CN103478509B - 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 the technical field of aquaculture in agriculture field and biological technical field, relate to a kind of production method of bacillus amyloliquefaciens antibacterial lipopeptid and the application in prawn feed thereof.
Background technology
Culture fishery uses antibiotic in production and process, mainly prevents (prophylactic) and treatment (curative) bacterial disease.Add antibiotic in feed can prevent and treat aquatic products disease, promote aquatic products output to increase and save feed.But, often use antibiotic can form drug resistance in the flora in breeding environment, once virulent bacterium obtains drug resistance to the antibiotic that people and animals share, after human infection, likely original effective antibiotic curative effect can reduce even invalid.In addition, the antibiotic remained in aquatic products enters human body, directly affects the health of the mankind.
Food and feed belongs to same range of management in the U.S., is applicable to same portion law.Government of Denmark, in order to ensure food security, has formulated in Feed Manufacturing and has prohibitted the use antibiotic regulation; European Union in 2006 has forbidden in animal feeding, use antibiotic as feed promoter.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 remain due to agricultural chemicals, antibiotic etc, and the technical indicators such as content of beary metal exceed the quality standard of European Union and main body entrance state, rejected, detain and termination of the contract etc.In recent years, the antibiotics residues problem in animal product is more and more subject to people's attention, and China strictly limits and some antibiotic is used for feed as health care and growth accelerator.2002, the Ministry of Agriculture issued " animal food herbal medicine MRL " bulletin, and the Ministry of Agriculture represents recently, and for promoting consumer to the trust of livestock products, 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 and causes outlet to be refused, and have impact on the foreign trade of China.This shows that conventional antibiotic will fade out the arena of history gradually, find Substitutes For Antibiotic safely and efficiently and just seem very important, in view of antibiotic adverse effect, develop reach growth promoting effects by adjustment animal gastrointestinal tract balance, maintain animal health, focus that the green feed additive that has no side effect has become people's research.Particularly in the development of the following aquaculture of China, the supervision that government will strengthen further to feed, veterinary drug and livestock products safe and sanitary, actively promotes development green animal husbandry.Therefore, the antibacterial additives of application modern high tech method research and development safety, efficient, environmental protection, ensure the health and safety of animals and humans self, Cai Nengshi China becomes breeding production by breeding production big country and makes the country prosperous, and realizes the modernization of aquaculture.And at present for aquaculture, market can be used for substitute antibiotics class feed addictive and be almost in space state.
Namely the present invention is for the problems referred to above, develops a kind of feeding additive aquatic animal of the antibacterial material based on bacillus amyloliquefaciens generation.Antibacterial peptide (Antibacterial Peptides) is a kind of polypeptide quasi-molecule with antibacterial action that organism produces, and general molecular weight is smaller, is usually less than 5kD, is the important molecule barrier of host defense pathogenic microorganism invasion.Up to now, discovery more than 600 kind of endogenous antibacterial active peptide in insect, birds, animal, plant and prokaryotes has been comprised at many biologies.These antibacterial materials not only have broad spectrum antibiotic ability, and have also have certain inhibitory or killing effect to fungi, virus and cancer cell.In addition, also have good stability, good water solubility, Antibacterial Mechanism unique, to features such as higher mammal not uninfected cells, belong to have no side effect, noresidue, without the class environment-friendly type preparation causing bacterial drug resistance.Have more research to antibacterial peptide abroad, in animal productiong reality, also started application, its result of use as feed addictive is confirmed in experiment, and has formed certain market demand.But the narrow antimicrobial spectrum of animal sources antibacterial material, can only suppress gram-positive bacteria usually, limit the extensive use in feed.The antibacterial lipopeptid in bacillus source is important a member in antibacterial lipopeptid family, and its specificity can kill 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, all has a large amount of microorganism in breeding water body, aquatic livestock body surface and enteron aisle, and the micro-ecological environment of this inside and outside all has direct or indirect impact to aquiculture animal health.Meanwhile, because microbial environment inside and outside dissimilar animal body is different, therefore, the demand for antiseptic feed additive is also different.Such as 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 object of the invention is the above-mentioned deficiency for prior art, a kind of antibacterial material feed addictive providing bacillus amyloliquefaciens to originate.
