antibiotic medicine residue problem in animal product more and more is subject to people's attention, China strictly limit using some antibiotic as the health care and growth accelerator for feed.2002, the Ministry of Agriculture issued " animal food herbal medicine MRL " bulletin, and the Ministry of Agriculture means recently, is the trust of lifting consumer to livestock products, and the plan plan is completely forbidden and add antibiotic in animal feed.China is aquaculture production big country, but the outlet of aquatic products usually runs into antibiotic residue, causes outlet to be refused, and has affected the foreign trade of China.This shows traditional antibiotic arena of history that will fade out gradually, find Substitutes For Antibiotic safely and efficiently and just seem very important, in view of antibiotic adverse effect, exploitation reaches by adjusting the animal gastrointestinal tract balance focus that the green feed additive that promotes growth, maintains animal health, has no side effect has become people's research.Particularly, in the development of Future in China aquaculture, government will further strengthen the supervision to feed, veterinary drug and livestock products safe and sanitary, actively promote the development green animal husbandry.Therefore, the antibacterial additives of application modern high tech method research and development safety, efficient, environmental protection, ensure and the health and safety of animals and humans self just can make China become breeding production power by breeding production big country, realizes the modernization of aquaculture.And, at present for aquaculture, can be used for substitute antibiotics class feed addictive on market almost in space state.
The present invention is for the problems referred to above, develops a kind of feeding additive aquatic animal of the antibacterial material produced based on bacillus amyloliquefaciens.Antibacterial peptide (Antibacterial Peptides) is a kind of polypeptide quasi-molecule with antibacterial action that organism produces, and general molecular weight is smaller, usually is less than 5kD, is the important molecule barrier of host defense pathogenic microorganism invasion.Up to now, at many biologies, comprise in insect, birds, animal, plant and prokaryotes and find more than 600 kind of endogenous antibacterial active peptide.These antibacterial materials not only have wide spectrum antibacterium ability, and have fungi, virus and cancer cell are also had to certain inhibitory or killing effect.In addition, also have good stability, good water solubility, Antibacterial Mechanism unique, to characteristics such as the higher mammal normal cell are harmless, belong to have no side effect, noresidue, without a class environment-friendly type preparation that causes bacterial drug resistance.To the existing more research of antibacterial peptide, also started application in animal production reality abroad, its result of use as feed addictive is confirmed by experiment, and has formed certain market demand.But the narrow antimicrobial spectrum of animal sources antibacterial material, can only suppress gram-positive bacteria usually, limited the extensive use in feed.The antibacterial lipopeptid in bacillus source is the important a member in antibacterial lipopeptid family, and it can specificity kill the competition bacterium and harmless to host itself, and its kind comprises cyclic peptide, glycopeptide and lipopeptid, as gramicidins, bacitracin, polymyxin etc.Aquiculture animal is lived in cultivation water environment all the life, in breeding water body, aquatic livestock body surface and enteron aisle, a large amount of microorganisms is all arranged, and the micro-ecological environment of this inside and outside all has direct or indirect impact to aquiculture animal health.Simultaneously, due to microbial environment difference inside and outside dissimilar animal body, therefore, also different for the demand of antiseptic feed additive.For example be applicable to mammiferous antiseptic feed additive, may not be applicable to aquatic livestock, and aquatic livestock is due to the difference of species, the feed addictive of use also likely has a great difference.
Summary of the invention
technical problem
The objective of the invention is the above-mentioned deficiency for prior art, a kind of antibacterial material feed addictive in bacillus amyloliquefaciens source is provided.
Another object of the present invention is to provide the preparation method of this antibacterial material feed addictive.
Another purpose of the present invention is to provide the application of this antibacterial material feed addictive.
