CN105861389B - For improving Bacillus strain and its screening technique and the application of aquatic livestock growth performance - Google Patents
For improving Bacillus strain and its screening technique and the application of aquatic livestock growth performance Download PDFInfo
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Abstract
The invention discloses for improving aquatic livestock growth performance Bacillus strain and its screening technique and application.BacillusBacillus sp.B1 bacterial strain, preservation registration number are as follows: CGMCC No:11752.The present invention is that feed addictive makes the edible feed of prawn using bacillus, the prawn that feeding eats addition bacillus feed as the result is shown greatly improves relative to the prawn speed of growth of edible normal diet and immunity increases obviously, utilize bacillus Fodder making, it is with short production cycle, production cost is low, easily controllable growth conditions, and antibacterial activity is high and many advantages, such as immunity can be remarkably reinforced, completely with the potentiality of industrialized production, there is preferable production application prospect.
Description
The present invention obtains 973 projects " immunological approach and key technology principle of white spot syndrome prevention and treatment ", project
Number 2012CB114405 and the outstanding young teacher's Funded Projects of Tianjin Normal University's biology, the money of item number ZX110QN008
It helps.
Technical field
The present invention relates to for improving aquatic livestock growth performance Bacillus strain and its screening technique and application, belong to
In technical field of bioengineering.
Background technique
Litopenaeus vannamei (Litopenaeus vannamei), also known as Penaeus Vannmei (White Prawn), vannamei boone pair
Shrimp delicious meat, and with Crustin and Penaeus monodon and referred to as three prawn of the world, accounted in the shrimp culture industry in China
There is extremely important effect.But with 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.To solve this problem, people's original adoption antibiotic controls disease of prawn, but
It is that the use of antibiotic can only play temporary control, with the increase of use time, a large amount of pathogens produce drug resistance, more
It is difficult to control.The use of antibiotic can also cause to damage to some microorganisms in litopenaeus vannamei enteron aisle and surrounding aqueous environment
Evil, breaks the balance of ring Tiny ecosystem, and can generate the food-safety problem as caused by antibiotic residue, to cause to dive to the mankind
Harm.Start relevant research in view of status of the bacterium in nature biotechnology monoid and effect, many scholars, by bacterium
It is added in feed with microbe additive, that is, probiotics living, by improving micropopulation relevant or surrounding to animal
It falls, it is ensured that increase the utilization of feed or enhance its nutritive value, enhance animal to the response of disease or improve its ambient enviroment
Water quality is expected to substitute antibiotics as an important research direction to be beneficial to growth of animal.
Summary of the invention
The present invention is easy to cause the farming disease harms to take place frequently and breeding water body to solve existing aquatic products high-density breeding mode
The problems such as severe exacerbation, provide one plant is with the bacterial strain and its screening technique that produce digestive enzyme activity and application, the bacterial strain
From one plant of new bacterial strain of Fan Nabin gut of shrimp separation screening, has and generate protease, amylase and lipase active.
The purpose of the present invention is be achieved by the following technical programs:
One plant has the bacterial strain for producing digestive enzyme activity, and the bacterial strain isBacillus sp.B1 bacterial strain, the bacterial strain preservation
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No:11752.When preservation
Between on November 27th, 2015, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode 100101.Bacterial strain of the invention is
The microorganism for separating and screening from Fan Nabin gut of shrimp has stronger production digestive enzyme activity, can be used for improving water
Produce the growth performance of animal.
Bacterial strain of the invention isBacillusCategory bacillus (Bacillus sp.), it is named asBacillus sp.B1 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation on November 27th, 2015
Number is CGMCC No:11752.
Of the inventionBacillus sp.The biological characteristics of B1 bacterial strain are as follows:
Bacillus (Bacillus sp.B1 bacterium colony) is opaque and has gauffer and protuberance, surface wettability, is in canescence,
Bacterium colony is clear-cut, and thallus is easily provoked, and colonial morphology is shown in Fig. 1, and microscope hypothallus is in the shape of a rod, and it is thin that gemma is born in trophosome
Among born of the same parents, cellular morphology is shown in Fig. 2.Bacillus sp.B1 bacterial strain belongs to gram-positive bacteria, 28 DEG C of optimum growth temperature, most suitable
Growing pH value is 7.Its physio-biochemical characteristics is as follows: can decomposition glucose, fructose, sucrose, do not decompose xylose, mannitol, I
Uncle's sugar, does not utilize urea, malonate, can liquefy gelatin, nitrate reductase and catalase positive, ornithine decarboxylation
Enzyme, lysine decarboxylase, oxidizing ferment, indole test are feminine gender.Do not produce hemotoxin, to common antibiotics mezlocillin, Ah
The sensitivities such as Lip river XiLin, cefotaxime, Cefepime, bacitracin, gentamicin, erythromycin, medecamycin, tetracycline, chloramphenicol,
Drug sensitive test uses agar diffusion method of the paper, and Antibiotic discs are purchased from Hangzhou microorganism reagent Co., Ltd.
