CN105942032A - Prawn feed containing rhodobacter capsulatus strain and application thereof - Google Patents

Abstract

The invention discloses prawn feed containing rhodobacter capsulatus strain. The prawn feed containing rhodobacter capsulatus strain is characterized by being prepared from prawn feed, rhodobacter capsulatus strain fermentation liquor and sodium alginate, wherein 10g of odium alginate and 20mL of rhodobacter capsulatus strain fermentation liquor are added into each 100g of prawn feed until concentration is 3-6*107CFU (Colony Forming Unit)/g; the rhodobacter capsulatus strain is a (i) Rhodobacter capsulatus(/i) R3 strain and has a preservation number of CGMCC (China General Microbiological Culture Collection Center) No:11753. The rhodobacter capsulatus is used as a feed additive to manufacture the prawn edible feed, a feeding result shows that the growth speed of the prawn which eats the feed in which the rhodobacter capsulatus is added is greatly improved compared with the prawn which eats common feed, and immunity is obviously improved. Since the rhodobacter capsulatus is used for manufacturing the feed, the production period is short, the production cost is low, a production condition can be easily controlled, the antimicrobial activity is high, the immunity can be obviously enhanced and the like, and the prawn feed has the potential of industrial production and has a good production application prospect.

Description

A kind of prawn feed containing Rhodobacter capsulatus bacterial strain and application thereof

The present invention obtains 973 projects " immunological approach of white spot syndrome preventing and treating and key technology principle ", project Number 2012CB114405 and outstanding young teacher Funded Projects Tianjin Normal University's biology, the money of item number ZX110QN008 Help.

Technical field

The present invention relates to a kind of prawn feed containing Rhodobacter capsulatus bacterial strain and application thereof, belong to biotechnology neck Territory.

Background technology

Litopenaeus vannamei (Litopenaeus vannamei), also known as Penaeus vannamei (White Prawn), vannamei boone pair Shrimp delicious meat, and with Fenneropenaeus chinensis Osbeck and Penaeus monodon the referred to as world three prawn, account in the shrimp culture industry of China There is extremely important effect.But being as the development of Litopenaeus vannamei intensive culture, the outburst of disease of prawn is received to all The cultivation of shore prawn constitutes serious harm.For solving this problem, people's original adoption antibiosis usually controls disease of prawn, but Being that the use of antibiotic can only play temporarily control, along with the growth of the time of use, a large amount of pathogen create Drug resistance, more It is difficult to control to.The use of antibiotic also can in Litopenaeus vannamei intestinal and surrounding aqueous environment some microorganism cause damage Evil, the balance of broken ring Tiny ecosystem, and the food-safety problem caused by antibiotic remains can be produced, thus the mankind are caused latent Harm.In view of antibacterial status in nature biotechnology monoid and effect, many scholars start the research being correlated with, by antibacterial With the microbe additive lived during i.e. probiotic bacteria adds feedstuff to, by improving relevant to animal or about micropopulation Fall, it is ensured that increase the utilization of feedstuff or strengthen its nutritive value, strengthen animal and to the response of disease or improve environment about Water quality thus be of value to growth of animal, and be expected to substitute antibiotics and become an important research direction.

Summary of the invention

The present invention is to solve that existing Aquatic product high-density breeding pattern is easily caused the farming disease harms and takes place frequently and breeding water body The problems such as severe exacerbation a, it is provided that strain has bacterial strain that is antibacterial and that produce digestive enzyme activity and screening technique thereof and application, should Bacterial strain is from the new bacterial strain of a strain of Fan Nabin gut of shrimp separation screening, has antibacterial activity and produces protease, amylase And lipase active.

It is an object of the invention to be achieved by the following technical programs:

A kind of prawn feed containing Rhodobacter capsulatus bacterial strain, it is characterised in that it is by prawn feed, Rhodobacter capsulatus bacterial strain Fermentation liquid, sodium alginate form, and wherein add 10g sodium alginate in every 100g prawn feed, add Rhodobacter capsulatus bacterial strain and send out Ferment liquid 20mL, to final concentration of 3 ~ 6 × 10⁷CFU/g；Described Rhodobacter capsulatus bacterial strain isRhodobacter capsulatus R3 bacterial strain, deposit number is CGMCC No:11753.

The present invention further discloses the prawn feed containing Rhodobacter capsulatus bacterial strain in terms of improving the prawn speed of growth Application.Particularly prawn feed application in terms of strengthening immunity of prawn.Described enhancing immunity of prawn refers to: right Shrimp is infected the cumulative mortality in 72h by pathogen Vibrio anguillarum.

Of the present inventionRhodobacter capsulatus R3The 16S rRNA sequence of bacterial strain is as shown in SEQ. NO1.

