CN102740707B - Endogenous antiviral complex-probiotic-preparation for prawn - Google Patents
Endogenous antiviral complex-probiotic-preparation for prawn Download PDFInfo
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Abstract
The present invention relates to an endogenous antiviral complex-probiotic-preparation for prawn. The endogenous antiviral complex-probiotic-preparation consists of 5-20% by weight of Bacillus firmus SSL065 powder, 5-10% by weight of Vibrio alginolyticus SSL073 powder and 70-90% by weight of auxiliary protection carriers, wherein, viable bacillus concentration of Bacillus firmus SSL065 in the Bacillus firmus SSL065 powder is not less than 10<12> CFU/g. The complex-probiotic-preparation of the present invention is composited by the Bacillus firmus SSL065 viable bacillus and the Vibrio alginolyticus SSL073 components, the viable bacillus and the bacteria components have synergistic effect. The present preparation has multiple biological functions such as obvious improvement of cultured prawn's nonspecific immunity level, great reinforcement of cultured prawn's resistance to pathogenic bacteria infection, and especially significant improvement of cultured prawn's resistance to viral pathogen infection.
Description
Technical field
The invention belongs to the probiotics preparation field, be specifically related to the antiviral composite probiotics preparations of a kind of endogenic prawn.
Background technology
Exist in shrimp culture environment that quantity is huge, miscellaneous bacterium microbe, they are not only the important composition link of prawn food chain/net, participate in material recycle and energy flow in cultivating system, and keep the ecological balance and the quality of optimizing the environment aspect taking on important role.Bacterium in water environment enters alimentary canal by the feeding behaviour of prawn, and different types of bacterium forms a microbial ecosystem complexity, dynamic in the prawn alimentary canal, and its structure and composition is controlled by biological itself and external environmental factor.In alimentary canal, the Main Function of beneficial bacterium comprises: keep the digestion power of alimentary canal microecological balance, raising feed, the non-specific immunity of enhancing body, the defence pathogenic microorganism is at gastral proliferation and spreading.But the anti-disease microbial in the development and utilization breeding environment, the microorganism that particularly has an antivirus action is rare report also.
The generation of viral disease and popular be one of the subject matter of puzzlement prawn culturing.The virus type cause of disease of prawn culturing is white spot syndrome virus (White spot syndrome virus most importantly, WSSV), the prawn fulminant epidemic disease beginning in 1993 that is caused by this virus is popular on a large scale in the Asia, spread to afterwards prawn culturing district, America, remain so far the most serious disease of whole world harm prawn culturing.These sick characteristics are that morbidity is anxious, dead speed is fast, the death rate is high, annual because of WSSV generation and popularly make global prawn output reduce half, the loss that China every year causes because of this disease is up to hundred million yuan of 30-50.Taura syndrome virus (Taura syndrome virus, TSV) has also caused important threat to cultivation phase prawn, infectious subcutaneous and hematopoietic tissue necrosis virus (Infectious hypodermal ﹠amp; Haematopoietic necrosis virus, IHHNV), hepatopancreatic parvovirus (Hepato pancereatic parvovirus, HPV) etc. also causes in various degree harm in prawn seed-rearing phase and juvenile prawn phase.Because virus has strict cytozoicus characteristic, there is no at present effectively, chemicals can prevent and treat prawn virus disease; And prawn lacks the specific immunity system, and vaccine can't play prevention effect effectively.Therefore, seek effective ecological prophylactico-therapeutic measures, particularly can strengthen the endogenous bacteria preparation of prawn resistance against diseases, just become and control the most promising strategy of prawn ' s virus disease.
Summary of the invention
The object of the invention is to for problems such as in the prawn culturing process, viral disease harm are serious, the prophylactic measures specific aim is poor, a kind of prawn cell and humoral immunity level of strengthening is provided, particularly can significantly improve prawn viral infection resisting ability, and derive from the antiviral composite probiotics preparations of the gastral a kind of endogenic prawn of prawn.
