CN105935108A - Prawn feed additive containing Rhodobacter Capsulatus and Bacillus subtilis strains, and application thereof - Google Patents
Prawn feed additive containing Rhodobacter Capsulatus and Bacillus subtilis strains, and application thereof Download PDFInfo
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Abstract
The invention discloses a prawn feed additive containing Rhodobacter Capsulatus and Bacillus subtilis strains. The prawn feed additive is composed of a prawn feed, a Rhodobacter Capsulatus strain fermentation liquid, a Bacillus subtilis strain fermentation liquid and sodium alginate, wherein 10g of sodium alginate is added to every 100g of the prawn feed, and culturing is carried out until the OD600 is 0.6, the total volume of the Rhodobacter Capsulatus strain fermentation liquid and the Bacillus subtilis strain fermentation liquid is 20mL and the final concentration is 3-6 * 10<7>/g. A result of feeding with prawn feeds prepared by using Rhodobacter Capsulatus and Bacillus subtilis as feed additives shows that the prawns fed with the feeds containing Rhodobacter Capsulatus and Bacillus subtilis have greatly higher growth speed and obviously high immunity than prawns fed with common feeds; and the feeds prepared by using Rhodobacter Capsulatus and Bacillus subtilis have the advantages of short production period, low production cost, easy control of growth conditions, high antibiotic activity, obviously enhanced immunity, high industrial production potential and good production application prospect.
Description
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 invention belongs to technical field of bioengineering, relate to that there is bacterial strain that is antibacterial and that produce digestive enzyme activity and screening side thereof
Method and application, be a kind of containing Rhodobacter capsulatus and the prawn feed of Bacillus strain and application in particular.
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, it is provided that a kind of containing Rhodobacter capsulatus and the prawn feed of Bacillus strain.Feed result to show
Show the edible prawn adding Rhodobacter capsulatus and bacillus feed relative to edible normal diet the prawn speed of growth significantly
Improving and immunity increases substantially, utilize Rhodobacter capsulatus and bacillus cereus Fodder making, with short production cycle, production cost is low,
Easily controllable growth conditions, antibacterial activity is high and can be remarkably reinforced the plurality of advantages such as immunity, has industrialized production completely
Potentiality, have preferable production application prospect
For achieving the above object, the invention discloses following technology contents:
A kind of prawn feed additive containing Rhodobacter capsulatus bacterial strain, it is characterised in that it is red carefully by prawn feed, pod membrane
Bacteria strain fermentation liquid, Bacillus strain fermentation liquid and sodium alginate composition, wherein add 10g Sargassum in every 100g prawn feed
Acid sodium, adds and cultivates to OD600=0.6(cell concentration 3 × 108Individual/mL) Rhodobacter capsulatus bacterial strain fermentation liquor and bacillus cereus
Fermentation liquid 20mL (volume ratio of Rhodobacter capsulatus and fermentation of bacillus liquid is 7:3) altogether, to final concentration of 3 ~ 6 × 107Individual/g；
Described Rhodobacter capsulatus bacterial strain isRhodobacter capsulatusR3 bacterial strain, deposit number is CGMCC No:11753,
Described Bacillus strain isBacillusB1 bacterial strain, deposit number is CGMCC No:11752.
The present invention further discloses the prawn feed additive containing Rhodobacter capsulatus bacterial strain and Bacillus strain to exist
Improve the application in terms of the prawn speed of growth.And the application in terms of digestive enzyme activity is produced in preparation, wherein said digestive enzyme
Refer to: protease, amylase and lipase.Particularly prawn feed improves it and supports pathogenic bacterium strengthening immunity of prawn
Application in terms of drag.Described enhancing immunity of prawn refers to: prawn is infected adding up in 72h by pathogen Vibrio anguillarum
Mortality rate.
The two strain bacterial strains of the present invention are respectivelyRhodobacterBelong to Rhodobacter capsulatus (Rhodobacter capsulatus), namedRhodobacter capsulatus R3, is preserved in Chinese micro-life on November 27th, 2015
Thing culture presevation administration committee's common micro-organisms center, deposit number is CGMCC No:11753；AndBacillus sp.
The bacillus cereus belonged toBacillusB1 bacterial strain, 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:11752.Preservation address: BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, Institute of Microorganism, Academia Sinica, postcode 100101.