CGGTGGCGCAGCTACACATGCAAGTCGAGCGGAACGAGTTATCTGAACCTTCGGGGAACGATAACGGCG TCGAGCGGCGGACGGGTGAGTAATGCCTAGGAAATTGCCCTGATGTGGGGGATAACCATTGGAAACGATGGCTAATA CCGCATGATGCCTACGGGCCAAAGAGGGGGACCTTCGGGCCTCTCGCGTCAGGATATGCCTAGGTGGGATTAGCTAG TTGGTGAGGTAAGGGCTCACCAAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTGAGAC ACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGT GTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCGTGAGGAAGGTGGGGACGTTAATAGCGGCTTCATTTGA CGTTAGCGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGA ATTACTGGGCGTAAAGCGCATGCAGGTGGTTTGTTAAGTCAGATGTGAAAGCCCGGGGCTCAACCTCGGAATAGCAT TTGAAACTGGCAGACTAGAGTACTGTAGAGGGGGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAG GAATACCGGTGGCGAAGGCGGCCCCCTGGACAGATACTGACACTCAGATGCGAAAGCGTGGGGAGCAAACAGGATTA GATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTTGGAGGTTGTGGCCTTGAGCCGTGGCTTTCGGAGCTAACG CGTTAAGTAGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTG GAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCAGAGAACTTTCCAGAGATGGAT TGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCC GCAACGAGCGCAACCCTTATCCTTGTTTGCCAGCGAGTAATGTCGGGAACTCCAGGGAGACTGCCGGTGATAAACCG GAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGCGCATACAG AGGGCGGCCAACTTGCGAAAGTGAGCGAATCCCAAAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCC ATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG TCACACCATGGGAGTGGGCTGCAAAAGAAGTAGGTAGTTTAACCTTCGGGGGGACGCTTACCACTTGTGGTCAGG

DescribedRhodobacter capsulatus R3 bacterial strain has the ability of antibacterial activity.The bacterial strain of the present invention is to Vibrio anguillarum (Vibrio anguillarum) there is preferable inhibition, fungistatic effect is shown in Fig. 3.

The bacterial strain of the present invention isRhodobacterBelong to Rhodobacter capsulatus (Rhodobacter capsulatus), name ForRhodobacter capsulatus R3, is preserved in Chinese microorganism strain preservation management on November 27th, 2015 and entrusts Member's meeting common micro-organisms center, deposit number is CGMCC No:11753.

The present invention'sRhodobacter capsulatus The biological characteristics of R3 bacterial strain is as follows:

Rhodobacter capsulatus (Rhodobacter capsulatus), namedRhodobacter capsulatusThe bacterium colony of R3 Opaque, surface wettability thickness, in crocus, positive and negative solid colour, bacterium colony is clear-cut, and thalline is easily provoked, its bacterium colony shape State is shown in that Fig. 1, microscope hypothallus are spherical, has the pod membrane of solid form, and cellular morphology is shown in Fig. 2.Rhodobacter capsulatusBacterial strain belongs to gram negative bacteria, and optimum growth temperature 28 DEG C, the most suitable growth pH value is between 7-8.Its physiology Biochemical characteristic is as follows: can decomposition glucose, fructose, sucrose, mannitol, do not decompose xylose and arabinose, do not utilize carbamide, third Diacid salt, can liquefy gelatin, nitrate reductase and catalase positive, ODC Ornithine decarboxylase, E.C. 4.1.1.18, oxidation Enzyme, indole test are feminine gender.Do not produce hemotoxin.To common antibiotics mezlocillin, azlocillin, cefotaxime, bacillus The sensitivities such as peptide, gentamycin, tetracycline, Lomefloxacin, chloromycetin nitrofurantoin, rifampicin, drug sensitive test uses scraps of paper agar Diffusion method, Antibiotic discs is purchased from Hangzhou microorganism reagent company limited.

DescribedRhodobacter capsulatus R3 bacterial strain has the ability of antibacterial activity.The bacterial strain of the present invention is to eel Vibrio (Vibrio anguillarum) there is preferable inhibition, fungistatic effect is shown in Fig. 3.

It addition, bacterial strain of the present inventionRhodobacter capsulatus R3 also has the stronger work producing digestive enzyme Property, wherein the activity of protease, amylase and lipase is the highest, and its three kinds of digestive enzyme activities are shown in Fig. 4.

The present invention further discloses the screening technique with bacterial strain that is antibacterial and that produce digestive enzyme activity, described bacterial strain isRhodobacter capsulatus R3 bacterial strain, the method comprises the following steps:

Taking the full intestinal of prawn under A, aseptic condition in homogenizer, ice bath grinds, and carries out gradient dilution with nine saline solution, by each dilute Releasing liquid to take 0.1mL and be coated in 2216E culture medium, after 28 DEG C of constant temperature culture, picking list bacterium colony is anti-on 2216E solid medium Multiple line is purified the single bacterium colony pure culture of acquisition.

B, the single bacterium colony pure culture obtained is seeded in the 2216E culture medium containing indicator bacteria and cultivates, screening, Obtain that there is antibacterial activityRhodobacter capsulatus R3 bacterial strain.

C, in the screening technique of the above-mentioned bacterial strain with antibacterial activity, described 2216E culture medium uses sea water to join System.Use sea water configuration culturing gene osmotic pressure not change, thus ensure that sea source microbial strains in screening not Can lose.

D, by the dibbling respectively of single bacterium colony of obtaining in fat culture medium, skimmed milk agar culture medium and starch culture-medium On, erythema, Proteolytic enzyme circle and the screening of Starch Hydrolysis circle can be produced according to bacterium colony and can secrete lipase, protease and starch EnzymeRhodobacter capsulatusR3 bacterial strain.

E, in the screening technique of the above-mentioned bacterial strain having and producing digestive enzyme activity, described fatty culture medium, defat cattle Breast agar culture medium and starch culture-medium all use nine saline solution preparations.Sea water preparation culturing gene osmotic pressure is used not occur Change, thus ensure that source, sea microbial strains will not be lost in screening.

In the screening technique of the above-mentioned bacterial strain with antibacterial and digestive enzyme activity, described 2216E culture medium uses Conventional 2216E culture medium.As preferably, described 2216E culture medium uses nine saline solution preparations, and described 2216E training Foster base includes the weight proportion of following component: peptone 5g/L, yeast extract 1g/L, iron phosphate 0.1g/L, agar 20g/L PH7.6～7.8.

