CN104630114A - Bacillus cereus J19 as well as induction medium and application thereof - Google Patents

Bacillus cereus J19 as well as induction medium and application thereof Download PDF

Info

Publication number
CN104630114A
CN104630114A CN201510070176.9A CN201510070176A CN104630114A CN 104630114 A CN104630114 A CN 104630114A CN 201510070176 A CN201510070176 A CN 201510070176A CN 104630114 A CN104630114 A CN 104630114A
Authority
CN
China
Prior art keywords
bacillus cereus
inducing culture
vibrio parahaemolyticus
water
application
Prior art date
Application number
CN201510070176.9A
Other languages
Chinese (zh)
Inventor
梁晶晶
王娇
谢金萍
张明俊
Original Assignee
青岛根源生物技术集团有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 青岛根源生物技术集团有限公司 filed Critical 青岛根源生物技术集团有限公司
Priority to CN201510070176.9A priority Critical patent/CN104630114A/en
Publication of CN104630114A publication Critical patent/CN104630114A/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RPROCESSES USING MICROORGANISMS
    • C12R1/00Processes using microorganisms
    • C12R1/01Processes using microorganisms using bacteria or actinomycetales
    • C12R1/07Bacillus
    • C12R1/085Bacillus cereus
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01NPRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10Animals; Substances produced thereby or obtained therefrom
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria

Abstract

The invention provides bacillus cereus J19 as well as an induction medium and application thereof. The preservation serial number of bacillus cereus J19 is CGMCC No.10052. The induction medium comprises the following components: 0.1-1% of glucose, 2.0-4.0% of bean pulp powder, 0.5-1% of yeast powder, 0.1-1% of sodium chloride and 0.1-1.0% of corn pulp. The induction medium can be used for inducing bacillus cereus J19 to generate activity of inhibiting vibrio parahaemolyticus, the number of pathogenic vibrio parahaemolyticus in an aquaculture environment can be effectively reduced, G+ bacteria are not inhibited, micro-ecological damage and bacterium drug resistance caused in the use process of antibiotics can be avoided, and by adopting an antibiotic liquid produced from bacillus cereus J19, the disease and death risk of aquaculture animals caused by excessive multiplication of vibrios can be reduced by means of ecological methods.