It is of the present inventionBacillus sp.The 16S rRNA sequence of B1 bacterial strain is as shown in SEQ. NO1.
TCTGTCACCTTAGGCGGCTGGCTCCAAAAAGGTTACCCCACCGACTTCGGGTGTTACAAACTCTCGTGG
TGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGC
TTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACGGTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCA
GCTCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCT
TCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTTAATGATGGCAACTAAGATCAAGGGTTGCGCTCGT
TGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCTCCCGAAGGAG
AAGCCCTATCTCTAGGGTTTTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATG
CTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTA
ATGCGTTAACTTCAGCACTAAAGGGCGGAAACCCTCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGG
TATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGT
GTTCCTCCATATCTCTACGCATTTCACCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTCTCCCAGTT
TCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCACCTGCGCGCGCTTTACGCCCAA
TAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGT
ACCGTCAAGGTGCCAGCTTATTCAACTAGCACTTGTTCTTCCCTAACAACAGAGTTTTACGACCCGAAAGCCTTCAT
CACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGG
GCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTC
ACCAACTAGCTAATGCGACGCGCGTCCATCCATAAGTGACAGCCGAAGCCGCCTTTCAATTTCGAACCATGCGGTTC
AAAATGTTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTATGGGCAGGTTACCCACGTGTTACTCAC
CCGTCCGCCGCTAACTTCATAAGAGCAAGCTCTTAATCCATTCGCTCGACTTGCATGTATTAGGCACGCCGCAGCGT
TCATCCTGAGCAGA
In addition, bacterial strain of the present inventionBacillus sp.B1 has the stronger activity for producing digestive ferment, wherein albumen
The activity of enzyme, amylase and lipase is higher, and three kinds of digestive enzyme activities are shown in Fig. 3.
The present invention further discloses the screening technique with the bacterial strain for producing digestive enzyme activity, the bacterial strain isBacillus sp.B1 bacterial strain, method includes the following steps:
A, take the full intestines of prawn in homogenizer under aseptic condition, ice bath grinding carries out gradient dilution with nine salting liquids, will
Each dilution takes 0.1mL to be coated on bacillus isolation medium, and after 28 DEG C of constant temperature incubations, picking single bacterium falls within bacillus
Scribing line carries out purifying and obtains single colonie pure culture repeatedly on separation solid medium.
B, by the difference dibbling of obtained single colonie in fatty culture medium, skimmed milk agar medium and starch culture-medium
On, can generate erythema, proteolysis circle and the screening of Starch Hydrolysis circle according to bacterium colony can secrete lipase, protease and starch
EnzymeBacillus sp.B1 bacterial strain.
C, in the above-mentioned screening technique with the bacterial strain for producing digestive enzyme activity, the fatty culture medium, degreasing ox
Newborn agar medium and starch culture-medium are all made of the preparation of nine salting liquids.Using seawater prepare culturing gene osmotic pressure there is no
Variation, to ensure that extra large source microbial strains will not be lost in screening.
In the screening technique of the above-mentioned bacterial strain with antibacterial and digestive enzyme activity, the bacillus is separately cultured
Base is using conventional bacillus isolation medium.Preferably, the bacillus isolation medium is molten using nine salt
Liquid is prepared, and the bacillus isolation medium includes the weight proportion of following component: peptone 10g/L, yeast extract 5g/L,
Glucose 3.5g/L, NaCl 5g/L, K2HPO40.5g/L, MgSO43g/L, MnSO40.025g/L, agar 15g/L
PH7.4~7.6.
In the above-mentioned screening technique with the bacterial strain for producing digestive enzyme activity, fat culture medium described in step C is used
The raw material proportioning of this field routine.Preferably, the fatty culture medium is prepared using nine salting liquids, and the fat
Culture medium includes the weight proportion of following component: nutrient agar 1000ml, peanut oil 10g, 1.6% dimethyl diaminophenazine chloride 1ml.
In the above-mentioned screening technique with the bacterial strain for producing digestive enzyme activity, the training of skimmed milk agar described in step C
Support the raw material proportioning that base uses this field routine.Preferably, the skimmed milk agar medium is molten using nine salt
Liquid is prepared, and the skimmed milk agar medium includes the weight proportion of following component: nutrient agar 1000ml, skimmed milk
100ml, pH7.4.
In the above-mentioned screening technique with the bacterial strain for producing digestive enzyme activity, starch culture-medium described in step C is used
The raw material proportioning of this field routine.Preferably, the starch culture-medium is prepared using nine salting liquids, and the starch
Culture medium includes the weight proportion of following component: soluble starch 10g/L, peptone 10g/L, yeast extract 5g/L, K2HPO4
1g/L, MgSO4·7H2O 0.2g/L, NaCl 10g/L, agar 18g/L.