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
Bacterium colony is opaque, surface wettability thickness, and in crocus, positive and negative solid colour, bacterium colony is clear-cut, and thalline is easily provoked, its bacterium
The form that falls 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'sBacillusThe biological characteristics of B1 bacterial strain is as follows:
Bacillus cereus (Bacillus sp.), namedBacillus The bacterium colony of B1 is opaque and has gauffer and protuberance, surface
Moistening thickness, in canescence, bacterium colony is clear-cut, and thalline is easily provoked, and its colonial morphology is shown in that Fig. 1, microscope hypothallus are shaft-like,
Spore and is born in the middle of trophosome cell, and cellular morphology is shown in Fig. 2.BacillusB1 bacterial strain belongs to gram negative bacteria, the suitableeest life
Long temperature 28 DEG C, the most suitable growth pH value is 7.Its physio-biochemical characteristics are as follows: energy decomposition glucose, fructose, sucrose, do not decompose wood
Sugar, mannitol, arabinose, do not utilize carbamide, malonate, and can liquefy gelatin, nitrate reductase and catalase sun
Property, ODC Ornithine decarboxylase, E.C. 4.1.1.18, oxidase, indole test are feminine gender.Do not produce hemotoxin, to conventional antibiosis
Element mezlocillin, azlocillin, cefotaxime, cefepime, bacitracin, gentamycin, erythromycin, midecamycin, tetracycline,
Chloromycetin etc. are the most sensitive, and drug sensitive test uses agar diffusion method of the paper, and it is limited that Antibiotic discs is purchased from Hangzhou microorganism reagent
Company.
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 andBacillusB1 is respectively provided with stronger
Produce digestive enzyme activity, wherein the active bacteria of protease, amylase and lipase is higher, 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 andBacillusB1 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
Release liquid to take 0.1mL and be respectively coated in 2216E culture medium and bacillus cereus isolation medium, after 28 DEG C of constant temperature culture, picking list
Bacterium colony is repeatedly rule on 2216E solid medium and 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 andBacillusB1 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 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 digestive enzyme activity, described bacillus cereus isolation medium uses
Conventional bacillus culture medium.As preferably, described bacillus cereus isolation medium includes that the weight of following component is joined
Ratio: 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 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 Rhodobacter capsulatus bacterial strainRhodobacter capsulatusR3 prepare antibacterial
Application in terms of Huo Xing；Described bacteriostatic activity refer to Vibrio anguillarum (Vibrio anguillarum) suppression.And pod
Film Rhodobacter strainRhodobacter capsulatusR3 and bacillus cereusBacillusB1 produces digestive enzyme activity in preparation
The application of aspect, wherein said digestive enzyme refers to: protease, amylase and lipase.Experimental result shows: the present invention's
Bacterial strain isRhodobacter capsulatusR3 andBacillusB1 bacterial strain, describedRhodobacter capsulatus
R3 andBacillusB1 bacterial strain is for preparing the microorganism mix preparation of aquatic animal.Due toRhodobacter capsulatusR3 andBacillusB1 bacterial strain has stronger antibacterial activity and yielding lipase, protease, diastatic work
Property.Therefore, it can for preparing the feed additive of aquaculture of aquatic animal and improving the microbial ecological agent of breeding water body.
Prawn feed additive containing Rhodobacter capsulatus bacterial strain and Bacillus strain disclosed by the invention and existing skill
Art compares having the active effect that of being had
The bacterial strain of the present inventionRhodobacter capsulatus R3 and BacillusB1, is that the present inventor is from gut of shrimp
The bacterial strain that two strains of middle screening are new, whereinRhodobacter capsulatus R3 bacterial strain has stronger antibacterial activity and product
Digestive enzyme activity,BacillusB1 bacterial strain has the stronger activity producing digestive enzyme, the invention provides a kind of new have anti-
Bacterium activity and improve digestion power extra large source animal intestinal microorganism mix preparation, for utilize animal endogeneous activity bacterial strain for
Disease control in aquaculture of aquatic animal, improve aquatic animal and to the digestibility of feedstuff and improve the aspects such as water quality and provide
New method, promotes the benign development of culture fishery.