Description

One strain bacillus cereus J19 and inducing culture thereof and application
Technical field
The invention belongs to fermentable and Application Areas, be specifically related to a strain bacillus cereus J19 and inducing culture thereof and application.
Background technology
The heavy losses that Culture of Penaeus Chinensis industry receives Deaths syndromes (EMS) are started from 2009, the first half of the year in 2011, the death loss that this disease of Hainan, Fujian, Guangdong and some areas of Guangxi causes is up to 80%, make shrimp 40/jin of specifications to form rate only have 5-10% to the multiple culture zone one of In South China in 2013, in make the most shrimp pool of shrimp and put and within seedling 10-20 days, occur row pool phenomenon, onset speed wide being more far more than that be fast, scope makes shrimp.The main cause of disease of EMS is highly pathogenic Vibrio parahaemolyticus, Algae toxins and the poor environment factor, and the conventional germ killing drugs such as microbiotic, sterilizing agent are to this sick poor effect.Adopt bacterium in biological preventions control water body to balance each other and become the new way of prevention and therapy EMS gradually.To bacteriostatic action be had to be used in aquaculture water without the fermentation of bacillus liquid of potential safety hazard again, can the use of partially or completely substitute antibiotics, not easily produce resistance, and to environment non-secondary pollution.Genus bacillus is as biological prevention and control agent mainly because its anti-microbial effect is powerful, but not all biocontrol microorganisms can produce antimicrobial substance.
Summary of the invention
The invention provides a strain bacillus cereus J19 and inducing culture thereof and application, utilize the inducing culture of bacillus cereus J19 of the present invention and correspondence thereof can obtain active substance Vibrio parahaemolyticus being had to bacteriostatic activity, significantly can reduce Vibrio parahaemolyticus content in water body.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
One strain bacillus cereus J19, its Classification And Nomenclature is bacillus cereus bacillus cereus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.10052.
Bacillus cereus J19 described in induction produces the inducing culture to the Substance of Vibrio parahaemolyticus, and described inducing culture comprises following component, by quality ratio:
Glucose 0.1% ~ 1%;
Bean cake powder 2.0% ~ 4.0%, described bean cake powder needs to use neutral protease enzymolysis in a water bath before using;
Yeast powder 0.5% ~ 1%;
Sodium-chlor 0.1% ~ 1%;
Corn steep liquor 0.1% ~ 1.0%.
Further improvement to technique scheme: described bean cake powder needs with the pre-enzymolysis 2-3h of neutral protease before using, and the consumption of neutral protease is 1500U/g bean cake powder, and hydrolysis temperature is 40 DEG C.
Further improvement to technique scheme: it comprises following component: glucose 0.1%, bean cake powder 2.0%, yeast powder 0.5%, sodium-chlor 0.2%, corn steep liquor 1.0%, surplus is water.
Present invention also offers described inducing culture to produce the application in the Substance of Vibrio parahaemolyticus at induction bacillus cereus J19.
Further improvement to technique scheme: described bacillus cereus J19 on inducing culture at 30 DEG C shaking table cultivate 12-16h and obtain bacillus cereus J19 fermented liquid.
Further improvement to technique scheme: be spread in water body by described fermented liquid, fermented liquid consumption is 1.2 ~ 1.8ml/m 3water body, the concentration of the Substance in described fermented liquid is 10 ± 0.2mg/L.
Further improvement to technique scheme: the pH 8.5 of described water body, salinity 8 ‰-10 ‰, temperature 27 DEG C-29 DEG C.
Advantage of the present invention and technique effect are: first the present invention screens a strain bacillus cereus J19, and and then obtain its inducing culture based formulas by experiment, described substratum can induce bacillus cereus J19 to produce the activity suppressing Vibrio parahaemolyticus, the quantity of pathogenic Vibrio parahaemolyticus in effective reduction culture environment of aquatic products, and to G +bacterium unrestraint effect, avoid Tiny ecosystem that microbiotic in use brings to destroy and the resistance of bacterium, adopt the bacillus cereus J19 fermented liquid that the present invention produces, reduce the ill and mortality risk of the cultivated animals brought because of vibrios excessive multiplication, and then effectively solve in recent years in aquaculture process because of high-density breeding, water environment pollution, the kinds of pathogenic vibrio amount reproduction that the factors such as the excessive use of microbiotic cause, the low-down problem of rate of forming, with the use of biotechnological formulation partially or completely substitute antibiotics, the object controlling kinds of pathogenic vibrio excessive multiplication is reached with the means of biological prevention and control.
Accompanying drawing explanation
Fig. 1 be in the present invention on 2216E flat board to the inhibition zone of Vibrio parahaemolyticus.
Fig. 2 is that the fungistatic effect of different fermentations time in the present invention measures.
Embodiment
The present invention is described in further detail the present invention in conjunction with following specific embodiment.
the acquisition of embodiment 1, bacillus cereus J19 bacterial strain
In November, 2010 gathers holothruian cultures district, Hai Yangsuo town, Rushan City, Weihai City bed mud by contriver, and carry out enrichment in laboratory, be separated acquisition one strain bacillus cereus, laboratory is numbered J19, i.e. bacillus cereus J19 of the present invention.
Described bacillus cereus J19 bacterial strain is carried out culture presevation, the title of depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC); Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.Bacillus cereus bacillus cereuspreservation date: on November 25th, 2014; Deposit number: CGMCC No. 10052.