The present invention further disclosesBacillus sp.Application of the B1 bacterial strain in terms of preparation produces digestive enzyme activity, with
And for improving aquatic livestock growth performance strengthen immunity in terms of application.Wherein the digestive ferment refers to: protease,
Amylase and lipase.Experimental result is shown: bacterial strain of the invention isBacillus sp.B1 bacterial strain, it is describedBacillus sp.B1 bacterial strain is used to prepare the microorganism formulation of aquatic livestock.Due toBacillus sp.B1 bacterial strain has stronger production fat
The activity of enzyme, protease, amylase.Therefore, it can be used for preparing the feed addictive of aquaculture of aquatic animal and improve cultivation water
The probiotics of body.
Bacillus strain disclosed by the invention is possessed compared with prior art to be had the active effect that
Bacterial strain with antibacterial activity of the invention is one plant of new bacterial strain that the present inventor screens from gut of shrimp,
The bacterial strain isBacillus sp.B1 bacterial strain has stronger production digestive enzyme activity, provides new the having of one kind and produces digestive ferment
Active sea source animal intestinal tract microorganism, to utilize animal endogeneous activity bacterial strain dynamic for improving aquatic products in aquaculture of aquatic animal
Object improves its immunity etc. to the digestibility and improvement water quality of feed in turn and provides new method, promotes aquaculture
The benign development of industry.
Detailed description of the invention:
Fig. 1 is the colonial morphology figure of Bacillus strain of the present invention；
Fig. 2 is micro- (100 ×) the photo figure of Bacillus strain of the present invention；
Fig. 3 is 3 kinds of digestive enzyme activity figures of Bacillus strain of the present invention；Wherein (a) albumen enzyme positive, (b) amylase
The positive, (c) fatty enzyme positive；
Fig. 4 (a-e) is the quantified results of related immune gene in prawn haemocyte；Wherein (a) superoxides discrimination
Change enzyme gene relative expression spirogram, (b) prophenoloxidase gene relative expression spirogram, (c) antibacterial peptide gene relative expression spirogram,
(d) lysozyme gene relative expression spirogram, (e) cell adhesion factor gene relative expression spirogram；
Fig. 5 (a-f) is prawn blood plasma related immune correlation enzyme activity determination result；Wherein (a) Acid Phosphatase Activity figure,
(b) activity of catalase figure, (c) antalzyme activity figure, (d) peroxidase activity figure, (e) superoxide dismutase is living
Try hard to, (f) Phenoloxidase Activities figure；
Fig. 6 (a-c) is the measurement result of gut of shrimp digestive enzyme activity；Wherein (a) protease activity is tried hard to, (b) starch
Enzyme activity is tried hard to, and (c) lipase activity is tried hard to；
Fig. 7 prawn weight gain figure；
Fig. 8 is prawn mortality statistics result after challenge test.
Specific embodiment
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.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, 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
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
Raw material sources used in the present invention are as follows:
Embodiment 1
Litopenaeus vannamei used in the present embodiment is derived from Tianjin Hangu District prosperous Yongfeng prawn culturing field；
Bacillus isolation medium used in the present embodiment is prepared using nine salting liquids；
Bacillus isolation medium used in the present embodiment includes the weight proportion of following component: peptone 10g/L,
Yeast extract 5g/L, glucose 3.5g/L, NaCl 5g/L, K2HPO40.5g/L, MgSO43g/L, MnSO40.025g/L, fine jade
PH7.4~7.6 rouge 15g/L.
The present embodimentBacillus sp.The screening technique of B1 bacterial strain:
Bacterial strain isolates and purifies: taking the full intestines of prawn in homogenizer under aseptic condition, ice bath grinding, with nine salting liquids pair
Lapping liquid carries out 10-1~10-8Gradient dilution；Choosing gradient is 10-3~10-7Each 0.1mL of dilution be coated on bacillus
On isolation medium；28 DEG C of 24~48h of constant temperature incubation；Single colonie is taken to carry out 3 ~ 5 times on bacillus isolation medium continuously
Scribing line isolates and purifies, and the bacterial strain after purification of acquisition carries out short-term and long-term preservation at 4 DEG C and -80 DEG C respectively.
Digestive enzyme activity test is carried out to bacterial strain after purification:
A, 28 DEG C of constant-temperature shaking cultures in bacterial strain switching fluid nutrient medium after purification are stayed overnight, is clamped with sterile tweezers
Aseptic filter paper piece, which is placed in culture solution, is impregnated with bacterium solution, is placed on the corresponding position of the skimmed milk culture medium prepared, 28 DEG C
For 24 hours, the bacterial strain for generating transparent circle to energy decomposing protein screens constant temperature incubation.
B, 28 DEG C of constant-temperature shaking cultures in bacterial strain switching fluid nutrient medium after purification are stayed overnight, is clamped with sterile tweezers
Aseptic filter paper piece, which is placed in culture solution, is impregnated with bacterium solution, is placed on the corresponding position of the starch culture-medium prepared, 28 DEG C of constant temperature
For 24 hours, the bacterial strain for generating transparent circle to energy starch-splitting after iodine solution covering screens for culture.