technical scheme
Purpose of the present invention can be achieved through the following technical solutions:
A kind of bacillus amyloliquefaciens (
bacillus amyloliquefaciens) preparation method of antibacterial material feed addictive, comprise following steps:
The bacillus amyloliquefaciens fmb-50 that the preserving number of take is CGMCC No.6249 is bacterial classification, by seed culture, expansion, cultivates successively and fermenting and producing acquisition zymotic fluid; Zymotic fluid is removed thalline through micro-filtration, adds 10%-15%(g/100ml) absorption of cornstarch or porous-starch, spray-drying obtains described antibacterial material feed addictive, and described antibacterial material feed addictive antibacterial potency is 40000 ~ 50000 IU/g; Perhaps zymotic fluid is after micro-filtration is removed thalline, then, through film ultrafiltration and the nanofiltration of 10KD, adds 10%-15% cornstarch or porous-starch absorption, spray-drying to obtain described antibacterial material feed addictive; Described antibacterial material feed addictive antibacterial potency is 250000 IU/g left and right; Wherein, the culture medium that described seed culture and expansion are cultivated is: glucose 3.5g/L, tryptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L, pH7.0; 115 ℃ of high pressure steam sterilization 30min; The liquid fermentation medium that described fermenting and producing is used is: Fructus Hordei Germinatus soaks powder 1g/L, analysis for soybean powder 15g/L, CaCO
36.68g/L, FeSO
40.4g/L, MgCl
22g/L, initial pH 7.0; 2 ~ 5% inoculate bacillus amyloliquefaciens fmb-50(CGMCC No.6249 by volume in fermenting and producing), 1000 L fermentation tank liquid deep layer fermenting 36～40 h, fermentation parameter is: 33～37 ℃ of temperature, speed of agitator 180 r ∕ min, throughput 80 m
3/ h, tank pressure 0.10 ~ 0.12 Mpa, pH7.0.
The bacillus amyloliquefaciens antibacterial material feed addictive prepared according to described preparation method.
The application of bacillus amyloliquefaciens antibacterial material feed addictive of the present invention in preparation fish or shrimp feed; The preferably application in preparation shrimp feed, the further preferably application in preparing feed for litopenaeus vannamei.
Add the bacillus amyloliquefaciens antibacterial material feed addictive containing antibacterial material in feed for litopenaeus vannamei, addition is to add 200000～400000IU antibacterial material in the per kilogram feed.
Beneficial effect:
1. the microbial antibacterial material in the present invention is compared with the animal sources antibacterial material, has the antibiotic advantage of broad-spectrum high efficacy.
2. the present invention utilizes the bacillus amyloliquefaciens fmb-50 that preserving number is CGMCC No.6249 to produce antibacterial material fat for fermenting raw materials, but large-scale industrial production, with short production cycle, production cost reduces greatly, and not being subject to the impact of the external environments such as season and climate change, is the optimal path that overcomes antibacterial material practical application technical bottleneck.
3. bacillus amyloliquefaciens antibacterial material additive of the present invention, for feed for litopenaeus vannamei, can reduce the death rate, improves efficiency of feed utilization, the promotion non-specific immune response of Environment of Litopenaeus vannamei Low of Environment of Litopenaeus vannamei Low and improves the energetic supersession level.
4. additive of the present invention is made an addition in the Environment of Litopenaeus vannamei Low mixed feed, do not use antibiotic, also can promote preferably growth and regulate immunity, the substitute feed antibiotic is added and ensures that the safety of food is significant.
biological preservation
Fmb-50 be bacillus amyloliquefaciens (
bacillus amyloliquefaciens), on June 21st, 2013 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, and Institute of Microorganism, Academia Sinica, preserving number is CGMCC NO.6249.
The specific embodiment
Embodiment 1
Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) fmb-50 is
bacillus amyloliquefacienseS-2-4 is through N
+the bacterial strain of the high yield antibacterial lipopeptid that the high productive mutant that the high productive mutant of ion mutagenic and breeding and nitrosoguanidine mutagenesis obtain etc. obtain through the protoplast fusion seed selection, called after fmb-50.
b. amyloliquefaciensfmb-50 be take corn flour and can be obtained the highest antibacterial lipopeptid output as carbon source, and its output is 3.4 times of parent strain ES-2-4.[the Taguchi method is optimized for Yao Shulin, Lu Zhaoxin etc.
b. amyloliquefaciensfmb-50 produces surfactin industrial fermentation culture medium, Beijing University of Chemical Technology's journal, and 2012,39(4): 77-83].Original bacterium with fmb-50
b. amyloliquefacienseS-2-4,
b. subtilis168 Hes
b. subtilisthe described bacterial strain of fmbJ(is public, sees yellow existing green grass or young crops etc., and bacillus subtilis fmbJ produces lipopeptid inhibition point mould effect and antiseptic experiment to peach thereof, 2008, EI; Sun Lijun etc., culture medium produces the impact of antibacterial lipopeptid, 2008, Scientia Agricultura Sinica on bacillus amyloliquefaciens ES-2 bacterial strain; Shanghai Inst. of Biochemistry, Chinese Academy of Sciences's genetic engineering group, application method for transformation breeding high-yield AMS from hay bacillus 168 produces bacterium, 1979, Acta Biochimica et Biophysica Sinica) in same fermentation condition fermentation, get respectively the zymotic fluid of 100ml, sink through bactofugation body, acid, sediment dissolves with methyl alcohol, obtain antibacterial material, then the bacterial strain of his-and-hers watches 1 carries out antibacterial experiment.Adopt TSA(tryptose soya agar culture medium) culture medium 37 ℃ of cultivation vibrio parahaemolytious, Vibrio harveyi, vibrio alginolyticus and Streptococcusagalactiaes, beat the 5mm hole and add above-mentioned antibacterial material 50 μ L aloft on agar plate, measure bacteriostatic diameter (table 1) after 24h.