In the screening technique of the above-mentioned bacterial strain with antibacterial activity, as preferably, indicator bacteria described in step B isVibrio anguillarum (Vibrio anguillarum is bought in Institute of Microorganism, Academia Sinica, in the aquatic life of Tianjin Normal University Thing laboratory preserves).

In the screening technique of the above-mentioned bacterial strain with antibacterial and digestive enzyme activity, fat culture medium described in step E Use the proportioning raw materials that this area is conventional.As preferably, described fatty culture medium uses nine saline solution preparations, and described Fat culture medium includes the weight proportion of following component: Nutrient agar 1000ml, Oleum Arachidis hypogaeae semen 10g, dimethyl diaminophenazine chloride 1ml of 1.6%.

In the screening technique of the above-mentioned bacterial strain with antibacterial and digestive enzyme activity, skimmed milk fine jade described in step E Fat culture medium uses the proportioning raw materials that this area is conventional.As preferably, described skimmed milk agar culture medium uses nine Saline solution is prepared, and described skimmed milk agar culture medium includes the weight proportion of following component: Nutrient agar 1000ml, defat Lac Bovis seu Bubali 100ml, pH7.4.

In the screening technique of the above-mentioned bacterial strain with antibacterial and digestive enzyme activity, starch culture-medium described in step E Use the proportioning raw materials that this area is conventional.As preferably, described starch culture-medium uses nine saline solution preparations, and described Starch culture-medium includes the weight proportion of following component: soluble starch 10g/L, peptone 10g/L, yeast extract 5g/L, K₂HPO₄1g/L, MgSO₄·7H₂O 0.2g/L, NaCl 10g/L, agar 18g/L.

The present invention further discloses the application in terms of preparing bacteriostatic activity of the Rhodobacter capsulatus bacterial strain；Described is antibacterial Activity refer to Vibrio anguillarum (Vibrio anguillarum) suppression.And Rhodobacter capsulatus bacterial strain produces digestive enzyme in preparation Application in terms of Huo Xing, wherein said digestive enzyme refers to: protease, amylase and lipase.Experimental result shows: this Bright bacterial strain isRhodobacter capsulatusR3 bacterial strain, describedRhodobacter capsulatusR3 bacterial strain is used for The microorganism formulation of preparation aquatic animal.Due toRhodobacter capsulatusR3 bacterial strain has stronger antibacterial activity And yielding lipase, protease, diastatic activity.Therefore, it can the feed additive for preparing aquaculture of aquatic animal and change The microbial ecological agent of kind breeding water body.

What Rhodobacter capsulatus bacterial strain disclosed by the invention was compared with prior art had has the active effect that

The bacterial strain with antibacterial activity of the present invention, is the new bacterial strain of a strain that the present inventor screens from gut of shrimp, this bacterium Strain isRhodobacter capsulatus R3 bacterial strain, has stronger antibacterial activity and produces digestive enzyme activity, it is provided that be a kind of The new extra large source animal intestinal microorganism with antibacterial activity, for utilizing animal endogeneous activity bacterial strain for aquaculture of aquatic animal In disease control, improve aquatic animal and to the digestibility of feedstuff and improve the aspects such as water quality new method is provided, promote The benign development of culture fishery.

Accompanying drawing illustrates:

Fig. 1 is the colonial morphology figure of Rhodobacter capsulatus bacterial strain of the present invention；

Fig. 2 is micro-(100 ×) photo figure of Rhodobacter capsulatus bacterial strain of the present invention；

Fig. 3 is the Vibrio anguillarum antagonism figure of Rhodobacter capsulatus bacterial strain of the present invention；

Fig. 4 is 3 kinds of digestive enzyme activity figures of Rhodobacter capsulatus bacterial strain of the present invention；Wherein (a) protease is positive, (b) amylase sun Property, (c) lipase is positive；

Fig. 5 (a-e) be in prawn hemocyte related immune gene quantified results；Wherein (a) superoxide dismutase Gene relative expression's spirogram, (b) prophenoloxidase gene relative expression's spirogram, (c) antibacterial peptide gene relative expression's spirogram, (d) Lysozyme gene relative expression's spirogram, (e) cell adhesion factor gene relative expression's spirogram；

Fig. 6 (a-f) is prawn blood plasma related immune relevant enzyme vitality test result；Wherein (a) Acid Phosphatase Activity figure, (b) Activity of catalase figure, (c) antalzyme activity figure, (d) peroxidase activity figure, (e) superoxide dismutase activity Figure, (f) Phenoloxidase Activities figure；

Fig. 7 (a-c) is the measurement result of gut of shrimp digestive enzyme activity；Wherein (a) protease activity is tried hard to, (b) diastatic activity Trying hard to, (c) lipase activity is tried hard to；

Fig. 8 prawn weight gain figure；

Fig. 9 is prawn mortality statistics result after challenge test.

Detailed description of the invention

The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention It is method known in those skilled in the art.It addition, embodiment is interpreted as illustrative, and the unrestricted present invention Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this On the premise of invention spirit and scope, the various changes carrying out the material component in these embodiments and consumption or change are also Belong to protection scope of the present invention.

Raw material sources used in the present invention are as follows:

Embodiment 1

Litopenaeus vannamei used in the present embodiment takes from prawn culturing field, Tianjin Hangu District prosperous Yongfeng；

2216E culture medium used in the present embodiment uses nine saline solution preparations；

2216E culture medium used in the present embodiment includes the weight proportion of following component: peptone 5g/L, yeast extract 1g/L, iron phosphate 0.1g/L, agar 20g/L pH7.6～7.8.