Another object of the present invention is to provide the preparation method of this antibacterial material feed addictive.
Another object of the present invention is to provide the application of this antibacterial material feed addictive.
technical scheme
Object of the present invention realizes by following technical scheme:
A kind of bacillus amyloliquefaciens (
bacillus amyloliquefaciens) preparation method of antibacterial material feed addictive, comprise following steps:
Take preserving number as the bacillus amyloliquefaciens fmb-50 of CGMCC No.6249 be bacterial classification, successively by seed culture, expand and to cultivate and fermenting and producing obtains zymotic fluid; Fermentation liquor micro-filtration removes thalline, adds 10%-15%(g/100ml) cornstarch or porous-starch absorption, spraying dry obtains described antibacterial material feed addictive, and described antibacterial material feed addictive antibacterial potency is 40000 ~ 50000 IU/g; Or fermentation liquor micro-filtration is removed after thalline, then through the film ultrafiltration of 10KD and nanofiltration, add 10%-15% cornstarch or porous-starch absorption, spraying dry obtains described antibacterial material feed addictive; Described antibacterial material feed addictive antibacterial potency is about 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; 115 DEG C of high pressure steam sterilization 30min; The liquid fermentation medium that described fermenting and producing uses is: Fructus Hordei Germinatus leaching 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% bacillus amyloliquefaciens fmb-50(CGMCC No.6249 are inoculated by volume in fermenting and producing), 1000 L fermentor liquid submerged fermentation 36 ~ 40 h, fermentation parameter is: temperature 33 ~ 37 DEG C, 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; Application preferably in preparation shrimp feed, is preferably further preparing the application in feed for litopenaeus vannamei.
In feed for litopenaeus vannamei, add the bacillus amyloliquefaciens antibacterial material feed addictive containing antibacterial material, addition is add 200000 ~ 400000IU antibacterial material in per kilogram feed.
Beneficial effect:
1. the microbial antibacterial material in the present invention is compared with animal sources antibacterial material, has the advantage that broad-spectrum high efficacy is antibacterial.
2. the bacillus amyloliquefaciens fmb-50 that the present invention utilizes preserving number to be CGMCC No.6249 is fermenting raw materials production antibacterial material fat, can large-scale industrial production, with short production cycle, production cost reduces greatly, and not by the impact of the external environment such as season and climate change, be the optimal path overcoming antibacterial material practical application technical bottleneck.
3. bacillus amyloliquefaciens antibacterial material additive of the present invention is used for, in feed for litopenaeus vannamei, can reducing the death rate, improves the efficiency of feed utilization of Environment of Litopenaeus vannamei Low, promotes the non-specific immune response of Environment of Litopenaeus vannamei Low and improve energetic supersession level.
4. additive of the present invention is made an addition in Environment of Litopenaeus vannamei Low mixed feed, do not use antibiotic, also can growth promoting effects and immunity moderation preferably, substitute feed antibiotic is added and ensures that the safety of food is significant.
biological deposits
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, Institute of Microorganism, Academia Sinica, and preserving number is CGMCC NO.6249.
Detailed description of the invention
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 protoplast fusion seed selection, called after fmb-50.
b. amyloliquefaciensfmb-50 is that carbon source can obtain the highest antibacterial lipopeptid output with corn flour, and its output is 3.4 times of parent strain ES-2-4.[Yao Shulin, Lu Zhaoxin etc., Taguchi method is optimized
b. amyloliquefaciensfmb-50 produces surfactin industrial fermentation culture medium, Beijing University of Chemical Technology's journal, 2012,39(4): 77-83].With the original bacteria of fmb-50
b. amyloliquefacienseS-2-4,
b. subtilis168 Hes
b. subtilisbacterial strain described in 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, and 2008, EI; Sun Lijun etc., culture medium produces the impact of antibacterial lipopeptid to bacillus amyloliquefaciens ES-2 bacterial strain, and 2008, Scientia Agricultura Sinica; Shanghai Inst. of Biochemistry, Chinese Academy of Sciences's genetic engineering group, application method for transformation breeding high-yield AMS producing strains from hay bacillus 168,1979, Acta Biochimica et Biophysica Sinica) ferment at same fermentation condition, get the zymotic fluid of 100ml respectively, sink through bactofugation body, acid, sediment methyl alcohol dissolves, obtain antibacterial material, then the bacterial strain of his-and-hers watches 1 carries out antibacterial experiment.Adopting TSA(tryptose soya agar culture medium) culture medium 37 DEG C cultivates vibrio parahaemolytious, Vibrio harveyi, vibrio alginolyticus and Streptococcusagalactiae, agar plate is beaten 5mm hole and adds above-mentioned antibacterial material 50 μ L aloft, after 24h, measure bacteriostatic diameter (table 1).