The inhibition (inhibition zone mm) of table 1 bacillus to aquatic pathogenic bacterium
Nd: have no inhibition zone
With bacillus amyloliquefaciens (
bacillus amyloliquefaciens) fmb-50, seed culture medium enlarges to be cultivated; Seed culture medium: glucose 3.5g/L, tryptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L, water 1000ml, pH7.0,115 ℃ of high pressure steam sterilization 30min, cultivate and enter exponential phase through 10-16h, is inoculated into fermentation tank.Liquid fermentation medium: fermentation medium: Fructus Hordei Germinatus soaks powder 1g/L, analysis for soybean powder 15g/L, CaCO
36.68g/L, MnSO
40.4g/L, MgCl
22g/L, initial pH 7.0; Volume ratio 2 ~ 5% inoculations, 1000 L fermentation tank liquid deep layer fermenting 36 ~ 40 h, fermentation parameter is: 33～37 ℃ of temperature, speed of agitator 180 r ∕ min, throughput 80 m
3/ h, tank pressure 0.10 ~ 0.12 Mpa, pH7.0.Zymotic fluid adopts the miillpore filter micro-filtration that aperture is 0.1 μ m to remove thalline, take that to remove the later fermented liquid supernatant of thalline be benchmark, adds 10-15%(g/100ml) cornstarch or porous-starch absorption, spray-drying obtain the antibacterial material additive.With
e. colifor measuring bacterial strain, Nisin is the antibacterial material benchmark, adopts the Oxford agar diffusion method to measure tiring of antibacterial material, and the dry products antibacterial potency is 40000 IU/g left and right.
Adopt membrane filter system (Jiangsu Jiuwu High-Tech Co., Ltd.) to fermentation liquor treatment, obtain the component of antibacterial material.Concrete operations are as follows: the first step is removed bacillus amyloliquefaciens thalline and the impurity that is greater than 0.1 μ m in zymotic fluid with ceramic micro filter film, adds the part of holding back of 10L clear water washing micro-filtration simultaneously, and cleaning solution passes through micro-filtration again, merges micro-filtration filtrate twice.Milipore filter for second step (milipore filters of 10 KDa) is removed the soluble impurity that molecular weight in micro-filtration filtrate is greater than 10 KDa, and the same 10L of adding clear water washing ultrafiltration is held back the part cleaning solution and passed through ultrafiltration again, collects two times of ultrafiltration filtrate.Then, ultrafiltrate is removed to soluble impurity and the most of moisture of little molecular weight through NF membrane (<200Da), collected the component that contains antibacterial material.Add appropriate cornstarch or porous-starch, spray-drying, obtain highly active antibacterial material additive powder.With
e. colifor measuring bacterial strain, Nisin is the antibacterial material benchmark, adopts the Oxford agar diffusion method to measure tiring of antibacterial material, and antibacterial activity reaches about 250000IU/g dry powder left and right.
Embodiment 2
By the pathogenic bacteria vibrio parahaemolytious that activated, Vibrio harveyi, vibrio alginolyticus, Streptococcusagalactiae, (described bacterial strain is public, sees Chen Jixiang etc., pathogenic Vibrio harveyi hemolysin gene clone and detecting, 2005, Chinese aquatic science;
zhai Xiumeideng, the impact of vibrio parahaemolytious on the Penaeus Vannmei physiological and biochemical index, 2007, Shanghai Aquatic Products Univ. 9CN)'s journal;
the Yan Qing Pideng, the varying environment condition adheres to the impact of large yellow croaker casing slime on vibrio alginolyticus, and 2006, aquatic product journal;
wu Yingruideng, 153 kinds of Chinese herbal medicines inhibition activity research to Tilapia mossambica Streptococcusagalactiae and Streptococcus iniae, Journal of Northwest Sci Tech University of Agriculture and Forestry, 2013) receive respectively TSB(tryptone soybean fluid nutrient medium) in, cultivate 4 ~ 6 h for 36 ± 1 ℃, with the TSB culture medium, adjust bacterium OD
600the value, to bacterial concentration be 1 * 10
8, after 100 times of dilutions standby (1 * 10
6).The fish serum preparation: approximately 2 jin heavy red snapper live fishes are bought in market, from the vertebral artery blood drawing, after centrifugal 5 min of 3000 rpm, get supernatant, and 4 ℃ of refrigerations are standby.