Indicator bacteria described in the present embodiment be Vibrio anguillarum (Vibrio anguillarum), buy in the Chinese Academy of Sciences micro- Biological study institute, preserves at Tianjin Normal University's aquatile laboratory.

The present embodimentRhodobacter capsulatus The screening technique of R3 bacterial strain:

Bacterial strain isolated and purified: taking the full intestinal of prawn under aseptic condition in homogenizer, ice bath grinds, with nine saline solution to grinding Liquid carries out the gradient dilution of 10-1～10-8；Choose each 0.1mL of diluent that gradient is 10-3～10-7 and be coated on 2216E cultivation On base；28 DEG C of constant temperature culture 24～48h；Taking single bacterium colony, to carry out 3 ~ 5 continuous line in 2216E culture medium isolated and purified, obtains The bacterial strain after purification obtained carries out short-term respectively at 4 DEG C and-80 DEG C and preserves for a long time.

Bacterial strain after purification is carried out bacteriostatic activity test: be inoculated in TSB fluid medium by indicator bacteria Vibrio anguillarum, 37 DEG C of constant-temperature tables 16～18h；By the indicator bacteria value of its OD600 of spectrophotometric determination after activation, when OD value 0.6～ Between 0.8, concentration is advisable；Fetching shows that bacteria suspension 150 μ L is spread evenly across in TSA solid medium, is divided into by flat board different Region labelling；Shaken cultivation in bacterial strain switching fluid medium after purification overnight, is gripped aseptic filter with aseptic tweezers The scraps of paper are placed in culture fluid and are impregnated with bacterium solution, are placed on the relevant position of culture dish, 28 DEG C of constant temperature culture 24h, observed and recorded antagonism The bacterial strain having bacteriostatic activity is screened by the production of speckle, obtains the bacterial strain having antibacterial activityRhodobacter capsulatus R3, it is common that this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms on November 27th, 2015 Microorganism center, deposit number is CGMCC No:11753.

Embodiment 2

The present embodiment has product digestive enzyme activityRhodobacter capsulatus The screening technique of R3 bacterial strain, it separates Purification process, with in embodiment 1, repeats no more here.

Bacterial strain after purification is carried out digestive enzyme activity test:

In A, fluid medium of being transferred by bacterial strain after purification, 28 DEG C of constant-temperature shaking culture are overnight, aseptic with aseptic tweezers gripping Filter paper is placed in culture fluid and is impregnated with bacterium solution, is placed on the correspondence position of the skimmed milk culture medium prepared, 28 DEG C of constant temperature Cultivating 24h, the bacterial strain that energy decomposing protein produces transparent circle screens.

In B, fluid medium of being transferred by bacterial strain after purification, 28 DEG C of constant-temperature shaking culture are overnight, with aseptic tweezers gripping Aseptic filter paper sheet is placed in culture fluid and is impregnated with bacterium solution, is placed on the correspondence position of the starch culture-medium prepared, 28 DEG C of constant temperature Cultivating 24h, the bacterial strain that energy starch-splitting produces after the covering of iodine liquid transparent circle screens.

In C, fluid medium of being transferred by bacterial strain after purification, 28 DEG C of constant-temperature shaking culture are overnight, with aseptic tweezers gripping Aseptic filter paper sheet is placed in culture fluid and is impregnated with bacterium solution, is placed on the correspondence position of the fatty culture medium prepared, 28 DEG C of constant temperature Cultivating 24h, the bacterial strain forming redness to reducing fat screens.

Skimmed milk culture medium used in the present embodiment includes the weight proportion of following component: Nutrient agar 1000ml, Skimmed milk 100ml, pH7.4.

Starch culture-medium used in the present embodiment includes the weight proportion of following component: soluble starch 10g/L, albumen Peptone 10g/L, yeast extract 5g/L, K₂HPO₄1g/L, MgSO₄·7H₂O 0.2g/L, NaCl 10g/L, agar 18g/L.

Fatty culture medium used in the present embodiment includes the weight proportion of following component: Nutrient agar 1000ml, Semen arachidis hypogaeae Oil 10g, dimethyl diaminophenazine chloride 1ml of 1.6%.Screen through said method, finally give and there is the bacterial strain producing three kinds of digestive enzyme activitiesRhodobacter capsulatus R3。

Embodiment 3

Choose the present invention that above-described embodiment obtains there is antibacterial activity and produce digestive enzyme activityRhodobacter capsulatus R3 bacterial strain is identified and analyzes, and concrete qualification and the method for analysis and corresponding qualification result are as follows.

What the method screening using above-described embodiment obtained has bacteriostatic activity and produces digestive enzyme activityRhodobacter capsulatus R3 inoculation, on 2216E culture medium flat plate, is inverted under the conditions of 28 DEG C and is cultivated 24h, observe its bacterium colony Growth conditions, strain morphology and physiological and biochemical test are with reference to industrial microorganism experimental technique handbook (Zhu Gejian, Wang Zhengxiang, industry Microbiological test technical manual [M], China Light Industry Press, 1994) and primary Jie Shi Bacteria Identification handbook (R.E Buchanan, N.E Ji Bensi, primary Jie Shi Bacteria Identification handbook [M], Science Press, Beijing, 1984).It is observed in constant incubator Growing state between 4 DEG C ~ 50 DEG C.