Accompanying drawing illustrates:
Fig. 1 (a) is Rhodobacter capsulatus colonial morphology figure of the present invention, and Fig. 1 (b) is Bacillus colonies aspect graph of the present invention；
Fig. 2 (a) is micro-(100 ×) photo figure of Rhodobacter capsulatus bacterial strain of the present invention, and Fig. 2 (b) is bacillus cereus bacterium of the present invention
Micro-(100 ×) photo figure of strain；
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 (Fig. 5,1-1 to 1-3) be in prawn hemocyte related immune gene quantified results；Wherein (a) super oxygen
Compound gasification enzyme gene relative expression's spirogram, (b) prophenoloxidase gene relative expression's spirogram, (c) antibacterial peptide gene is relative to table
Reach spirogram, (d) lysozyme gene relative expression's spirogram, (e) cell adhesion factor gene relative expression's spirogram；
Fig. 6 (Fig. 6,1-1 to 1-3) is prawn blood plasma related immune relevant enzyme vitality test result；Wherein (a) acid phosphatase is lived
Try hard to, (b) activity of catalase figure, (c) antalzyme activity figure, (d) peroxidase activity figure, (e) superoxide dismutase
Enzyme activity figure, (f) Phenoloxidase Activities figure；
Fig. 7 (Fig. 7,1-1 to 1-2) is the measurement result of gut of shrimp digestive enzyme activity；Wherein (a) protease activity is tried hard to, (b)
Diastatic activity is tried hard to, and (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 10-1～10-8Gradient dilution；Choosing gradient is 10-3～10-7The each 0.1mL of diluent be coated on 2216E culture medium
On；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, it is thus achieved that
Bacterial strain after purification at 4 DEG C and-80 DEG C, carry out short-term respectively and preserve 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 R3 bacterial strain andBacillusB1 bacterial strain
Screening technique, its isolation and purification method, with in embodiment 1, differs only in and changes the 2216E culture medium in embodiment 1 into bud
Spore bacillus isolation medium, repeats no more here.Wherein bacillus cereus isolation medium includes the weight proportion of following component: egg
White peptone 10g/L, yeast extract 5g/L, glucose 3.5g/L, NaCl 5g/L, K2HPO40.5g/L, MgSO43g/L, MnSO4
0.025g/L, agar 15g/L pH7.4～7.6.
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:
As it is shown in figure 1, the present inventionRhodobacter capsulatus The bacterium colony of R3 bacterial strain is opaque, surface wettability thickness,
In crocus, positive and negative solid colour, bacterium colony is clear-cut, and thalline is easily provoked, and its colonial morphology is shown in Fig. 1, microscope hypothallus
In spherical, having the pod membrane of solid form, cellular morphology is shown in Fig. 2.Rhodobacter capsulatusBacterial strain belongs to gram-negative
Property bacterium, optimum growth temperature 28 DEG C, the most suitable growth pH value is between 7-8.Energy decomposition glucose, fructose, sucrose, mannitol, no
Decomposing xylose and arabinose, do not utilize carbamide, malonate, can liquefy gelatin, nitrate reductase and catalase sun
Property, ODC Ornithine decarboxylase, E.C. 4.1.1.18, oxidase, indole test are feminine gender.Do not produce hemotoxin.To conventional antibiosis
Element mezlocillin, azlocillin, cefotaxime, bacitracin, gentamycin, tetracycline, Lomefloxacin, chloromycetin nitrofurantoin,
The sensitivities such as rifampicin.
Embodiment 4
Choose the present invention that above-described embodiment obtains to have and produce digestive enzyme activityBacillusB1 bacterial strain carries out identifying and dividing
Analysis, concrete qualification and the method for analysis are with embodiment 3, and here is omitted, and corresponding qualification result is as follows.
Above-mentionedBacillusThe qualification result of B1 strain morphology and physiological and biochemical property shows:
As it is shown in figure 1, the present inventionBacillusB1 bacterial strain bacterium colony is opaque and has gauffer and protuberance, and surface wettability, in greyish white
Color, bacterium colony is clear-cut, and thalline is easily provoked, and microscope hypothallus is shaft-like, and spore and is born in trophosome cell intermediate cell shape
State is shown in Fig. 2.Bacillus sp.B1 bacterial strain belongs to gram positive bacteria, optimum growth temperature 28 DEG C, and the most suitable growth pH value is 7.
Energy decomposition glucose, fructose, sucrose, do not decompose xylose, mannitol, arabinose, do not utilize carbamide, malonate, can liquefy
Gelatin, nitrate reductase and catalase positive, ODC Ornithine decarboxylase, E.C. 4.1.1.18, oxidase, indole test are equal
For feminine gender.Do not produce hemotoxin, to common antibiotics mezlocillin, azlocillin, cefotaxime, cefepime, bacitracin, celebrating
The sensitivities such as big mycin, erythromycin, midecamycin, tetracycline, chloromycetin.