Table 1 bacillus is to the inhibition (inhibition zone mm) of aquatic pathogenic bacterium
Nd: have no inhibition zone
With bacillus amyloliquefaciens (
bacillus amyloliquefaciens) fmb-50, seed culture medium expands 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 DEG C of high pressure steam sterilization 30min, cultivate through 10-16h and enter exponential phase, be inoculated into fermentation tank.Liquid fermentation medium: fermentation medium: Fructus Hordei Germinatus leaching 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% is inoculated, and 1000 L fermentor liquid submerged fermentation 36 ~ 40 h, fermentation parameter is: temperature 33 ~ 37 DEG C, speed of agitator 180 r ∕ min, throughput 80 m
3/ h, tank pressure 0.10 ~ 0.12 Mpa, pH7.0.Zymotic fluid adopts aperture to be that the miillpore filter micro-filtration of 0.1 μm removes thalline, to remove the later fermented liquid supernatant of thalline for benchmark, adds 10-15%(g/100ml) cornstarch or porous-starch absorption, spraying dry obtains antibacterial material additive.With
e. colifor measuring bacterial strain, Nisin is antibacterial material benchmark, and adopt Odontothrips loti to measure tiring of antibacterial material, dry products antibacterial potency is about 40000 IU/g.
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: bacillus amyloliquefaciens thalline and the impurity being greater than 0.1 μm in first step ceramic micro filter film removing zymotic fluid, what add 10L clear water washing micro-filtration retains part simultaneously, and cleaning solution, again through micro-filtration, merges twice micro-filtration filtrate.Second step milipore filter (milipore filters of 10 KDa) removes the soluble impurity that micro-filtration filtrate middle-molecular-weihydroxyethyl is greater than 10 KDa, and the same 10L of adding clear water washing ultrafiltration retains portion washes liquid again through ultrafiltration, collects two times of ultrafiltration filtrate.Then, ultrafiltrate is removed soluble impurity and the most of moisture of small-molecular-weight through NF membrane (<200Da), collect the component containing antibacterial material.Add appropriate cornstarch or porous-starch, spraying dry, obtain highly active antibacterial material additive powder.With
e. colifor measuring bacterial strain, Nisin is antibacterial material benchmark, adopts Odontothrips loti to measure tiring, about antibacterial activity reaches about 250000IU/g dry powder of antibacterial material.
Embodiment 2
By activated pathogenic bacteria vibrio parahaemolytious, Vibrio harveyi, vibrio alginolyticus, (described bacterial strain is public Streptococcusagalactiae, sees Chen Jixiang etc., pathogenic Vibrio harveyi hemolysin gene clone and detecting, 2005, Chinese aquatic science;
zhai Xiumeideng, vibrio parahaemolytious on the impact of Penaeus Vannmei physiological and biochemical index, 2007, Shanghai Aquatic Products Univ. 9CN)'s journal;
yan Qing Pideng, varying environment condition adheres to the impact of large yellow croaker casing slime to vibrio alginolyticus, and 2006, aquatic product journal;
wu Yingruideng, the inhibit activities of 153 kinds of Chinese herbal medicines to Tilapia mossambica Streptococcusagalactiae and Streptococcus iniae is studied, Journal of Northwest Sci Tech University of Agriculture and Forestry, 2013) receive TSB(Triptic soya fluid nutrient medium respectively) in, cultivate 4 ~ 6 h for 36 ± 1 DEG C, with TSB culture medium adjustment bacterium OD
600value is 1 × 10 to bacterial concentration
8, through 100 times dilution after for subsequent use (1 × 10
6).Prepared by fish serum: about 2 jin heavy red snapper live fishes are bought in market, and from vertebral artery blood drawing, get supernatant after centrifugal 5 min of 3000 rpm, 4 DEG C of refrigerations are for subsequent use.