C, 28 DEG C of constant-temperature shaking cultures in bacterial strain switching fluid nutrient medium after purification are stayed overnight, is clamped with sterile tweezers
Aseptic filter paper piece, which is placed in culture solution, is impregnated with bacterium solution, is placed on the corresponding position of the fatty culture medium prepared, 28 DEG C of constant temperature
Culture for 24 hours, forms red bacterial strain and screens to that can reduce fat.
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.
Fat culture medium used in the present embodiment includes the weight proportion of following component: nutrient agar 1000ml, peanut
Oily 10g, 1.6% dimethyl diaminophenazine chloride 1ml.It screens, is finally obtained with the bacterial strain for producing three kinds of digestive enzyme activities through the above methodBacillus sp.B1.It is common that the bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms on November 27th, 2015
Microorganism center, deposit number are CGMCC No:11752.
Embodiment 2
Choosing the present invention that above-described embodiment obtains has production digestive enzyme activityBacillus sp.B1 bacterial strain reflects
Fixed and analysis, specific identification and analysis method and corresponding qualification result are as follows.
Digestive enzyme activity is produced using having of screening of the method for above-described embodimentBacillus sp.B1 bacterial strain connects
In kind to bacillus isolation medium plate, culture is inverted for 24 hours under the conditions of 28 DEG C, observes the growth conditions of its bacterium colony, bacterium
(Zhu Gejian, Wang Zhengxiang, industrial microorganism test skill referring to industrial microorganism experimental technique handbook for plant shape state and physiological and biochemical test
Art handbook [M], China Light Industry Press, 1994) and primary Jie Shi Bacteria Identification handbook (Buchanan R.E, N.E Ji Bensi, primary
Jie Shi Bacteria Identification handbook [M], Science Press, Beijing, 1984).It is observed in constant incubator between 4 DEG C ~ 50 DEG C
Growing state.
It is above-mentionedBacillus sp.The qualification result of B1 strain morphology and physiological and biochemical property shows:
As shown in Figure 1, of the inventionBacillus sp.B1Bacterial strain bacterium colony is opaque and has gauffer and protuberance, and surface is wet
Profit is in canescence, and bacterium colony is clear-cut, and thallus is easily provoked, and microscope hypothallus is in the shape of a rod, and gemma is born in nutrition body cell
Between, cellular morphology is shown in Fig. 2.Bacillus sp.B1 bacterial strain belongs to gram-positive bacteria, and 28 DEG C of optimum growth temperature, the most suitable growth
PH value is 7.Energy decomposition glucose, fructose, sucrose, do not decompose xylose, mannitol, arabinose, do not utilize urea, malonic acid
Salt, can liquefy gelatin, nitrate reductase and catalase positive, ornithine decarboxylase, lysine decarboxylase, oxidizing ferment,
Indole test is feminine gender.Hemotoxin is not produced, to common antibiotics mezlocillin, azlocillin, cefotaxime, cephalo pyrrole
The sensitivities such as oxime, bacitracin, gentamicin, erythromycin, medecamycin, tetracycline, chloramphenicol.
Embodiment 3
Of the inventionBacillus sp.The molecular biology research of B1 bacterial strain, i.e., the 16s rRNA's of bacterial strain of the present invention
PCR amplification is adopted with the conventional methods in the field, it should be noted that the acquisition of PCR reaction template.
The acquisition of template: digestive enzyme activity is produced using having of screening of the method for above-described embodimentBacillus sp.The pure culture of B1 bacterial strain is inoculated into bacillus separation fluid nutrient medium, and for 24 hours, preparation bacterium is outstanding for 28 DEG C of shaken cultivations
Liquid.The ultrapure water of the bacteria suspension and 70 μ L that take 30 μ L mixes, and 100 DEG C of heating 5min, 2000rpm centrifugation 10min take supernatant conduct
The template of PCR reaction.
The primer that PCR amplification uses in above-described embodiment is synthesized for universal primer by Huada gene company, draws sequence such as
Under:
5'-ACAAGCCCTGGAAACGGGGT-3'；
5'-CACCAGGAATTCCGATCT-3'；
The condition of pcr amplification reaction are as follows: 94 DEG C of initial denaturation 4min, into circulation, 94 DEG C of denaturation 30s, 50 DEG C of annealing 1min,
72 DEG C of extension 2min, 30 recycle, and 4 DEG C of states are saved after 72 DEG C of extension 10min.PCR product is completed to survey by Huada gene company
Sequence, sequencing result is as shown in SEQ ID NO:1.The 16s rRNA gene order of Bacillus strain of the invention is carried out homologous
Property analysis, the bacterial strain withBacillus sp.Homology highest, homology is up to 100%.
Embodiment 4
Of the inventionBacillus sp.The production application of B1 bacterial strain is studied.