Get 40000IU/g antibacterial material additive (embodiment 1 first method preparation) 2 g, doubling dilution, then get successively 50 μ L and add in 96 microwell plate 1 ~ 11 holes respectively, and the 12nd hole does not add antibacterial material.Then add respectively the bacterium 10 every hole of μ L(final concentrations 1 * 10 in 1 ~ 12 hole
5cFU), 40 μ L dilutions (distilled water, seawater, physiological saline, 2% sodium chloride or fish serum), the 12nd hole is contrast, and the dilution of adding 50 μ L replaces the antibacterial material additive, and every hole cumulative volume is 100 μ L.Enzyme mark version after above-mentioned application of sample is put into to aseptic sylphon, be placed in incubator, cultivate 24 h for 28 ℃.Then measure each micropore OD with ELIASA
600value, the results are shown in Table 1.
The minimum inhibitory concentration of table 1. bacillus amyloliquefaciens antibacterial material additive to several seafood fish pathogenic bacteria
From table 1, the bacillus amyloliquefaciens antibacterial material has antibacterial action preferably to seawater fish common pathogen vibrio parahaemolytious, Vibrio harveyi, vibrio alginolyticus, Streptococcusagalactiae.To the resistance MIC such as pathogenic Vibrio harveyi, vibrio parahaemolytious, vibrio alginolyticus and Streptococcusagalactiae in seawater
50reach respectively 25 μ g/kg, 30 μ g/kg, 35 μ g/kg, 38 μ g/kg (table 1).
The antibiotic property effect to pathogenic Vibrio harveyi, vibrio parahaemolytious, vibrio alginolyticus and Streptococcusagalactiae in fish serum of bacillus amyloliquefaciens antibacterial material is stronger, MIC
50reach respectively 18,28,30,30 μ g/kg, illustrate that the bacillus antibacterial material can bring into play better antibacterial action in serum.
The bacillus amyloliquefaciens antibacterial material still keeps higher antibacterial activity in seawater, and in distilled water, activity, significantly lower than other group, shows that certain density salting liquid is conducive to keep the activity of antibacterial material.
Embodiment 3
Select outward appearance normal, the physique stalwartness, the Environment of Litopenaeus vannamei Low that the equal weight of tail is 1. 02 ± 0.10 g is used shrimp as test.Take fish meal, dregs of beans, peanut meal and brewer's yeast as protein sources, and fish oil and soya-bean oil (1: 1) are fat source, and flour is carbon source, and add suitable minerals and vitamins etc. as Environment of Litopenaeus vannamei Low mixed feed (table 2).According to adding the 6g(A group in the per kilogram mixed feed), the 8g(B group), the 10g(C group) described antibacterial material feed addictive (first method preparation in embodiment 1,40000 IU/g tire), do not add the CK group (subordinate list 2) of antibacterial material.3 repetitions are established in each processing, and each Glass fibre reinforced plastic tub is put Environment of Litopenaeus vannamei Low 30 tails in a suitable place to breed, and the Glass fibre reinforced plastic tub water capacity is 0. 3 m
3.Water temperature is (27. 5 ± 1. 5) ℃, and sea water specific gravity is 1. 012 ± 0. 002, and culture experiment continues 6 weeks.Test claims initial weight after first supporting 7 days temporarily with shrimp, and then average packet, adopt and quantitatively throw something and feed.The feeding volume initial stage, the later stage threw something and fed by 8 % by 10 % of shrimp body weight.Feeding time is respectively 06: 00,10: 00,15: 00,19: 00 and 23: 00 o'clock, and early 6 feeding volumes with 19 of evenings are more, accounts for 70 % of day feeding volume.Claim whole opisthosoma quality to stop throwing something and feeding in first 24 hours.
The average weight gain of A group cultivation after 6 weeks that adds 6g/kg improves 12.3 % than the CK group; Survival rate has improved 7.5% than CK; Feed efficiency has improved 14.5% than CK.The average weight gain of B group cultivation after 6 weeks that adds 8g/kg improves 21.3 % than the CK group; Survival rate has improved 20.8% than CK; Feed efficiency has improved 44.1% than CK.The average weight gain of C group cultivation after 6 weeks that adds 10g/kg improves 26.2 % than the CK group; Survival rate has improved 13.8% than CK; Feed efficiency has improved 39.2% than CK.