Get 40000IU/g antibacterial material additive (preparation of embodiment 1 first method) 2 g, respectively doubling dilution, then get 50 μ L successively and add in 96 microwell plate 1 ~ 11 holes, the 12nd hole does not add antibacterial material.Then in 1 ~ 12 hole, add bacterium 10 μ L(every hole final concentration 1 × 10 respectively
5cFU), 40 μ L dilutions (distilled water, seawater, physiological saline, 2% sodium chloride or fish serum), the 12nd hole is contrast, and the dilution adding 50 μ L replaces antibacterial material additive, and every hole cumulative volume is 100 μ L.Enzyme mark version after above-mentioned application of sample is put into aseptic sylphon, is placed in incubator, cultivate 24 h for 28 DEG C.Then each micropore OD is measured with ELIASA
600value, the results are shown in Table 1.
Table 1. bacillus amyloliquefaciens antibacterial material additive is to the minimum inhibitory concentration of several seafood fish pathogenic bacteria
From table 1, bacillus amyloliquefaciens antibacterial material has good antibacterial action to seawater fish common pathogen vibrio parahaemolytious, Vibrio harveyi, vibrio alginolyticus, Streptococcusagalactiae.To resistance MIC such as Vibrio harveyi pathogenic in seawater, vibrio parahaemolytious, vibrio alginolyticus and Streptococcusagalactiaes
50reach 25 μ g/kg, 30 μ g/kg, 35 μ g/kg, 38 μ g/kg (table 1) respectively.
Bacillus amyloliquefaciens antibacterial material is stronger to the antibiotic property effect of pathogenic Vibrio harveyi, vibrio parahaemolytious, vibrio alginolyticus and Streptococcusagalactiae in fish serum, MIC
50reach 18,28,30,30 μ g/kg respectively, illustrate that bacillus antibacterial material can play antibacterial action better in serum.
Bacillus amyloliquefaciens antibacterial material still keeps comparatively high antibacterial activity in the seawater, and active significantly lower than other group in distilled water, shows that certain density salting liquid is conducive to keeping the activity of antibacterial material.
Embodiment 3
Select outward appearance normal, physique is healthy and strong, and the equal weight of tail is that the Environment of Litopenaeus vannamei Low of 1. 02 ± 0.10 g uses shrimp as test.With fish meal, dregs of beans, peanut meal and brewer's yeast for protein sources, 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).Add 6g(A group according in per kilogram mixed feed), 8g(B group), 10g(C group) described in antibacterial material feed addictive (in embodiment 1 first method preparation, tire 40000 IU/g), do not add the CK group (subordinate list 2) of antibacterial material.3 repetitions are established in each process, and each Glass fibre reinforced plastic tub puts Environment of Litopenaeus vannamei Low 30 tail in a suitable place to breed, and Glass fibre reinforced plastic tub water capacity is 0. 3 m
3.Water temperature is (27. 5 ± 1. 5) DEG C, and sea water specific gravity is 1. 012 ± 0. 002, and culture experiment continues 6 weeks.Test after first supporting 7 days temporarily with shrimp, claim initial weight, 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.When Feeding time is respectively 06: 00,10: 00,15: 00,19: 00 and 23: 00, the feeding volume in morning 6 and evening 19 is more, accounts for 70 % of day feeding volume.Whole opisthosoma quality is claimed within first 24 hours, to stop throwing something and feeding.
Add average weight gain comparatively CK group raising 12.3 % of A group cultivation after 6 weeks of 6g/kg; Survival rate improves 7.5% than CK; Feed efficiency improves 14.5% than CK.Add average weight gain comparatively CK group raising 21.3 % of B group cultivation after 6 weeks of 8g/kg; Survival rate improves 20.8% than CK; Feed efficiency improves 44.1% than CK.Add average weight gain comparatively CK group raising 26.2 % of C group cultivation after 6 weeks of 10g/kg; Survival rate improves 13.8% than CK; Feed efficiency improves 39.2% than CK.