It is describedBacillus sp.B1 bacterial strain in production application beBacillus sp.B1 bacterial strain is added to feed original
In powder, it is fed to the litopenaeus vannamei regular period, observes and measure the opposite variation of its growth conditions and autoimmunity.Specifically
Method is as follows:
Of the inventionBacillus sp.B1 bacterial strain is expanded culture in liquid bacillus isolation medium step by step,
The fermentation liquid of a large amount of bacterial strains is prepared, while measuring the cell concentration of fermentation liquid.
The fermentation liquid that will be enlarged by culture is added to Fodder making in prawn feed original powder, and the method for Fodder making is as follows:
The fermentation liquid of the bacterial strain of preparation is proportionally added in feed original powder, wherein being added in every 100g prawn feed
10g sodium alginate, additionBacillus sp.B1 bacterial strain fermentation liquor 20mL, until final concentration of 3 ~ 6 × 107CFU/g.Keep feed former
Powder and bacterium solution and sodium alginate are sufficiently mixed uniformly.Feed processing.The feed mixed is kneaded into bulk, it is straight with granule mechanism
Diameter is the particle of 2mm.Preparation in feed every 7 days is primary, and preparation amount is continuously increased with the growth of litopenaeus vannamei.
Feeding:
The feed feeding prawn that will be prepared is divided into test group and control group, all receiving of every group of 600 cabrage of dispensing about 3.5g
Shore prawn.Feed is launched by the 5% of litopenaeus vannamei weight, is fed daily 4 times.The variation of water quality is recorded, and changes water in time, is prevented
Control residual bait overlong time causes change of water quality to influence litopenaeus vannamei in water growth and health.
Sampling and processing: every 7 days are a cycle, carry out primary sample, take 30 ~ 40 tail vannamei boones at random for every group when sampling
Prawn and the weight for recording litopenaeus vannamei；Hemolymph is extracted from litopenaeus vannamei cardiocoelom, is added according to the ratio of 1:1 anti-
Solidifying agent, and at 4 DEG C, 800g is centrifuged 10min, obtains haemocyte and serum.- 80 DEG C are placed in by 1mL Trizol is added in haemocyte
It saves, serum is placed in 4 DEG C for use；The enteron aisle of litopenaeus vannamei is taken, the enzyme Extraction buffer in kit is used after removal excrement, is ground
Mill prepares tissue sample.
Anti-coagulants used in the present embodiment includes the proportion of following component:
KCl:10 mM；NaCl:450 mM；HEPES:10 mM；EDTA(Na2): 10 mM；PH:7.45.
FeedingBacillus sp.The measurement of B1 bacterial strain prawn items physical signs: it is extracted with resulting haemocyte is sampled
RNA is reversed to cDNA, measures prawn related immune gene pro-phenoloxidase with the method for real time fluorescent quantitative using it as template
Gene, superoxide dismutase gene, lysozyme gene, cell adhesion molecules gene and antibacterial peptide gene quantitative expression, tool
Body measuring method is shown in embodiment 5.To feedingBacillus sp.The blood and enteron aisle of B1 bacterial strain group prawn and control group prawn into
Row sampling, resulting serum and intestinal tissue lapping liquid, are measured with kit (Suzhou Ke Mingshi biological reagent Co., Ltd) and are tried
A group prawn nospecific immunity related enzyme activity is tested, is specifically included: phenol oxidase, peroxidase, acid phosphatase, super oxygen
The measurement of object mutase SOD, catalase and antalzyme activity, measuring method are shown in embodiment 6.To feedingBacillus sp.The enteron aisle of B1 bacterial strain group prawn and control group prawn is sampled, resulting intestinal tissue lapping liquid, with kit (Suzhou
Ke Mingshi biological reagent Co., Ltd) measurement test group gut of shrimp protease, amylase and lipase activity variation, specifically
Measuring method is shown in embodiment 7.In addition, also measured were feedingBacillus sp.The weight of B1 bacterial strain group prawn and control group prawn
Variation, and in the case where being stimulated by pathogen the death rate difference, specific measuring method is shown in 8 He of embodiment respectively
Embodiment 9.
Embodiment 5
FeedingBacillus sp.After B1 bacterial strain, the variation of prawn related immune gene expression amount:
The extraction of RNA: by the prawn haemocyte of above-mentioned sampling, 1mL Trizol is added；4 DEG C, 12000g centrifugation 10
min；Supernatant is taken, 5 minutes is stood, cracks it completely.Then plus 200 μ L chloroforms, 15s is acutely vibrated, 2-3min is stored at room temperature.