Two kinds of bacterial strain information involved in the present invention are as follows:
Bacillus firmus SSL065 (Bacillus firmus) bacterial strain separates from Health China prawn alimentary canal, determine to safety such as Chinese prawn (Penaeus chinensis), Environment of Litopenaeus vannamei Low (Litopenaeus vannamei), japonicus (Penaeusjaponicus), without pathogenicity through high dose injection or the experiment of throwing something and feeding.In Wuhan Chinese Typical Representative culture collection center preservation (Wuhan, China Wuhan University), deposit number was bacterial strain: CCTCC NO:M 2010192 on July 30th, 2010.The 16rDNA base sequence of bacillus firmus SSL065 bacterial strain is as shown in SEQ ID NO:1.
Vibrio alginolyticus SSL073 (Vibrio alginolyticus) bacterial strain separates from Health China prawn alimentary canal, determines to safety such as Chinese prawn, Environment of Litopenaeus vannamei Low, japonicus, without pathogenicity through high dose injection or the experiment of throwing something and feeding.In Wuhan Chinese Typical Representative culture collection center preservation (Wuhan, China Wuhan University), deposit number was bacterial strain: CCTCC NO:M 2010193 on July 30th, 2010.The 16rDNA base sequence of vibrio alginolyticus SSL073 bacterial strain is as shown in SEQ ID NO:2.
The antiviral composite probiotics preparations of a kind of endogenic prawn of the present invention; it is characterized in that: mixed by bacillus firmus SSL065 bacterium powder, vibrio alginolyticus SSL073 pulvis, auxiliary protection carrier, its gross mass percentage is: bacillus firmus SSL065 bacterium powder 5-20%, vibrio alginolyticus SSL073 pulvis 5-10%, auxiliary protection carrier 70-90%.
Described bacillus firmus SSL065 bacterium powder is that freeze drying is made, wherein bacillus firmus SSL065 viable bacteria concentration can reach 〉=10
12The CFU/ gram; Spray-dried the forming of described vibrio alginolyticus SSL073 pulvis can not detect vibrio alginolyticus SSL073 viable bacteria.
Described auxiliary protection carrier is chosen any one kind of them in glucan powder, wheat dextrin, cornstarch or is several.
Bacillus firmus SSL065 bacterium powder, preparation method thereof is as follows: line on a small quantity the 2216E solid medium from the bacterial classification picking of-80 ℃ of preservations, cultivate 24h at 37 ℃ and obtain single bacterium colony, get a single colony inoculation in the bacillus firmus seed culture fluid, 37 ℃ of shaking flasks are cultivated 24h and are obtained seed liquor, again seed liquor is inoculated in fermented and cultured in the bacillus firmus zymotic fluid, fermentation temperature is controlled at 30-38 ℃; As zymotic fluid OD
600Value reaches at 1.5～2.0 o'clock, stops fermentation, obtains zymocyte liquid.Get zymocyte liquid centrifugal concentrating 80-100 and doubly make bacterium mud, adding mass ratio in bacterium mud is 6% commercially available Trehalosc protection agent, carries out freeze drying after mixing, and makes bacillus firmus SSL065 bacterium powder.
Wherein, bacillus firmus seed culture fluid composition quality percentage is: dextrin 1.5%, peptone 0.5%, ferrous sulfate 0.05 ‰; The pH value is 5.7.
The zymotic fluid composition quality percentage of bacillus firmus is: peptone 0.5%, yeast extract 0.1%, ferrous sulfate 0.02 ‰.
The preparation method is as follows for vibrio alginolyticus SSL073 pulvis: line on a small quantity the 2216E solid medium from the bacterial classification picking of-80 ℃ of preservations, cultivate 24h at 28 ℃ and obtain single bacterium colony, get a single colony inoculation in the vibrio alginolyticus seed culture fluid, 28 ℃ of shaking flasks are cultivated 24h and are obtained seed liquor, again seed liquor is inoculated in fermented and cultured in the vibrio alginolyticus zymotic fluid, fermentation temperature is controlled at 25-28 ℃; Zymotic fluid OD600 value reaches at 1.6 o'clock, stops fermentation; Obtain zymocyte liquid; Getting zymocyte liquid carries out centrifugal concentrating 20-50 and doubly makes the turbid liquid of bacterium.By the turbid liquid volume ratio 1 of bacterium: 5-1: 10 add the resuspended thalline of lysate, 37 ℃ of digestion 24 hours; 300w, 10s/10s ultrasonic disruption 20 minutes, the bacterium liquid spray-drying after fragmentation (air intake and leaving air temp are respectively at 195 ℃ and 85 ℃) makes vibrio alginolyticus SSL073 pulvis.