Embodiment 5
The present invention'sRhodobacter capsulatus R3 and BacillusThe production application research of B1 bacterial strain.
DescribedRhodobacter capsulatus R3 and BacillusThe application aborning of B1 bacterial strain be byRhodobacter capsulatus R3 and BacillusB1 bacterial strain mixes by a certain percentage and adds in the former powder of feedstuff, feeds
The change relative with autoimmunity of its growth conditions is observed and measured to food, to the Litopenaeus vannamei regular period,.Concrete grammar is such as
Under:
The present invention'sRhodobacter capsulatus R3 and BacillusB1 bacterial strain is respectively at liquid 2216E culture medium
And bacillus cereus isolation medium carries out amplification culture step by step, prepare the fermentation liquid of a large amount of bacterial strain, measure fermentation liquid simultaneously
Cell concentration.
Amplification culture the most step by step, the method for the fermentation liquid preparing a large amount of bacterial strain is as follows:
First inclined-plane is preservedRhodobacter capsulatus R3 and BacillusB1 strain is inoculated in 2216E respectively
And in bacillus cereus liquid culture medium (culture medium prescription is shown in embodiment 1), 28 DEG C of constant-temperature table shaken cultivation 16 ~ 18h, connect by 1%
The amount of kind is transferred respectively in 50mL respective liquid culture medium, fills fluid medium 50mL, shaking speed in 500mL triangular flask
200rpm, 28 DEG C of constant-temperature table shaken cultivation 16h, then transfer respectively amplification culture in 10L fermentation tank by 2% inoculum concentration, with
The canned amount of 70%, initial pH is set as 7.0-7.2, cultivation temperature 28 DEG C, ventilation 1:0.8, the knot when cultivation to OD600 is 0.8
Bundle fermentation, gained bacterium solution is used for preparing prawn feed.
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:
Carry out mixing by the fermentation liquid of the bacterial strain of preparation and mix according to the ratio of 7:3 and add in the former powder of feedstuff, make in feedstuff
Final concentration of 3 ~ 6 × the 10 of antibacterial7Individual/g, according to adding 10g sodium alginate in every 100g feedstuff in the preparation process of feedstuff
Ratio add sodium alginate, beneficially feedstuff molding.The former powder of feedstuff is made to be sufficiently mixed uniformly with bacterium solution and sodium alginate.
The method of feed manufacturing is as follows:
The feedstuff mixed is kneaded into bulk, with the granule of the granule a diameter of 2mm of mechanism.Feedstuff preparation in every 7 days once, is prepared
Amount is continuously increased with the growth of Litopenaeus vannamei.
The method fed is as follows:
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.
The mensuration of the every physical signs of test group prawn: extract RNA with the hemocyte of sampling gained, be reversed to cDNA, with
It uses the method for real time fluorescent quantitative to measure prawn related immune gene prophenoloxidase gene, superoxide dismutase for template
Enzyme gene, lysozyme gene, cell adhesion molecules gene and the quantitative expression of antibacterial peptide gene, concrete assay method is shown in embodiment
8.Test group prawn and the blood of matched group prawn and intestinal are sampled, the serum of gained and intestinal tissue lapping liquid, use
Test kit (Suzhou Ke Mingshi biological reagent company limited) determination test group prawn nonspecific immunity related enzyme activity, specifically wraps
Include: phenol oxidase, peroxidase, acid phosphatase, superoxide dismutase SOD, catalase and antalzyme activity
Measuring, assay method is shown in embodiment 9.The intestinal of test group prawn and matched group prawn is sampled, the intestinal tissue of gained
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 10.Additionally, also measured were test group prawn and matched group prawn
Body weight change difference, and the difference of mortality rate in the case of being stimulated by pathogen, concrete assay method is shown in enforcement respectively
Example 11 and embodiment 12.