4 DEG C, 12000g, it is centrifuged 15min, divide three-phase after centrifugation: the pale red azoic coupling component of lower layer, chloroform are mutually protein, and interphase is DNA, on
Layer colourless aqueous phase is RNA.Colourless aqueous phase is taken, adds 500 μ L isopropanols, mixes gently, stands 15min, precipitates RNA.4 DEG C,
12000g is centrifuged 10 min, abandons supernatant inversion and drains；The ethyl alcohol of 1mL75% is added, piping and druming is gently rubbed with pipette tips floats RNA；
4 DEG C, 7500g 6 min of centrifugation, abandon supernatant inversion and drain about 15 min；Suitable DEPC water is added, 5 min of water-bath in 55 DEG C
It is placed in -80 DEG C of refrigerators and saves.The haemocyte total serum IgE of extraction through 1% formaldehyde agarose gel electrophoresis and NANODrop2000 into
Row qualitative and quantitative detection, confirm RNA integrality after be used for cDNA synthesis.
The synthesis of first chain cDNA: using 2 μ g litopenaeus vannamei haemocyte total serum IgEs as reverse transcription template, AOLP (5 '-
GGCCACGCGTCGACTAGTAC (T) 16-3 ') it is reverse primer, the first chain cDNA is synthesized according to following reaction:
1 first chain cDNA reaction system of table
The quantitative determination of related immune gene: using cDNA obtained above as the template of Real-time PCR, withβ-
Actin is as internal control primer, the reaction system of Real-time PCR are as follows: 95 DEG C of initial denaturation 30s, a circulation；95 DEG C of changes
Property 5s, 60 DEG C of annealing 30s, 40 circulation；95 DEG C most are warming up to through 60 DEG C afterwards, each circulation rises 0.5 DEG C of progress melt curve analysis
Analysis.It is calculated after data statistical analysis obtained using 2- △ △ Ct method, it is poor using t check analysis target gene conspicuousness
The primer sequence of different different immunogenes used is as follows.
Primer is used in the experiment of table 2
3 real-time quantitative PCR reaction system of table
Test result shows that feeding adds of the inventionBacillus sp.The immunogene of the prawn of B1 bacterial strain feed
Expression quantity is in up-regulation trend, while test group is obviously increased than the immunogene expression quantity of control group, and concrete outcome is shown in Fig. 4,
From the level of molecule demonstrate feeding add it is of the inventionBacillus sp.The immunity of the prawn of B1 bacterial strain feed obviously mentions
It is high.
Embodiment 6
The measurement of cultured prawn related immune enzyme activity: it is biological with the inscription examination of Suzhou section is purchased to sample determining the protein quantity in this
The Coomassie brilliant blue kit of reagent Co., Ltd.
Protein content (μ g/mL)=standard protein concentration × (A measurement-A blank)/(A standard-A blank).
The measurement of Phenoloxidase Activities: the reagent of biological Co., Ltd is engraved in the measurement of Phenoloxidase Activities using Suzhou section
Box is carried out according to kit specification operation.This test, which defines every mg albumen, in the reaction system changes light absorption value at 525nm
0.01 is an enzyme activity unit.
Phenoloxidase Activities (U/mg prot)=reaction total volume/sample volume/reaction time/0.01 × (- A pairs of A measurement
According to)/protein concentration (mg/mL).
The measurement of peroxidase activity: the examination of biological Co., Ltd is engraved in the measurement of peroxidase activity using Suzhou section
Agent box is carried out according to kit specification operation.This test defines every mL serum and does not have a minute A470 variation in the reaction system
0.01 is an enzyme activity unit.
Peroxidase activity (U/mL)=reaction total volume/sample volume/0.01 × Δ A
The measurement of Acid Phosphatase Activity: the examination of biological Co., Ltd is engraved in the measurement of Acid Phosphatase Activity using Suzhou section
Agent box is carried out according to kit specification operation.This test defines every milliliter of blood in 37 DEG C and is catalyzed 1 μm of ol phenol of generation per minute
For an enzyme activity unit.
Acid Phosphatase Activity (U/mL)=[C standard items × (A measures pipe-A control tube) ÷ (A standard pipe-A blank tube)
× V is anti-total] the ÷ V sample × total ÷ T of V sample.
The measurement of superoxide dismutase SOD vigor: the measurement of superoxide dismutase activity engraves biology using Suzhou section
The kit of Co., Ltd is carried out according to kit specification operation.This test is defined on xanthine oxidase coupling reaction body
When inhibiting rate is 50% in system, the SOD enzyme activity in reaction is an enzyme activity unit.
Inhibit percentage=(A control tube-A measurement pipe)/A control tube × 100%
SOD activity (U/mL)=inhibit percentage/(1- inhibits percentage)
The measurement of activity of catalase: the examination of biological Co., Ltd is engraved in the measurement of activity of catalase using Suzhou section
Agent box is carried out according to kit specification operation.This test defines the amount of every milliliter of serum hydrogen peroxide per second for decomposing 1 μm of ol
For a unit of activity.
Activity of catalase (U/mL)=(A control-A measurement) × 271/(60 × sampling amount) it dilutes before × test sample
Multiple.