Be that a strain has the probiotics suppressing gram negative bacterium potentiality through the known bacillus cereus J19 of experimental verification of the present invention, but in different culture media, fungistatic effect differ greatly, and does not even have fungistatic effect in some substratum.So the stable generation of medium component to bacillus cereus antibacterial substance has significant influence.
the selection of embodiment 2, different inducing culture based formulas
Selective agar medium component glucose 0.1% ~ 1% in the present embodiment; Bean cake powder 2.0% ~ 4.0%; Yeast powder 0.5% ~ 1%; Sodium-chlor 0.1% ~ 1%; Corn steep liquor 0.1% ~ 1.0% carries out optimized designing for fortnulation, and the per-cent in the present invention in culture medium prescription is mass ratio.Described bean cake powder need carry out pre-treatment, and pre-treating process is more than or equal to 40 object bean cake powder 2g for getting granularity, and add 3000U neutral protease, add water 100ml, after stirring evenly, is placed in 40 DEG C of shaking baths, 100 turns/min enzymolysis 2h.
Whether in test, yeast powder, corn steep liquor and sodium-chlor select conventional amount used, be respectively 0.5%, 1.0%, 0.2%, need enzymolysis to explore further to different glucose consumption and dregs of beans.Bacteriostatic test is carried out according to the formula of the various components shown in table 1.
Table 1: different culture media formula is on the impact of fungistatic effect
Formula number Glucose Bean cake powder Yeast powder Sodium-chlor Corn steep liquor Inhibition zone
1 0 2% enzymolysis 0.5% 0.2% 1.0% 12±0.21mm
2 0.1% 2% enzymolysis 0.5% 0.2% 1.0% 19±0.18mm
3 0.3% 2% enzymolysis 0.5% 0.2% 1.0% 19±0.25mm
4 1% 2% enzymolysis 0.5% 0.2% 1.0% 12±0.12mm
5 0.1% 2% not enzymolysis 0.5% 0.2% 1.0% 13±0.17mm
Find in test that the consumption of glucose and the processing mode of dregs of beans have remarkably influenced to inhibiting product, the growth that yeast powder, corn steep liquor and sodium-chlor are mainly bacillus cereus provides indispensable amino acid and VITAMIN, nucleic acid, trace element etc., and provide certain osmotic pressure, to the generation of antibacterial substance without significance.
Prove that glucose is a kind of inducibility nutritive substance that inhibiting product is expressed, when not adding glucose during the fermentation, when fermented liquid inhibition zone is significantly less than interpolation 0.1% glucose by test; In addition added glucosan (0.3%-1%), the growth cycle prolongation of bacterium, whole bacterium amount improve, but inhibition zone reduces, and too much glucose carbon source makes bacterium be in vegetative state but not expression status, and product antibacterial substance reduces; When other nutrition sources are identical, the fermented liquid fungistatic effect that the pre-enzymolysis group of fermentation bean cake powder is produced obviously is better than not enzymolysis group, it can thus be appreciated that the content of monose and micromolecule polypeptide can affect generation (the dregs of beans employing neutral protease enzymolysis 2h of inhibiting product in fermention medium, before enzymolysis, content of peptides is 1.97g/L, is 10.4g/L after enzymolysis).From the viewpoint of cost and effect two, best culture medium prescription and glucose 0.1%, bean cake powder 2%, yeast powder 0.5%, sodium-chlor 0.2%, corn steep liquor 1.0%, surplus is water.
embodiment 3, utilize described substratum to ferment after the bacteriostatic activity of tunning
The present embodiment is from infection EMS(prawn Deaths syndromes, Early Mortality Syndrome) Penaeus vannamei be separated and obtain pathogenic bacterium Vibrio parahaemolyticus 3#, be inoculated in 2216E liquid nutrient medium after activation, 30 DEG C, 12-24h cultivated by 180 turns/min shaking table, and being cultured to cell concentration is 10 8cFU/ml, gets 0.1ml and coats on 2216E flat board, on flat board, then place the Oxford cup of sterilizing, draw 0.2ml testing sample in the cup of Oxford, be placed in 30 DEG C of incubators and cultivate 12-24h, observe inhibition zone, 2216E flat board is greater than 15mm to Vibrio parahaemolyticus inhibition zone.
Test-results is as shown in Figure 1, Figure 2 with shown in table 2, and as can be seen from different fermentation time sample fungistatic effect test, fermented liquid occurs fungistatic effect in this formula from 12h, tends towards stability after 16h, and maximum antibacterial circle diameter is 19mm.Fermented liquid still has stable fungistatic effect after adopting the ultrafiltration of 3kDa Millipore super filter tube, and illustrate that the fermented liquid adopting this formula, fermenting to produce has the activity significantly suppressing Vibrio parahaemolyticus, Substance molecular weight is less than 3kDa.
Table 2: the fungistatic effect of different piece
Sample Fungistatic effect
J19 fermented liquid +
J19 12000rpm centrifuged supernatant +
J19 3kDa super filter tube ultrafiltrated +
embodiment 4, the present invention are on the impact of Vibrio parahaemolyticus in culture of Penaeus vannamei
Adopt the inducing culture of above-mentioned optimum, in 50L fermentor tank, carry out small-scale fermentation, fermentation volume is 30L, and initial pH 6.89, ventilation 1:0.5, tank pressure 0.05MPa, inoculum size is a Kolle flask, and fermentation period is 22h.The fermented liquid obtained carries out pond application test.The happy bright selection 16m in Zhanjiang 3the leg Shrimp Litopenaeus vannamei Culture Raceway-type Ponds pool, shrimp seedling specification is 0.7-1.0cm, water body pH 8.5, salinity 8 ‰-10 ‰, temperature 27 DEG C-29 DEG C, and splash 24ml/ pond J19 fermented liquid after 12 hours, the Vibrio parahaemolyticus quantity in water body can reduce 30%-40%; Splash after twice continuously, in water body, Vibrio parahaemolyticus quantity is reduced to about 30% of vibrios sum in original water body.Fermented liquid consumption is 1.5ml/m 3vibrio parahaemolyticus content in water body significantly can be reduced during water body.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.