Above-mentioned vibrio alginolyticus seed culture fluid composition quality percentage is: glucose 1.2%, peptone 1.0%, beef extract 0.3%, yeast extract 0.5%, trisodium citrate 0.8 ‰, K
2HPO
40.2 ‰, KH
2PO
40.1 ‰, MgSO
40.4 ‰; PH is 7.2.
Above-mentioned vibrio alginolyticus zymotic fluid composition quality percentage is glucose 1.2%, peptone 0.2%, beef extract 1.5%, NaCl2.5%.
Above-mentioned lysate is: 50mMTris-HCl, pH8.0; 2mM EDTA, 100mM NaCl, 0.1%Triton x-100 adds lysozyme to 100 μ g/ml.
Composite probiotics preparations of the present invention is composited bacillus firmus SSL065 viable bacteria and vibrio alginolyticus SSL073 composition, the viable bacteria that it is contained and bacterium composition are mutually promoted, synergy, has obvious raising cultured prawn innate immune activity, greatly strengthen cultured prawn anti-microbial pathogen appeal, particularly can significantly improve prawn and resist the various biological functions such as virus causing disease infection ability.
The specific embodiment
Embodiment 1:
1, composite probiotics preparations preparation of the present invention:
Bacillus firmus SSL065 bacterium powder, preparation method thereof is as follows: line on a small quantity the 2216E solid medium from the bacterial classification picking of-80 ℃ of preservations, be inoculated in seed liquor after 37 ℃ are cultivated the single bacterium colony of 24h acquisition in, shaking flask is cultivated, again seed liquor is inoculated in fermented and cultured in zymotic fluid, fermentation temperature is controlled at 30-38 ℃; As zymotic fluid OD
600Value reaches at 1.6 o'clock, stops fermentation, obtains zymocyte liquid.Get zymotic fluid and carry out centrifugal concentrating 80-100 and doubly make bacterium mud, adding mass ratio in bacterium mud is 6% Trehalosc protection agent, carries out freeze drying after mixing, and makes viable bacteria concentration higher than 10
12The bacillus firmus SSL065 bacterium powder of CFU/ gram.
The preparation method is as follows for vibrio alginolyticus SSL073 pulvis: line on a small quantity the 2216E solid medium from the bacterial classification picking of-80 ℃ of preservations, be inoculated in seed liquor after 28 ℃ are cultivated the single bacterium colony of 24h acquisition in, shaking flask is cultivated, again seed liquor is inoculated in fermented and cultured in zymotic fluid, fermentation temperature is controlled at 25-28 ℃; Zymotic fluid OD
600Value reaches at 1.6 o'clock, stops fermentation; Obtain zymocyte liquid; Getting zymocyte liquid carries out centrifugal concentrating 20-50 and doubly makes the turbid liquid of bacterium.The 1/5-1/10 long-pending by the turbid liquid of bacterium adds the resuspended thalline of lysate, and 37 ℃ digested 24 hours; 300w, 10s/10s ultrasonic disruption 20 minutes, the bacterium liquid spray-drying after fragmentation (air intake and leaving air temp are respectively at 195 ℃ and 85 ℃) makes vibrio alginolyticus SSL073 pulvis.
Above-mentioned bacillus firmus bacterium powder 20 grams that make, vibrio alginolyticus pulvis 5 grams, auxiliary protection carrier cornstarch 75 grams evenly are mixed and made into composite probiotics preparations.
2, the effect detection of composite probiotics preparations:
This composite probiotics preparations is added in 10 kilograms of basic prawn feeds (containing fish meal 30%, shrimp med 10%, bean powder 25%, peanut powder 25%, flour 3%, corn flour 3%, B B-complex 1%), is mixed with the immunization feedstuff that contains composite probiotics preparations.Various raw materials were pulverized respectively 60 mesh sieves, abundant mixing step by step after accurately weighing, with sodium alginate as adhesive, add suitable quantity of water and be crushed to pellet with small meat mincer, dry to moisture under 40 ℃ of conditions below 10%, bag distribution packaging saves backup in 4 ℃ of refrigerators.