Embodiment 6
FeedingRhodobacter capsulatus R3 and BacillusAfter B1 bacterial strain, prawn related immune gene expression amount
Change:
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 R3 and BacillusB1 mixes
The expression of the immunogene of the test group prawn of bacterial strain feedstuff is in the trend of rise, and test group is than the immunogene of matched group simultaneously
Expression substantially increases, and concrete outcome is shown in Fig. 5, demonstrates feeding from the level of molecule and adds the present invention'sBacillus sp.B1
The immunity of the prawn of bacterial strain feedstuff 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 and Bacillus B1
The phenol oxidase of test group prawn of bacterial strain mixed fodder, peroxidase, acid phosphatase, superoxide dismutase SOD, mistake
Hydrogen oxide enzyme 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, and the level lived from enzyme demonstrates the feeding interpolation present invention'sRhodobacter capsulatus R3 andBacillusThe immunity of the prawn of B1 bacterial strain feedstuff significantly improves.
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 and BacillusB1 mixes
The protease of test group gut of shrimp, amylase and the lipase activity of bacterial strain feedstuff all has different journey relative to matched group prawn
The raising of degree, concrete outcome is shown in Fig. 7, shows that feeding adds the present invention'sRhodobacter capsulatus R3 andBacillusThe abilities of digestive and absorption of feedstuff is significantly improved by the prawn of B1 hybrid bacterial strain feedstuff.
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 R3 and Bacillus B1
The body weight of the test group prawn of hybrid bacterial strain feedstuff dramatically increases with than compared with matched group, and 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 feeding adds the present invention'sRhodobacter capsulatus
R3 and BacillusThe test group prawn of B1 hybrid bacterial strain feedstuff, its cumulative mortality, significantly lower than blank group, illustrates to add
The feedstuff of hybrid bacterial strain is improving immunity of prawn and then is improving it and play a role the resistivity of pathogenic bacterium.
Embodiment 11
Prawn feed additive containing Rhodobacter capsulatus bacterial strain, it is by prawn feed, Rhodobacter capsulatus bacterial strain fermentation liquor, bud
Spore bacillus strain fermentation liquid and sodium alginate composition, wherein add 10g sodium alginate in every 100g prawn feed, add and cultivate extremely
OD600=0.6(cell concentration 3 × 108Individual/mL) Rhodobacter capsulatus bacterial strain fermentation liquor and fermentation of bacillus liquid altogether 20mL (pod
The volume ratio of the red antibacterial of film and fermentation of bacillus liquid is 7:3), to final concentration of 3 × 107Individual/g；Described Rhodobacter capsulatus
Bacterial strain isRhodobacter capsulatusR3 bacterial strain, deposit number is CGMCC No:11753, described bacillus cereus bacterium
Strain isBacillusB1 bacterial strain, deposit number is CGMCC No:11752.
The feedstuff mixed is kneaded into bulk, with the granule of the granule a diameter of 2mm of mechanism.Feedstuff is prepared once for every 7 days,
Preparation amount is continuously increased with the growth of Litopenaeus vannamei.
Embodiment 12
A kind of prawn feed additive containing Rhodobacter capsulatus and Bacillus strain, it is red carefully by prawn feed, pod membrane
Bacteria strain fermentation liquid, Bacillus strain fermentation liquid and sodium alginate composition, wherein add 10g Sargassum in every 100g prawn feed
Acid sodium, adds and cultivates to OD600=0.6, cell concentration 3 × 108The Rhodobacter capsulatus bacterial strain fermentation liquor of individual/mL and bacillus cereus are sent out
Ferment liquid 20mL altogether, wherein the volume ratio of Rhodobacter capsulatus and fermentation of bacillus liquid is 7:3, to final concentration of 6 × 107Individual/g；
Described Rhodobacter capsulatus bacterial strain isRhodobacter capsulatusR3 bacterial strain, deposit number is CGMCC No:11753,
Described Bacillus strain isBacillusB1 bacterial strain, deposit number is CGMCC No:11752.
Specific embodiment described in the present invention is only to illustrate spirit of the present invention, the technical field of the invention
Described specific embodiment can be made corresponding amendment and supplement or use similar mode to substitute by technical staff, but not
The spirit of the present invention can be deviateed or surmount scope defined in institute's person's appended claims.Although the present invention has made in detail
Description and quoted some specific embodiments as proof, but for this area institute those of skill in the art, as long as without departing from this
Bright spirit and scope can do respective change or correction is obvious.