Claims (8)

1. a strain bacillus cereus J19, is characterized in that: its Classification And Nomenclature is bacillus cereus bacillus cereus, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.10052.
2. induction bacillus cereus J19 according to claim 1 produces the inducing culture to the Substance of Vibrio parahaemolyticus, it is characterized in that described inducing culture comprises following component, by quality ratio:
Glucose 0.1% ~ 1%;
Bean cake powder 2.0% ~ 4.0%, described bean cake powder needs to use neutral protease enzymolysis in a water bath before using;
Yeast powder 0.5% ~ 1%;
Sodium-chlor 0.1% ~ 1%;
Corn steep liquor 0.1% ~ 1.0%.
3. inducing culture according to claim 2, is characterized in that: described bean cake powder needs with the pre-enzymolysis 2-3h of neutral protease before using, and the consumption of neutral protease is 1500U/g bean cake powder, and hydrolysis temperature is 40 DEG C.
4. inducing culture according to claim 3, is characterized in that: described inducing culture comprises following component: glucose 0.1%, bean cake powder 2.0%, yeast powder 0.5%, sodium-chlor 0.2%, corn steep liquor 1.0%, and surplus is water.
5. inducing culture according to claim 2 produces the application in the Substance of Vibrio parahaemolyticus at induction bacillus cereus J19.
6. inducing culture according to claim 5 produces the application in the Substance of Vibrio parahaemolyticus at induction bacillus cereus J19, it is characterized in that, described bacillus cereus J19 on inducing culture at 30 DEG C shaking table cultivate 12-16h and obtain bacillus cereus J19 fermented liquid.
7. inducing culture according to claim 6 produces the application of material in the bacteriostatic activity of Vibrio parahaemolyticus at induction bacillus cereus J19, and it is characterized in that, be spread in water body by described J19 fermented liquid, fermented liquid consumption is 1.2 ~ 1.8ml/m 3water body, the concentration of the Substance in described fermented liquid is 10 ± 0.2mg/L.
8. the application of inducing culture according to claim 6 material in induction bacillus cereus J19 generation is to the bacteriostatic activity of Vibrio parahaemolyticus, is characterized in that, the pH 8.5 of described water body, salinity 8 ‰-10 ‰, temperature 27 DEG C-29 DEG C.
CN201510070176.9A 2015-02-11 2015-02-11 Bacillus cereus J19 as well as induction medium and application thereof CN104630114A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510070176.9A CN104630114A (en) 2015-02-11 2015-02-11 Bacillus cereus J19 as well as induction medium and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510070176.9A CN104630114A (en) 2015-02-11 2015-02-11 Bacillus cereus J19 as well as induction medium and application thereof

Publications (1)