The measurement of antalzyme activity: with micrococcus lysodeikticus (Micrococcus lysoleikticus), freeze-dried powder (build by Nanjing
Produced at Bioengineering Research Institute) make OD570nm with the kaliumphosphate buffer preparation substrate suspension of 0.1M pH6.4 for substrate
≈ 0.3~0.5.Serum and bacteria suspension are added in 96 orifice plates, serum: equal blood=1:10, the light absorption value that measurement 570nm goes out are
For initial light absorption value A0.37 DEG C of water-bath 15min, ice bath 3min, the light absorption value that measurement 570nm goes out is A.Antalzyme activity (U/
ML)=(A0-A)/A.
Test result shows that feeding adds of the inventionBacillus sp.The phenol oxidase of B1 bacterial strain feed prawn, mistake
Oxide enzyme, acid phosphatase, superoxide dismutase SOD, catalase and antalzyme activity etc. are special and non-specific to exempt from
Epidemic disease enzyme activity has different degrees of raising relative to control group prawn, and concrete outcome is shown in Fig. 5, demonstrates feeding from the level of enzyme activity
It adds of the inventionBacillus sp.The immunity of the prawn of B1 bacterial strain feed significantly improves.
Embodiment 7
The measuring method of cultured prawn enteron aisle digestive enzyme activity is as follows:
A, the measurement of prolease activity: the kit of biological Co., Ltd is engraved in the measurement of prolease activity using Suzhou section,
It is carried out according to kit specification operation.This test 30 DEG C of every milligram of albumen of definition hydrolyze 1 μm of ol tyrosine of generation per minute
One unit of activity.
Prolease activity (U/mg prot)=C standard items × (A measurement-A blank)/(A standard-A blank) × extension rate/
(protein content × determinand volume).
B, the measurement of amylase activity: the kit of biological Co., Ltd is engraved in the measurement of amylase activity using Suzhou section,
It is carried out according to kit specification operation.It is one that this test, which defines every mg histone and is catalyzed generation 1mg reduced sugar per minute,
Enzyme activity unit.
Amylase activity (U/mg prot)=89.4 × (A measures pipe-A control tube+0.022)/protein content
C, the measurement of lipase activity:
The kit of biological Co., Ltd is engraved in the measurement of lipase activity using Suzhou section, is carried out according to kit specification
Operation.It is an enzyme activity list that this test, which defines every milligram of albumen Hydrolysis of Olive Oil per minute in 37 DEG C and generates 1 μm of ol fatty acid,
Position.
Lipase activity (U/mg prot)=[C standard items × V standard items × (A measures pipe-A blank tube)/(A standard pass-A
Blank tube)]/(the supernatant volume that protein content × supernatant total volume/is added to reaction system)/reaction time
Test result shows that feeding adds of the inventionBacillus sp.The protease of B1 bacterial strain feed gut of shrimp,
Amylase and lipase activity have different degrees of raising relative to control group prawn, and concrete outcome is shown in Fig. 6, show that feeding adds
Add of the inventionBacillus sp.The prawn of B1 bacterial strain feed significantly improves the abilities of digestive and absorption of feed.
Embodiment 8
The measurement of cultured prawn weight gain: before on-test, 50 tail vannamei boones pair are taken out in every group of aquaculture pond at random
Shrimp measures its weight, and carries out record M0, after feeding experiment, takes 50 tails to measure its body in every group of aquaculture pond at random and goes forward side by side
Row record M.Pass through the weight gain that every group of prawn is calculated: weight gain (WGR, %)=(M-M0)/M0 × 100%.Examination
Test the results show that feeding add it is of the inventionBacillus sp.The weight of the prawn of B1 bacterial strain feed is compared with than control group
It dramatically increases, showsBacillus sp.The addition of B1 bacterial strain to the abilities of digestive and absorption of feed and then improves body to raising prawn
The incrementss of weight have certain facilitation, and concrete outcome is shown in Fig. 7.
Embodiment 9
The measurement of the death rate after cultured prawn challenge test: after feeding experiment, every group takes 90 tails of uniform size
Litopenaeus vannamei carries out challenge test, and every group of 90 tail prawns are randomly divided into three in parallel.The pathogenic bacteria Vibrio anguillarum that will be prepared,
Intramuscular injection is carried out by the second uromere in prawn and carries out challenge test, and the concentration for injecting bacterium is 3.58 × 107 cell /
ML, injection volume are 15 μ L.It attacks after the completion of poison, observes prawn survival condition, record the The dead quantity of prawn, calculate in 72h
The prawn death rate.Test result is shown: being measured it to the prawn grab sample by feeding and is infected by pathogen Vibrio anguillarum
Cumulative mortality in 72h, as a result as shown in Figure 8, it can be deduced that addition is waitedBacillus sp.After B1 bacterial strain, test group prawn
Cumulative mortality be significantly lower than blank group, illustrate additionBacillus sp.B1 bacterial strain is improving immunity of prawn and then is mentioning
High its plays a role to the resistivity of pathogenic bacteria.