Above-mentionedRhodobacter capsulatus The qualification result of R3 strain morphology and physiological and biochemical property shows:

Bacterium colony is opaque, surface wettability thickness, in crocus, and positive and negative solid colour, bacterium colony is clear-cut, and thalline is easily provoked, Its colonial morphology is shown in that Fig. 1, microscope hypothallus are spherical, has the pod membrane of solid form, and cellular morphology is shown in Fig. 2.Rhodobacter capsulatusBacterial strain belongs to gram negative bacteria, and optimum growth temperature 28 DEG C, the most suitable growth pH value is between 7-8.Bacterial strain energy Decomposition glucose, fructose, sucrose, mannitol, do not decompose xylose and arabinose, do not utilize carbamide, malonate, can liquefy bright Glue, nitrate reductase and catalase positive, ODC Ornithine decarboxylase, E.C. 4.1.1.18, oxidase, indole test are Negative.Do not produce hemotoxin.To common antibiotics mezlocillin, azlocillin, cefotaxime, bacitracin, gentamycin, Fourth Ring The sensitivities such as element, Lomefloxacin, chloromycetin nitrofurantoin, rifampicin.

Embodiment 4

The present invention'sRhodobacter capsulatus The molecular biology research of R3 bacterial strain, the 16s of bacterial strain the most of the present invention The PCR amplification of rRNA uses the method that this area is conventional, it should be noted that the acquisition of PCR reaction masterplate.

The acquisition of template: use that the method screening of above-described embodiment obtains has antibacterial activity and digestive enzyme activityRhodobacter capsulatus The pure culture of R3 bacterial strain is inoculated in bacillus cereus separation fluid medium, 28 DEG C Shaken cultivation 24h, prepares bacteria suspension.Take the bacteria suspension of 30 μ L and the ultra-pure water mixing of 70 μ L, 100 DEG C of heating 5min, 2000rpm Centrifugal 10min, takes the template that supernatant reacts as PCR.

The primer that in above-described embodiment, PCR amplification uses is universal primer, and by Hua Da, genome company synthesizes, and draws sequence such as Under:

5’-ACAAGCCCTGGAAACGGGGT-3’；

5’-CACCAGGAATTCCGATCT-3’；

The condition of pcr amplification reaction is: 94 DEG C of denaturations 4min, enters and circulates, 94 DEG C of degeneration 30s, 50 DEG C of annealing 1min, 72 DEG C Extending 2min, 30 circulations, 72 DEG C extend 4 DEG C of states of preservation after 10min.PCR primer is completed order-checking by Hua Da genome company, surveys Sequence result is as shown in SEQ ID NO:1.The 16s rRNA gene order of the Rhodobacter capsulatus bacterial strain of the present invention is carried out homology Analyze, this bacterial strain withRhodobacter capsulatusHomology is the highest, and homology is up to 99%.

Embodiment 5

The present invention'sRhodobacter capsulatus The production application research of R3 bacterial strain.

DescribedRhodobacter capsulatus The application aborning of R3 bacterial strain isRhodobacter capsulatus R3 bacterial strain adds in the former powder of feedstuff, is fed to the Litopenaeus vannamei regular period, observes and measure its growth shape State change relative with autoimmunity.Concrete grammar is as follows:

The present invention'sRhodobacter capsulatus R3 bacterial strain carries out amplification culture step by step in liquid 2216E culture medium, Prepare the fermentation liquid of a large amount of bacterial strain, measure the cell concentration of fermentation liquid simultaneously.

The fermentation liquid that will be enlarged by cultivating adds Fodder making in the former powder of prawn feed to, and the method for Fodder making is as follows:

The fermentation liquid of the bacterial strain of preparation is proportionally added in the former powder of feedstuff, make the concentration of antibacterial in feedstuff be 3 ~ 6 × 10⁷CFU/g, adds sodium alginate according to the ratio adding 10g sodium alginate in every 100g feedstuff in the preparation process of feedstuff, It is beneficial to feedstuff molding.The former powder of feedstuff is made to be sufficiently mixed uniformly with bacterium solution and sodium alginate.

Feed manufacturing.The feedstuff mixed is kneaded into bulk, with the granule of the granule a diameter of 2mm of mechanism.Every 7 days of feedstuff Once, preparation amount is continuously increased with the growth of Litopenaeus vannamei in preparation.

Feed:

The feedstuff feeding prawn that will prepare, is divided into test group and matched group, and often group throws in the vannamei boone pair of 600 cabrages about 3.5g Shrimp.By 5% input feedstuff of Litopenaeus vannamei body weight, throw something and feed every day 4 times.The change of record water quality, and change water in time, prevent and treat residual Bait overlong time in water causes change of water quality to affect growth and the health of Litopenaeus vannamei.

Sampling is with process: every 7 days is a cycle, carries out primary sample, often organizes and take 30 ~ 40 tail vannamei boones at random during sampling Prawn also records the body weight of Litopenaeus vannamei；At Litopenaeus vannamei pricardial coelom, extract hemolymph, add anti-according to the ratio of 1:1 Solidifying agent, and at 4 DEG C, 800g is centrifuged 10min, it is thus achieved that hemocyte and serum.It is placed in-80 DEG C by hemocyte adds 1mL Trizol Preserve, serum be placed in 4 DEG C stand-by；Take the intestinal of Litopenaeus vannamei, use the enzyme extraction buffer in test kit after removing feces, grind Grind standby tissue sample.

Anticoagulant used in the present embodiment includes the proportioning of following component:

KCl:10 mM；NaCl:450 mM；HEPES:10 mM；EDTA(Na₂): 10 mM；PH:7.45.