Publication Number Publication Date
CN104630114A true CN104630114A (en) 2015-05-20

Family

ID=53209366

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510070176.9A CN104630114A (en) 2015-02-11 2015-02-11 Bacillus cereus J19 as well as induction medium and application thereof

Country Status (1)

Country Link
CN (1) CN104630114A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106591206A (en) * 2017-03-08 2017-04-26 中国海洋大学 Bacillus cereus, compound bacteria preparation thereof and preparation method
CN106635933A (en) * 2017-02-28 2017-05-10 湖南省中科农业有限公司 Mixed bacterial agent capable of degrading antibiotics in soil and preparation method thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293707A (en) * 2014-09-26 2015-01-21 国家海洋局第三海洋研究所 Bacillus cereus strain and application thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104293707A (en) * 2014-09-26 2015-01-21 国家海洋局第三海洋研究所 Bacillus cereus strain and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
动物肠道益生菌腊样芽孢杆菌的筛选及培养基的优化;江敏等;《食品安全监管与法制建设国际研讨会暨第二届中国食品研究生论坛论文集》;20090921;第1033页 *
益生菌腊样芽孢杆菌的筛选及培养基的优化;江敏等;《中国奶牛》;20061231(第02期);第12页 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106635933A (en) * 2017-02-28 2017-05-10 湖南省中科农业有限公司 Mixed bacterial agent capable of degrading antibiotics in soil and preparation method thereof
CN106591206A (en) * 2017-03-08 2017-04-26 中国海洋大学 Bacillus cereus, compound bacteria preparation thereof and preparation method
CN106591206B (en) * 2017-03-08 2019-06-28 中国海洋大学 One plant of bacillus cereus and its composite bacteria preparation and preparation method

Similar Documents

Publication Publication Date Title
Kongnum et al. Effect of Lactobacillus plantarum isolated from digestive tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi
CN103497906B (en) Microecological preparation, preparation method, and applications thereof
CN103395890B (en) Microbial preparation for improving freshwater aquaculture water body and preparation method thereof
CN101580799B (en) Micro-ecological preparation and application thereof
CN103667133B (en) A kind of mixing microorganisms preparation used for aquiculture and preparation method thereof
CN102583776B (en) Compound bacteria enzyme preparation for improving cultivation watery environment and preparation method
CN101275119B (en) Application of bdellovibrio to eliminating pathogen in freshwater product and culture water thereof
CN102178057B (en) Bacillus subtilis and feed additive and fermenting agent thereof
CN102805209B (en) Synbiotics feed additive for beasts and birds and application thereof
CN100588624C (en) Microorganism renovation agent of water environment and preparation method thereof
CN101278702B (en) Novel microbial feed additive and method of producing the same
CN104396779A (en) Pigpen fermentation bed packing
CN104045164B (en) Microorganism formulation for freshwater aquiculture water improvement
CN102286376B (en) Microbial inoculum for high-efficiency fermenting bed and preparation method thereof
CN103160455B (en) Preparation method of spore preparation of bacillus coagulans
CN101220342B (en) FQ15 enterococcus faecalis and method for producing somatotrophic feed additive with the bacteria
CN100394926C (en) Production process of bdellophage preparation
CN102864111B (en) Schizochytrium limacinum strain for producing docosahexaenoic acid
CN103614307B (en) A kind of solid ocean rhodotorula preparation and its preparation method and application
CN102067941B (en) Method for producing compound microbial preparation serving as fodder
CN102293340B (en) Compound premix able to improve immune function for pigs
CN102660461B (en) Microbial preparation for shortening tobacco fermentation period and application of microbial preparation
CN104195074B (en) A kind of deep brown Bacillus strain of Alpine Grasslands herbage endogenetic bacteria, microbial bacterial agent and its preparation method and application
CN104894098B (en) A kind of immobilization probiotics leaven and preparation method thereof
CN104388339B (en) Microecological preparation suitable for marine culture and application thereof

Legal Events

Date Code Title Description
PB01 Publication
C06 Publication
SE01 Entry into force of request for substantive examination
C10 Entry into substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20150520

RJ01 Rejection of invention patent application after publication