Just initial body is long is (5.70 ± 1.18) cm for healthy Environment of Litopenaeus vannamei Low more than 600 tails, experiment prawn, is 2 processed group of experimental group and control group at random, each processed group have 3 parallel, put prawn 60 tails in a suitable place to breed for each parallel group.Change water every day 1 time, a day quantity of exchanged water is 1/3; Throw something and feed every day feed 4 times, daily ration of feeding is about 0.9-1.2 gram/10 tails, sucks residual feed after the 1h that throws something and feeds, and according to the daily ration, feeding quantity of how much regulating of residual bait; Wherein the immunization experiment group adopts the interval feeding method, the namely continuous 4 days immunization feedstuffs of throwing something and feeding, and 3 days basal feeds of throwing something and feeding, circulation in 7 days is until experiment finishes.Water temperature 24-26 ℃, 7d is supported in continuous charge temporarily before experiment.Control group is at the basal feed of throwing something and feeding of whole experimental stage always.Respectively 0,5,10,15 and 20d take a sample, measure disease-resistant microorganism preparation to the impact of Environment of Litopenaeus vannamei Low innate immune activity.Result shows: compare with control group, the disease-resistant microorganism preparation oral administration can significantly improve the activity of the Immune interrelation enzymes such as acid phosphatase, alkaline phosphatase, superoxide dismutase and lysozyme in prawn serum, has namely improved the immune level (table 1) of prawn.
Add the immunoenzymatic activity of disease-resistant microorganism preparation Serum of Penaeus vannamei in table 1 feed
Embodiment 2:
1, composite probiotics preparations preparation:
Bacillus firmus SSL065 bacterium powder and vibrio alginolyticus SSL073 pulvis preparation method are with embodiment 1; with bacillus firmus SSL065 bacterium powder 5 grams, vibrio alginolyticus SSL073 pulvis 10 grams, auxiliary protection carrier cornstarch 65 grams, glucan powder 20 grams evenly are mixed and made into composite probiotics preparations.
2, the effect detection of composite probiotics preparations:
This composite probiotics preparations is added in 10 kilograms of basic prawn feed raw materials (containing fish meal 30%, shrimp med 10%, bean powder 25%, peanut powder 25%, flour 3%, corn flour 3%, B B-complex 1%), is mixed with the immunization feedstuff that contains disease-resistant microorganism preparation.Various raw materials were pulverized respectively 60 mesh sieves, abundant mixing step by step after accurately weighing, with sodium alginate as adhesive, add suitable quantity of water and be crushed to pellet with small meat mincer, dry to moisture under 40 ℃ of conditions below 10%, bag distribution packaging saves backup in 4 ℃ of refrigerators.
Healthy Environment of Litopenaeus vannamei Low more than 800 tails, long (6.80 ± 0.38) cm of body, random packet is raised in 550L fiberglass tank.Change water every day 1 time, a day quantity of exchanged water is 1/3; Throw something and feed every day feed 4 times, daily ration of feeding is about 1.5-2.0 gram/10 tails, sucks residual feed after the 1h that throws something and feeds, and according to the daily ration, feeding quantity of how much regulating of residual bait; 22～25 ℃ of water temperatures, pH8.2 ± 0.2, and continuous charge are supported 7d temporarily before experiment.
The phase of supporting temporarily later will be tested prawn and will be divided at random experimental group and 2 processed group of control group, each processed group have 3 parallel, put prawn 90 tails in a suitable place to breed for each parallel group.Control group is at the basal feed of throwing something and feeding of whole experimental stage always, the experimental group interval immunization feedstuff of throwing something and feeding, and the namely 1 day basal feed of throwing something and feeding, 1 day immunization feedstuff of throwing something and feeding is until experiment finishes.22d after the immunization feedstuff of throwing something and feeding, press the dosage of 4.2g/kg body weight, directly oral infection the prawn pathological material of disease of white spot syndrome virus, record the prawn quantity of virus infections stage death every day, and calculate the cumulative mortality that WSSV infects Environment of Litopenaeus vannamei Low in 14d.Result shows: compare with control group, the disease-resistant microorganism preparation oral administration can significantly reduce the death rate that prawn infects WSSV, and immune protective rate reaches 65.52%, has namely improved the anti-WSSV infection ability (table 2) of prawn.