FeedingRhodobacter capsulatus The mensuration of the every physical signs of R3 bacterial strain prawn: with sampling gained Hemocyte extracts RNA, is reversed to cDNA, uses the method for real time fluorescent quantitative to measure prawn related immune gene with it for template Determining of prophenoloxidase gene, superoxide dismutase gene, lysozyme gene, cell adhesion molecules gene and antibacterial peptide gene Scale reaches, and concrete assay method is shown in embodiment 6.To feedingRhodobacter capsulatus R3 bacterial strain group prawn and comparison Blood and the intestinal of group prawn are sampled, the serum of gained and intestinal tissue lapping liquid, with test kit (Suzhou section inscription examination biology Reagent company limited) determination test group prawn nonspecific immunity related enzyme activity, specifically include: phenol oxidase, peroxide Enzyme, acid phosphatase, superoxide dismutase SOD, catalase and the mensuration of antalzyme activity, assay method is shown in embodiment 7.To feedingRhodobacter capsulatus The intestinal of R3 bacterial strain group prawn and matched group prawn is sampled, gained Intestinal tissue lapping liquid, with test kit (Suzhou Ke Mingshi biological reagent company limited) determination test group gut of shrimp protease, Amylase and the change of lipase activity, concrete assay method is shown in embodiment 8.Additionally, also measured were feedingRhodobacter capsulatus R3 bacterial strain group prawn and the body weight change difference of matched group prawn, and in situation about being stimulated by pathogen The difference of lower mortality rate, concrete assay method is shown in embodiment 9 and embodiment 10 respectively.

Embodiment 6

FeedingRhodobacter capsulatus After R3 bacterial strain, the change of prawn related immune gene expression amount:

The extraction of RNA: by the prawn hemocyte of above-mentioned sampling, adds 1mL Trizol；4 DEG C, 12000g is centrifuged 10 min；Take Supernatant, stands 5 minutes so that it is crack completely.Then adding 200 μ L chloroforms, acutely vibrate 15s, and room temperature stands 2-3min.4 DEG C, 12000g, centrifugal 15min, centrifugal after point three-phase: the pale red azoic coupling component of lower floor, chloroform is protein mutually, and mesophase is DNA, upper strata without Color aqueous phase is RNA.Take colourless aqueous phase, add 500 μ L isopropanols, mix gently, stand 15min, precipitate RNA.4 DEG C, 12000g from The heart 10 min, abandons supernatant inversion and drains；Add the ethanol of 1mL75%, gently rub piping and druming with rifle head and make RNA float； 4℃、 7500g is centrifuged 6 min, abandons supernatant inversion and drains about 15 min；Adding appropriate DEPC water, in 55 DEG C, water-bath 5 min is rearmounted Preserve in-80 DEG C of refrigerators.The hemocyte total serum IgE extracted carries out determining with NANODrop2000 through 1% formaldehyde agarose gel electrophoresis Property and detection by quantitative, confirm the synthesis for cDNA after RNA integrity.

The synthesis of the first chain cDNA: with 2 μ g Litopenaeus vannamei hemocyte total serum IgE for reverse transcription template, AOLP (5 '- GGCCACGCGTCGACTAGTAC (T) 16-3 ') it is reverse primer, according to the following first chain cDNA that is synthesized:

Table 1 first chain cDNA reaction system

The quantitative determination of related immune gene: using cDNA obtained above as the template of Real-time PCR, withβ-actin As internal reference primer, the reaction system of Real-time PCR is: 95 DEG C of denaturations 30s, a circulation；95 DEG C of degeneration 5s, 60 DEG C annealing 30s, 40 circulations；Being warmed up to 95 DEG C through 60 DEG C after, each circulation rises 0.5 DEG C and carries out melt curve analysis analysis.Institute Use 2-△ △ Ct method to calculate after the data statistical analysis obtained, use t check analysis genes of interest significant difference, used The primer sequence of different immunogenes is as follows.

Table 2 experiment primer

Table 3 real-time quantitative PCR reaction system

Result of the test shows, feeding adds the present invention'sRhodobacter capsulatus Exempting from of the prawn of R3 bacterial strain feedstuff The expression of epidemic disease gene is in the trend of rise, and test group substantially increases than the immunogene expression of matched group simultaneously, concrete outcome See Fig. 5, demonstrate feeding from the level of molecule and add the present invention'sRhodobacter capsulatus R3 bacterial strain feedstuff right The immunity of shrimp significantly improves.

Embodiment 7

The mensuration that cultured prawn related immune enzyme is lived: sampled determining the protein quantity in this and try biological reagent with being purchased from the inscription of Suzhou section The Coomassie brilliant blue test kit of company limited.

Protein content (μ g/mL)=standard protein concentration × (it is blank that A measures-A)/(A standard-A is blank).

The mensuration of Phenoloxidase Activities: the reagent measuring the biological company limited of inscription of use Suzhou section of Phenoloxidase Activities Box, operates according to test kit description.This test defines every mg albumen and makes light absorption value change at 525nm in reaction system 0.01 is an enzyme activity unit.

Phenoloxidase Activities (U/mg prot)=reaction cumulative volume/sample volume/response time/0.01 × (A measures-A pair According to)/protein concentration (mg/mL).

The mensuration of peroxidase activity: the examination measuring the biological company limited of inscription of use Suzhou section of peroxidase activity Agent box, operates according to test kit description.This test defines every mL serum and does not has a minute A470 change in reaction system 0.01 is an enzyme activity unit.

Peroxidase activity (U/mL)=reaction cumulative volume/sample volume/0.01 × Δ A

The mensuration of Acid Phosphatase Activity: the reagent measuring the biological company limited of inscription of use Suzhou section of Acid Phosphatase Activity Box, operates according to test kit description.During this test defines 37 DEG C, every milliliter of blood catalysis per minute generation 1 μm ol phenol is One enzyme activity unit.

Acid Phosphatase Activity (U/mL)=[C standard substance × (A measures pipe-A control tube) ÷ (A standard pipe-A blank tube) × V is the most total] ÷ V sample × V sample total ÷ T.