Get 8 ~ 10 tail prawns for every group, with 1 mL syringe, from heart extracting blood, be placed in the Eppendorf pipe, centrifugal after standing 24 h in 4 ℃ of refrigerators, get supernatant standby.Superoxide dismutase activity (SOD) is pressed the pyrogallol autoxidation method mensuration of the improvement such as Deng Biyu.Phenol oxidase (PO) vigor be take with reference to Ashida the method that L-dopa is the substrate colorimetric and is measured.Serum ACP, AKP activity, the test box that adopts Nanjing to build up production is measured.Adopt the method mensuration peroxidase (POD) of Guan Huashi and the vigor of lysozyme (LSZ).
The test A group alkaline phosphatase (AKP) relevant with energetic supersession and nutrition that adds 6g/kg improved 104% than CK group; The lysozyme relevant with antibacterial action (LSZ) improved 37.4% than CK; Improved 113% with the relevant phenol oxidase of non-specific immune response (PO) of crustacean than CK group; Improved 24.1% with anti-oxidant, disease-resistant relevant superoxide dismutase (SOD) than CK group; But acid phosphatase (ACP) and peroxidase (POD) do not have significant difference.
The test B group alkaline phosphatase (AKP) relevant with energetic supersession and nutrition that adds 8g/kg improved 158% than CK group; The lysozyme relevant with antibacterial action (LSZ) improved 72.1% than CK; Improved 152% with the relevant phenol oxidase of non-specific immune response (PO) of crustacean than CK group; Improved 72.3% with anti-oxidant, disease-resistant relevant superoxide dismutase (SOD) than CK group; But acid phosphatase (ACP) and peroxidase (POD) do not have significant difference.
The test C group alkaline phosphatase (AKP) relevant with energetic supersession and nutrition improved 110% than CK group; The lysozyme relevant with antibacterial action (LSZ) improved 79.5% than CK; Improved 138% with the relevant phenol oxidase of non-specific immune response (PO) of crustacean than CK group; Improved 25.8% with anti-oxidant, disease-resistant relevant superoxide dismutase (SOD) than CK group; But acid phosphatase (ACP) and peroxidase (POD) do not have significant difference.
Table 2. prawn test feed formula and experimental design
* in table 2, each raw material unit is g.
Claims (5)
1. the production method of a bacillus amyloliquefaciens antibacterial material feed addictive, is characterized in that, comprises following steps:
The bacillus amyloliquefaciens that the preserving number of take is CGMCC No.6249 (
bacillus amyloliquefaciens) fmb-50 is bacterial classification, successively by seed culture, enlarge and cultivate and fermenting and producing obtains zymotic fluid; Zymotic fluid is removed thalline through micro-filtration, adds 10%-15%(w/v) absorption of cornstarch or porous-starch, spray-drying obtains described antibacterial material feed addictive; Perhaps zymotic fluid is after micro-filtration is removed thalline, then, through film ultrafiltration and the nanofiltration of 10KD, adds 10-15%(w/v) cornstarch or porous-starch absorption, spray-drying obtain described antibacterial material feed addictive; Described antibacterial material feed addictive antibacterial potency is 40000 ~ 250000 IU/g; Wherein, the culture medium that described seed culture and expansion are cultivated is: glucose 3.5g/L, tryptone 10g/L, yeast extract 5g/L, sodium chloride 5g/L, pH7.0; The liquid fermentation medium that described fermenting and producing is used is: Fructus Hordei Germinatus soaks powder 1g/L, analysis for soybean powder 15g/L, CaCO
36.68g/L, FeSO
40.4g/L, MgCl
22g/L, initial pH 7.0; 2 ~ 5% inoculate bacillus amyloliquefaciens fmb50 by volume in fermenting and producing, 1000 L fermentation tank liquid deep layer fermenting 36～40 h, fermentation parameter is: 35～37 ℃ of temperature, speed of agitator 180 r ∕ min, throughput 80 m
3/ h, tank pressure 0.10 ~ 0.12 Mpa, pH7.0.
2. the bacillus amyloliquefaciens antibacterial material feed addictive prepared according to production method claimed in claim 1.
3. the application of bacillus amyloliquefaciens antibacterial material feed addictive claimed in claim 2 in preparation fish or shrimp feed.
4. application according to claim 3, is characterized in that, described shrimp is Environment of Litopenaeus vannamei Low.
5. application according to claim 4, is characterized in that, adds the bacillus amyloliquefaciens antibacterial material feed addictive containing antibacterial material in feed for litopenaeus vannamei, and addition is to add 200000～400000U antibacterial material in the per kilogram feed.
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