Often group gets 8 ~ 10 tail prawns, with 1 mL syringe from heart extracting blood, is placed in Eppendorf pipe, centrifugal leave standstill 24 h in 4 DEG C of refrigerators after, gets supernatant for subsequent use.The pyrogallol autoxidation method that superoxide dismutase activity (SOD) is improved by Deng Biyu etc. measures.The method that phenol oxidase (PO) vigor take L-dopa as substrate colorimetric with reference to Ashida measures.Serum ascites albumin gradient, AKP are active, and the test box adopting Nanjing to build up production measures.The method of Guan Huashi is adopted to measure the vigor of peroxidase (POD) and lysozyme (LSZ).
The alkaline phosphatase (AKP) that the test A group of interpolation 6g/kg is relevant with energetic supersession and nutrition improves 104% than CK group; The lysozyme (LSZ) relevant with antibacterial action improves 37.4% than CK; 113% is improve than CK group with the relevant phenol oxidase of non-specific immune response (PO) of crustacean; 24.1% is improve than CK group with anti-oxidant, disease-resistant relevant superoxide dismutase (SOD); But acid phosphatase (ACP) and peroxidase (POD) do not have significant difference.
The alkaline phosphatase (AKP) that the test B group of interpolation 8g/kg is relevant with energetic supersession and nutrition improves 158% than CK group; The lysozyme (LSZ) relevant with antibacterial action improves 72.1% than CK; 152% is improve than CK group with the relevant phenol oxidase of non-specific immune response (PO) of crustacean; 72.3% is improve than CK group with anti-oxidant, disease-resistant relevant superoxide dismutase (SOD); But acid phosphatase (ACP) and peroxidase (POD) do not have significant difference.
The alkaline phosphatase (AKP) testing C group relevant with energetic supersession and nutrition improves 110% than CK group; The lysozyme (LSZ) relevant with antibacterial action improves 79.5% than CK; 138% is improve than CK group with the relevant phenol oxidase of non-specific immune response (PO) of crustacean; 25.8% is improve than CK group with anti-oxidant, disease-resistant relevant superoxide dismutase (SOD); 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 feed material is g.
Claims (5)
1. a production method for bacillus amyloliquefaciens antibacterial material feed addictive, is characterized in that, comprises following steps:
Take preserving number as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) fmb-50 of CGMCC No.6249 be bacterial classification, successively by seed culture, expand and to cultivate and fermenting and producing obtains zymotic fluid; Fermentation liquor micro-filtration removal thalline, adds 10%-15% (w/v) cornstarch or porous-starch adsorbs, spraying dry obtains described antibacterial material feed addictive; Or fermentation liquor micro-filtration is removed after thalline, then through the film ultrafiltration of 10KD and nanofiltration, add 10-15% (w/v) cornstarch or porous-starch absorption, spraying dry obtains described antibacterial material feed addictive; Described antibacterial material feed addictive antibacterial potency is 40000 ~ 250000IU/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 uses is: Fructus Hordei Germinatus leaching 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, 1000L fermentor liquid submerged fermentation 36 ~ 40h, fermentation parameter is: temperature 35 ~ 37 DEG C, speed of agitator 180r/min, throughput 80m
3/ h, tank pressure 0.10 ~ 0.12Mpa, pH7.0.
2. the bacillus amyloliquefaciens antibacterial material feed addictive for preparing of production method according to claim 1.
3. the application of bacillus amyloliquefaciens antibacterial material feed addictive according to 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 add 200000 ~ 400000U antibacterial material in per kilogram feed.
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| Taguchi 法优化B. amyloliquefaciens fmb-50 产surfactin 工业发酵培养基;姚树林等;《北京化工大学学报》;20120831;第39卷(第4期);第77－83页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI636990B (en) * | 2017-06-07 | 2018-10-01 | 國立屏東科技大學 | Antimicrobial peptide and use thereof |
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