The mensuration of superoxide dismutase SOD vigor: the mensuration of superoxide dismutase activity uses Suzhou section inscription biology The test kit of company limited, operates according to test kit description.This test is defined on xanthine oxidase coupling reaction body When in system, suppression ratio is 50%, the SOD enzyme activity in reaction is an enzyme activity unit.

Inhibition percentage=(A control tube-A measures pipe)/A control tube × 100%

SOD activity (U/mL)=inhibition percentage/(1-inhibition percentage)

The mensuration of activity of catalase: the reagent measuring the biological company limited of inscription of use Suzhou section of activity of catalase Box, operates according to test kit description.The amount of the hydrogen peroxide of this test every milliliter of serum decomposition per second 1 μm ol of definition is One unit of activity.

Activity of catalase (U/mL)=(A comparison-A measure) × 271/(60 × sampling amount) dilute before × test sample Multiple.

The mensuration of antalzyme activity: lyophilized powder (build by Nanjing with micrococcus lysodeikticus (Micrococcus lysoleikticus) Bioengineering Research Institute is become to produce) it is substrate, prepare substrate suspension with the kaliumphosphate buffer of 0.1M pH6.4, make OD570nm ≈ 0.3～0.5.Adding serum and bacteria suspension in 96 orifice plates, serum: all blood=1:10, the light absorption value that mensuration 570nm goes out is i.e. For initial light absorption value A0.37 DEG C of water-bath 15min, ice bath 3min, the light absorption value that mensuration 570nm goes out is A.Antalzyme activity (U/ ML)=(A0-A)/A.

Result of the test shows, feeding adds the present invention'sRhodobacter capsulatus R3 bacterial strain feedstuff prawn Phenol oxidase, peroxidase, acid phosphatase, superoxide dismutase SOD, catalase and antalzyme activity etc. are special And nonspecific immunity enzyme work all has raising in various degree relative to matched group prawn, concrete outcome is shown in Fig. 6, the level lived from enzyme Demonstrate feeding and add the present invention'sRhodobacter capsulatus The immunity of the prawn of R3 bacterial strain feedstuff substantially carries High.

Embodiment 8

The assay method of cultured prawn intestinal digestive enzyme activity is as follows:

A, the mensuration of prolease activity: the test kit measuring the biological company limited of inscription of use Suzhou section of prolease activity, according to Test kit description operates.This test defines 30 DEG C of every milligram of albumen hydrolysis per minute and produces 1 μm ol tyrosine is one Unit of activity.

Prolease activity (U/mg prot)=C standard substance × (it is blank that A measures-A)/(A standard-A is blank) × extension rate/ (volume of protein content × determinand).

B, the mensuration of amylase activity: the test kit measuring the biological company limited of inscription of use Suzhou section of amylase activity, Operate according to test kit description.This test defines the catalysis per minute of every mg histone and produces 1mg reducing sugar is one Enzyme activity unit.

Amylase activity (U/mg prot)=89.4 × (A measures pipe-A control tube+0.022)/protein content

C, the mensuration of lipase activity: the test kit measuring the biological company limited of inscription of use Suzhou section of lipase activity, according to Test kit description operates.During this test defines 37 DEG C, every milligram of albumen Hydrolysis of Olive Oil per minute generates 1 μm ol fat Acid is an enzyme activity unit.

Lipase activity (U/mg prot)=[C standard substance × V standard substance × (A measures pipe-A blank tube)/(A standard pass-A Blank tube)]/(the supernatant volume of protein content × supernatant cumulative volume/join reaction system)/response time.

Result of the test shows, feeding adds the present invention'sRhodobacter capsulatus R3 bacterial strain feedstuff gut of shrimp Protease, amylase and lipase activity all have raising in various degree relative to matched group prawn, and concrete outcome is shown in Fig. 7, table Bright feeding adds the present invention'sRhodobacter capsulatus The prawn of the R3 bacterial strain feedstuff abilities of digestive and absorption to feedstuff Significantly improve.

Embodiment 9

The mensuration of cultured prawn weight gain: before on-test, often random 50 tail Litopenaeus vannamei of taking out, survey in group culturing pool Its body weight fixed, and carry out recording M0, after feeding experiment terminates, often group culturing pool takes 50 tails at random and measure its body and carry out record M.By calculating the weight gain often organizing prawn: weight gain (WGR, %)=(M-M0)/M0 × 100%.

Result of the test shows, feeding adds the present invention'sRhodobacter capsulatus The prawn of R3 bacterial strain feedstuff Body weight dramatically increase with than compared with matched group, concrete outcome is shown in Fig. 8.

Embodiment 10

The mensuration of mortality rate after cultured prawn challenge test: after feeding experiment terminates, often group takes that 90 tails are of uniform size all to be received Shore prawn carries out challenge test, the 90 tail prawns often organized be randomly divided into three parallel.The pathogenic bacterium Vibrio anguillarum that will prepare, passes through The second uromere prawn carries out intramuscular injection and carries out challenge test, and the concentration of injection bacterium is 3.58 × 10⁷Cell/mL, injection Amount is 15 μ L.After counteracting toxic substances completes, observe prawn survival condition, the dead quantity of record prawn, calculate the prawn in 72h dead Rate.

Result of the test shows: the prawn randomization through feeding measures it and is being infected 72h by pathogen Vibrio anguillarum Interior cumulative mortality, result is as shown in Figure 9, it can be deduced that add the present invention'sRhodobacter capsulatus R3 bacterium After strain, the cumulative mortality of test group prawn, significantly lower than blank group, illustrates interpolationRhodobacter capsulatus R3 Bacterial strain is improving immunity of prawn and then is improving it and play a role the resistivity of pathogenic bacterium.