Table 2WSSV attacks the cumulative mortality (%) of the rear Environment of Litopenaeus vannamei Low of poison
Embodiment 3:
Antiviral composite probiotics preparations is at all effect of prawn culturing in producing of receiving:
After evenly being mixed, the antiviral composite probiotics preparations of prawn (containing bacillus firmus bacterium powder 5-20%, vibrio alginolyticus pulvis 5-10%, auxiliary protection carrier 70-90%) adds routine techniques granulation feed (crude protein 〉=41% in common prawn feed to; crude fat 4-8%; crude fibre≤5%; coarse ash 16%, Ca
2+1-3%, total phosphorus 1-2%, moisture content≤11%).Test arrangement carries out in Shanghai Jinshan cultivation company, selects 6, rectangle soil pond, each 7 mu of left and right.The cultivation kind is Environment of Litopenaeus vannamei Low, and test was since July 5.3 of test tanks, the immunization feedstuff of respectively throwing something and feeding 1 time in 6 o'clock every mornings, at 6 o'clock in afternoon, after the 7 days immunization feedstuffs of throwing something and feeding continuously, throwing something and feeding contrasted prawn feed in 7 days, so repeated, until September 16, culture experiment finished; Contrast 3, pond test tank, 6 o'clock every mornings, at 6 o'clock in afternoon raise the time for throwing, and the whole process common prawn feed of throwing something and feeding.Cultivation begins until by the end of August, and all culturing pools comprise that test tank is all good with the prawn growth that contrasts the pond, and vicinity this moment cultured prawn on every side had quite a few morbidity at the beginning of 8 months.To by the end of August, due to the pond morbidity aggravation of contiguous shrimp, the seabirds such as sea-gull predation horizontal transmission makes this test also begin to be affected, and especially contrasts the pond and begins successively morbidity, detects through nucleic acid probe, confirms that the morbidity cause of disease is WSSV.One of them pond is impaired the heaviest, survival rate less than 20%.And the test tank morbidity is obviously light, only finds indivedual dead shrimps.To September 16, still continuing owing to contrasting the pond incidence, determine to finish test, the receipts shrimp.From result of the test (table 3) as can be known:
The antiviral composite probiotics preparations of table 3 is received the effect of prawn culturing in producing relatively all
Test group prawn average survival is 65.4%, improve 34.6% than the throw something and feed culturing pool prawn average survival of basal feed of other whole process, survival rate difference is (P＜0.01) extremely significantly, to the cultivation end of term, experiment pool output per hectare average out to 8084.9Kg, output improves 33.4%, remarkable in economical benefits.Result shows: the prawn disease-resistant microorganism preparation has good prevention effect to the infection of virosis.
Industrial applicibility
Composite probiotics preparations of the present invention is composited bacillus firmus SSL065 viable bacteria and vibrio alginolyticus SSL073 composition, the viable bacteria that it is contained and bacterium composition are mutually promoted, synergy, have obvious raising cultured prawn innate immune activity, greatly strengthen cultured prawn anti-microbial pathogen appeal.This composite probiotics preparations can be used for all receiving during prawn culturing produces, and strengthens prawn cell and humoral immunity level.
Claims (10)
1. the antiviral composite probiotics preparations of endogenic prawn, is characterized in that this composite probiotics preparations is mixed by bacillus firmus (Bacillus firmus) SSL065 bacterium powder, vibrio alginolyticus (Vibrio alginolyticus) SSL073 pulvis, auxiliary protection carrier; Each material quality percentage is: bacillus firmus SSL065 bacterium powder 5-20%, vibrio alginolyticus SSL073 pulvis 5-10%, auxiliary protection carrier 70-90%;
Wherein the depositary institution of bacillus firmus (Bacillus firmus) SSL065 bacterial strain is Wuhan Chinese Typical Representative culture collection center preservation, and deposit number is: CCTCC NO:M 2010192;
The depositary institution of vibrio alginolyticus (Vibrio alginolyticus) SSL073 bacterial strain is Wuhan Chinese Typical Representative culture collection center preservation, and deposit number is: CCTCC NO:M 2010193.
2. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 1, is characterized in that bacillus firmus SSL065 viable bacteria concentration 〉=10 in above-mentioned bacillus firmus SSL065 bacterium powder
12The CFU/ gram.
3. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 1, is characterized in that above-mentioned auxiliary protection carrier is one or more in glucan powder, wheat dextrin, cornstarch.
4. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 1, the preparation process that it is characterized in that above-mentioned bacillus firmus SSL065 bacterium powder is as follows: the bacterial classification of-80 ℃ of preservations is lined the 2216E solid medium, cultivate 24h at 37 ℃ and obtain single bacterium colony, get a single colony inoculation in the bacillus firmus seed culture fluid, 37 ℃ of shaking flasks are cultivated and are obtained seed liquor, again seed liquor is inoculated in fermented and cultured in the bacillus firmus zymotic fluid, fermentation temperature is controlled at 30-38 ℃; As zymotic fluid OD
600Value reaches at 1.5～2.0 o'clock, stops fermentation and obtains zymocyte liquid; Zymocyte liquid is carried out centrifugal concentrating 80-100 doubly make bacterium mud, adding mass ratio in bacterium mud is 6% Trehalosc protection agent, carries out freeze drying after mixing and makes bacillus firmus SSL065 bacterium powder.
5. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 4 is characterized in that above-mentioned bacillus firmus seed culture fluid composition quality percentage is: dextrin 1.5%, and peptone 0.5%, ferrous sulfate 0.05 ‰, pH value is 5.7.
6. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 4, is characterized in that above-mentioned bacillus firmus zymotic fluid composition quality percentage is: peptone 0.5%, yeast extract 0.1%, ferrous sulfate 0.02 ‰.
7. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 1, the preparation process that it is characterized in that above-mentioned vibrio alginolyticus SSL073 pulvis is as follows: picking lines the 2216E solid medium on a small quantity from the bacterial classification of-80 ℃ of preservations, cultivate 24h at 28 ℃ and obtain single bacterium colony, get a single colony inoculation in the vibrio alginolyticus seed culture fluid, 28 ℃ of shaking flasks are cultivated 24h and are obtained seed liquor, again seed liquor is inoculated in fermented and cultured in the vibrio alginolyticus zymotic fluid, fermentation temperature is controlled at 25-28 ℃; Zymotic fluid OD
600Value reaches at 1.6 o'clock, stops fermentation and obtains zymocyte liquid; Getting zymocyte liquid carries out centrifugal concentrating 20-50 and doubly makes the turbid liquid of bacterium; 1:5-1:10 adds the resuspended thalline of lysate by the turbid liquid volume ratio of bacterium, and 37 ℃ digested 24 hours; 300w, 10s/10s ultrasonic disruption 20 minutes, the bacterium liquid spray-drying after fragmentation is made vibrio alginolyticus SSL073 pulvis.
8. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 7, is characterized in that above-mentioned vibrio alginolyticus seed culture fluid composition quality percentage is: glucose 1.2%, peptone 1.0%, beef extract 0.3%, yeast extract 0.5%, trisodium citrate 0.8 ‰, K
2HPO
40.2 ‰, KH
2PO
40.1 ‰, MgSO
40.4 ‰, pH is 7.2.
9. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 7, is characterized in that above-mentioned vibrio alginolyticus zymotic fluid composition quality percentage is: glucose 1.2%, peptone 0.2%, beef extract 1.5% and NaCl 2.5%.
10. the antiviral composite probiotics preparations of a kind of endogenic prawn as claimed in claim 7, is characterized in that above-mentioned lysate is: 50mMTris-HCl, pH8.0; 2mM EDTA, 100mM NaCl, and 0.1%Triton X-100 add lysozyme to 100 μ g/ml.
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|---|---|---|---|
| PCT/CN2010/076082 WO2012022032A1 (en) | 2010-08-17 | 2010-08-17 | Endogenous antiviral complex-probiotic-preparation for prawn |
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| CN111091872B (en) * | 2019-12-06 | 2023-05-16 | 宁波大学 | Method for screening probiotics combination based on multi-pathogen of prawn diseases |
| CN111349592B (en) * | 2020-05-11 | 2022-06-03 | 河南大学 | Spore production culture medium and preparation method of bacterial spores |
| CN111714526A (en) * | 2020-07-06 | 2020-09-29 | 浙江德丰生态农业科技发展有限公司 | Application of metabolic product of chytrid in improving antifungal effect of Yangcheng lake hairy crab |
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| 溶藻弧菌疫苗对凡纳滨对虾免疫功能的影响;曹剑香等;《湛江海洋大学学报》;20041231;第24卷(第06期);11-17 * |
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| Publication number | Publication date |
|---|---|
| WO2012022032A1 (en) | 2012-02-23 |
| CN102740707A (en) | 2012-10-17 |
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