Embodiment 10
A kind of prawn feed containing Bacillus strain, it is by prawn feed, Bacillus strain fermentation liquid, seaweed
Sour sodium composition adds Bacillus strain fermentation liquid 20mL, until end wherein adding 10g sodium alginate in every 100g prawn feed
Concentration is 3 × 107CFU/g；The Bacillus strain isBacillus sp.B1 bacterial strain, deposit number are CGMCC No:
11752。
Embodiment 11
A kind of prawn feed containing Bacillus strain, it is by prawn feed, Bacillus strain fermentation liquid, seaweed
Sour sodium composition adds Bacillus strain fermentation liquid 20mL, until end wherein adding 10g sodium alginate in every 100g prawn feed
Concentration is 6 × 107CFU/g；The Bacillus strain isBacillus sp.B1 bacterial strain, deposit number are CGMCC No:
11752。
Specific embodiment described in the present invention is done to spirit of that invention for example, technology belonging to the present invention is led
The technical staff in domain can make corresponding modification and supplement or is substituted in a similar manner to described specific embodiment, but
Range defined in institute's person's the appended claims is not deviated from the spirit of the invention or surmounts.Although the present invention has made
Detailed description has simultaneously been cited some specific embodiments, but for this field institute those of skill in the art, as long as without departing from
It is obvious that the spirit and scope of the present invention, which can do corresponding change or amendment,.
SEQUENCE LISTING
<110>Tianjin Normal University
<120>for improving Bacillus strain and its screening technique and the application of aquatic livestock growth performance
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1469
<212> DNA
<213>artificial sequence
<400> 1
tctgtcacct taggcggctg gctccaaaaa ggttacccca ccgacttcgg gtgttacaaa 60
ctctcgtggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct 120
gatccgcgat tactagcgat tccagcttca tgtaggcgag ttgcagccta caatccgaac 180
tgagaacggt tttatgagat tagctccacc tcgcggtctt gcagctcttt gtaccgtcca 240
ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct 300
tcctccggtt tgtcaccggc agtcacctta gagtgcccaa cttaatgatg gcaactaaga 360
tcaagggttg cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa 420
ccatgcacca cctgtcactc tgctcccgaa ggagaagccc tatctctagg gttttcagag 480
gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc 540
ttgtgcgggc ccccgtcaat tcctttgagt ttcagccttg cggccgtact ccccaggcgg 600
agtgcttaat gcgttaactt cagcactaaa gggcggaaac cctctaacac ttagcactca 660
tcgtttacgg cgtggactac cagggtatct aatcctgttt gctccccacg ctttcgcgcc 720
tcagtgtcag ttacagacca gaaagtcgcc ttcgccactg gtgttcctcc atatctctac 780
gcatttcacc gctacacatg gaattccact ttcctcttct gcactcaagt ctcccagttt 840
ccaatgaccc tccacggttg agccgtgggc tttcacatca gacttaagaa accacctgcg 900
cgcgctttac gcccaataat tccggataac gcttgccacc tacgtattac cgcggctgct 960
ggcacgtagt tagccgtggc tttctggtta ggtaccgtca aggtgccagc ttattcaact 1020
agcacttgtt cttccctaac aacagagttt tacgacccga aagccttcat cactcacgcg 1080
gcgttgctcc gtcagacttt cgtccattgc ggaagattcc ctactgctgc ctcccgtagg 1140
agtctgggcc gtgtctcagt cccagtgtgg ccgatcaccc tctcaggtcg gctacgcatc 1200
gttgccttgg tgagccgtta cctcaccaac tagctaatgc gacgcgcgtc catccataag 1260
tgacagccga agccgccttt caatttcgaa ccatgcggtt caaaatgtta tccggtatta 1320
gccccggttt cccggagtta tcccagtctt atgggcaggt tacccacgtg ttactcaccc 1380
gtccgccgct aacttcataa gagcaagctc ttaatccatt cgctcgactt gcatgtatta 1440
ggcacgccgc agcgttcatc ctgagcaga 1469
Claims (3)
1. a bacillus bacterial strain, it is characterised in that the bacterial strain isBacillus sp.B1 bacterial strain, the bacterial strain in
On November 27th, 2015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is
CGMCC No:11752；It is describedBacillus sp.The 16S rRNA sequence of B1 bacterial strain is as shown in SEQ. NOl.
2. Bacillus strain described in claim 1Bacillus sp.Application of the B1 bacterial strain in terms of preparing digestive ferment, wherein
The digestive ferment refers to: protease, amylase and lipase.
3. Bacillus strain described in claim 1Bacillus sp.B1 bacterial strain is in preparation for improving aquatic livestock growth
Application in terms of energy strengthen immunity reagent.
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| CN103602608B (en) * | 2013-08-02 | 2015-07-01 | 国家海洋局第三海洋研究所 | Application of bacillus pumilus in prawn culture |
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