Embodiment 11

A kind of prawn feed containing Rhodobacter capsulatus bacterial strain, it is by prawn feed, Rhodobacter capsulatus bacterial strain fermentation liquor, Sargassum Acid sodium composition, wherein adds 10g sodium alginate in every 100g prawn feed, adds Rhodobacter capsulatus bacterial strain fermentation liquor 20mL, extremely Final concentration of 3 × 10⁷CFU/g；Described Rhodobacter capsulatus bacterial strain isRhodobacter capsulatusR3 bacterial strain, preservation Numbered CGMCC No:11753.

Embodiment 12

A kind of prawn feed containing Rhodobacter capsulatus bacterial strain, it is by prawn feed, Rhodobacter capsulatus bacterial strain fermentation liquor, Sargassum Acid sodium composition, wherein adds 10g sodium alginate in every 100g prawn feed, adds Rhodobacter capsulatus bacterial strain fermentation liquor 20mL, extremely Final concentration of 6 × 10⁷CFU/g；Described Rhodobacter capsulatus bacterial strain isRhodobacter capsulatusR3 bacterial strain, preservation Numbered CGMCC No:11753.

Specific embodiment described in the present invention is only to illustrate spirit of the present invention, technology neck belonging to the present invention Described specific embodiment can be made corresponding amendment and supplement or use similar mode to substitute by the technical staff in territory, but Without departing from the spirit of the present invention or surmount scope defined in institute's person's appended claims.Although the present invention makes Detailed description has also quoted some specific embodiments as proof, but for this area institute those of skill in the art, as long as without departing from The spirit and scope of the present invention can do respective change or correction is obvious.

SEQUENCE LISTING

<110>Tianjin Normal University

<120>a kind of prawn feed containing Rhodobacter capsulatus bacterial strain and application thereof

<130> 1

<170> PatentIn version 3.5

<210> 1

<211> 1453

<212> DNA

<213>artificial sequence

<400> 1

cggtggcgca gctacacatg caagtcgagc ggaacgagtt atctgaacct tcggggaacg 60

ataacggcgt cgagcggcgg acgggtgagt aatgcctagg aaattgccct gatgtggggg 120

ataaccattg gaaacgatgg ctaataccgc atgatgccta cgggccaaag agggggacct 180

tcgggcctct cgcgtcagga tatgcctagg tgggattagc tagttggtga ggtaagggct 240

caccaaggcg acgatcccta gctggtctga gaggatgatc agccacactg gaactgagac 300

acggtccaga ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgcaagcctg 360

atgcagccat gccgcgtgtg tgaagaaggc cttcgggttg taaagcactt tcagtcgtga 420

ggaaggtggg gacgttaata gcggcttcat ttgacgttag cgacagaaga agcaccggct 480

aactccgtgc cagcagccgc ggtaatacgg agggtgcgag cgttaatcgg aattactggg 540

cgtaaagcgc atgcaggtgg tttgttaagt cagatgtgaa agcccggggc tcaacctcgg 600

aatagcattt gaaactggca gactagagta ctgtagaggg gggtagaatt tcaggtgtag 660

cggtgaaatg cgtagagatc tgaaggaata ccggtggcga aggcggcccc ctggacagat 720

actgacactc agatgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac 780

gccgtaaacg atgtctactt ggaggttgtg gccttgagcc gtggctttcg gagctaacgc 840

gttaagtaga ccgcctgggg agtacggtcg caagattaaa actcaaatga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgatgca acgcgaagaa ccttacctac 960

tcttgacatc cagagaactt tccagagatg gattggtgcc ttcgggaact ctgagacagg 1020

tgctgcatgg ctgtcgtcag ctcgtgttgt gaaatgttgg gttaagtccc gcaacgagcg 1080

caacccttat ccttgtttgc cagcgagtaa tgtcgggaac tccagggaga ctgccggtga 1140

taaaccggag gaaggtgggg acgacgtcaa gtcatcatgg cccttacgag tagggctaca 1200

cacgtgctac aatggcgcat acagagggcg gccaacttgc gaaagtgagc gaatcccaaa 1260

aagtgcgtcg tagtccggat tggagtctgc aactcgactc catgaagtcg gaatcgctag 1320

taatcgtgga tcagaatgcc acggtgaata cgttcccggg ccttgtacac accgcccgtc 1380

acaccatggg agtgggctgc aaaagaagta ggtagtttaa ccttcggggg gacgcttacc 1440

acttgtggtc agg 1453

Claims (4)

1. the prawn feed containing Rhodobacter capsulatus bacterial strain, it is characterised in that it is by prawn feed, Rhodobacter capsulatus bacterium Strain fermentation liquid, sodium alginate form, and wherein add 10g sodium alginate in every 100g prawn feed, add Rhodobacter capsulatus bacterial strain Fermentation liquid 20mL, to final concentration of 3 ~ 6 × 10⁷CFU/g；Described Rhodobacter capsulatus bacterial strain isRhodobacter capsulatusR3 bacterial strain, deposit number is CGMCC No:11753.

2. prawn feed containing Rhodobacter capsulatus bacterial strain described in claim 1 is improving the application in terms of the prawn speed of growth.

3. prawn feed containing Rhodobacter capsulatus bacterial strain described in claim 1 is strengthening the application in terms of immunity of prawn.

4. the application described in claim 3, enhancing immunity of prawn therein refers to: prawn is infected by pathogen Vibrio anguillarum Cumulative mortality in 72h.