SEQUENCE LISTING
<110>Tianjin Normal University
<120>Rhodobacter capsulatus and the prawn feed additive of Bacillus strain and application thereof are contained
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
aagatgtgtg acgacgaagt ag 22
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atacctcgct tgctctgg 18
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<400> 3
tgtgaggttc aagggcgt 18
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<211> 19
<212> DNA
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<400> 4
gtattgtcgt acagcaggc 19
<210> 5
<211> 18
<212> DNA
<213>artificial sequence
<400> 5
gtgtgagcac catccagc 18
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<211> 18
<212> DNA
<213>artificial sequence
<400> 6
acgtagcaat ggcgtggt 18
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<211> 19
<212> DNA
<213>artificial sequence
<400> 7
gttccgatct gatgtccga 19
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<400> 9
aaggacggga tggagtt 17
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<400> 10
agaagccgtt cgtgcc 16
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<400> 11
aggcggtgat tctgtgc 17
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<400> 12
tcgcttgtgc cagttcc 17
<210> 13
<211> 20
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tgccatacat caaggctaca 20
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Claims (5)
1. one kind contains Rhodobacter capsulatus and the prawn feed additive of Bacillus strain, it is characterised in that it is to be raised by prawn
Material, Rhodobacter capsulatus bacterial strain fermentation liquor, Bacillus strain fermentation liquid and sodium alginate composition, wherein in every 100g prawn feed
Add 10g sodium alginate, add and cultivate to OD600=0.6, cell concentration 3 × 108The Rhodobacter capsulatus bacterial strain fermentation liquor of individual/mL
And fermentation of bacillus liquid 20mL altogether, wherein the volume ratio of Rhodobacter capsulatus and fermentation of bacillus liquid is 7:3, to final concentration of
3~6×107Individual/g；Described Rhodobacter capsulatus bacterial strain isRhodobacter capsulatusR3 bacterial strain, deposit number is
CGMCC No:11753, described Bacillus strain isBacillusB1 bacterial strain, deposit number is CGMCC No:11752.
2. the prawn feed additive containing Rhodobacter capsulatus bacterial strain and Bacillus strain described in claim 1 is improving prawn
Application in terms of the speed of growth.
3. prawn feed additive containing Rhodobacter capsulatus bacterial strain and Bacillus strain described in claim 1 disappears preparing product
Changing the application in terms of enzymatic activity, wherein said digestive enzyme refers to: protease, amylase and lipase.
4. the prawn feed additive containing Rhodobacter capsulatus bacterial strain and Bacillus strain described in claim 1 is strengthening prawn
Immunity improves it to the application in terms of the resistance of pathogenic bacterium.
5. the application described in claim 3, enhancing immunity of prawn therein refers to: prawn is infected by pathogen Vibrio anguillarum
Cumulative mortality in 72h.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610363254.9A CN105935108A (en) | 2016-05-30 | 2016-05-30 | Prawn feed additive containing Rhodobacter Capsulatus and Bacillus subtilis strains, and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384732A (en) * | 2018-02-09 | 2018-08-10 | 上海海洋大学 | A kind of hydrogenlike silicon ion and its application for reducing metrifonate toxicity |
| CN114271410A (en) * | 2021-12-24 | 2022-04-05 | 盐城恒兴饲料有限公司 | Feather meal-containing prawn feed and preparation method thereof |
| CN116656579A (en) * | 2023-07-27 | 2023-08-29 | 海南热带海洋学院 | Novel bacterial strain for producing enzymes from ocean and application thereof |
-
2016
- 2016-05-30 CN CN201610363254.9A patent/CN105935108A/en active Pending
Non-Patent Citations (1)
| Title |
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| 窦春萌等: "凡纳滨对虾肠道内产消化酶益生菌的分离与筛选", 《水产学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384732A (en) * | 2018-02-09 | 2018-08-10 | 上海海洋大学 | A kind of hydrogenlike silicon ion and its application for reducing metrifonate toxicity |
| CN108384732B (en) * | 2018-02-09 | 2021-03-02 | 上海海洋大学 | Rhodobacter sphaeroides for reducing trichlorphon toxicity and application thereof |
| CN114271410A (en) * | 2021-12-24 | 2022-04-05 | 盐城恒兴饲料有限公司 | Feather meal-containing prawn feed and preparation method thereof |
| CN116656579A (en) * | 2023-07-27 | 2023-08-29 | 海南热带海洋学院 | Novel bacterial strain for producing enzymes from ocean and application thereof |
| CN116656579B (en) * | 2023-07-27 | 2023-10-27 | 海南热带海洋学院 | Novel bacterial strain for producing enzymes from ocean and application thereof |
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Application publication date: 20160914 |