CN1699585A - Process for preparing biological antibacterial agent - Google Patents
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- CN1699585A CN1699585A CN 200410022562 CN200410022562A CN1699585A CN 1699585 A CN1699585 A CN 1699585A CN 200410022562 CN200410022562 CN 200410022562 CN 200410022562 A CN200410022562 A CN 200410022562A CN 1699585 A CN1699585 A CN 1699585A
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Abstract
The invention belongs to the field of biological engineering and is aimed for solving the problem of mass scale plant diseases caused by fungus. The preparing process comprises subjecting a strong of Bacillus firmus (CGMCC No.0924) to second level fermentation culture under suitable condition, then carrying out filtering, salting out and column chromatography treatment.
Description
One, affiliated field
The invention belongs to bioengineering field, be specifically related to a kind of method for preparing active substance with microbial fermentation.
Two, background technology
Plant diseases is usually brought heavy losses to agriculture production, is the one of the main reasons of the world food underproduction.And in the Plant diseases 80% by plant pathogenic fungi cause how to prevent and treat fungal diseases of plants effectively, be one of problem demanding prompt solution in the agriculture production always.Normally prevent and treat both at home and abroad by the agricultural chemicals goods of using chemosynthesis, although certain effect is arranged, but long-term a large amount of the use, the ecotope system has been caused very big destruction, and because diversity, variability and the resistance of pathogenic bacteria have brought difficulty also for the chemical prevention of fungal disease.
People begin to pay close attention to other environment amenable fungal diseases and prevent and treat method, as the biological pesticide of exploitation control fungal diseases of plants, microbial pesticide etc.The domestic and foreign literature report, bacterium, fungi and actinomycetes all can produce the environment amenable antimicrobial substance that can be developed to biological pesticide.
The inventor finds the research process that a strain is separated the bacterium " bacillus firmus " that obtains from the Xinjiang cotton plant, a kind of antimicrobial substance one prompt peace peptide element that this bacterial strain produces, to multiple pathogenic fungi, as phytophthora (Phytophthora sp.), cotton seedling blight bacterium (Rhizoctonia solani KChn), Rhizoctonia solani Kuhn (Thanatephorus cucumeris Donk), pepper anthracnose bacterium (Colletotrichumgossypii Southw), Fusarium graminearum (Fusarium graminearum schw.), botrytis cinerea (Botrytis cinerea), cotton-wilt fusarium (Fusarium oxysporum f.sp.vasinfectum) etc. has very strong restraining effect, is used for the generation that field crop can prevent fungal diseases of plants effectively.The contriver is at the fermentation preparation that continues victory peace peptide element, and antimicrobial spectrum etc. have been finished the present invention after having carried out studying in great detail." batch liquid nutrient medium flow feeding technology " fermentation method is adopted in invention, preparation antibacterial agent one prompt peace peptide element, and prompt peace peptide element can effectively be prevented and treated fungal diseases of plants.Used bacterium is bacillus firmus (Bacillus firmus handed over common micro-organisms center preservation, culture presevation CGMCCNo.0924 on April 21st, 2003).
Three, summary of the invention
Purpose of the present invention:
One of purpose of the present invention provides a kind of " batch liquid nutrient medium flow feeding technology ", and the peptide element is pacified in one victory of fermentative production antiseptic-germicide;
Two of purpose of the present invention provides a kind of new bacterial strain " bacillus firmus " (Bacillus firmus) that is used to produce prompt peace peptide element;
Three of purpose of the present invention provides a kind of new substratum of the present invention that is applicable to;
Four of purpose of the present invention provides the method for extracting prompt peace peptide element from fermented liquid;
Five of purpose of the present invention provides the chemical constitution of prompt peace peptide element.
More particularly, the invention provides a kind of method for preparing the prompt peace of antiseptic-germicide peptide element, comprise following steps:
The bacillus firmus that can produce prompt peace peptide element at the level liquid substratum (for example, culture medium A hereinafter described) cultivates in, as primary seed solution, primary seed solution (for example is inoculated in second stage liquid nutrient medium, substratum B hereinafter described) in, after cultivating one suitable period, begin to carry out second stage liquid nutrient medium fed batch fermentation and cultivate.After the fermentation ends, from above-mentioned fermentation culture, collect prompt peace peptide element.
Bacillus firmus of the present invention (Bacillus firmus, handed over common micro-organisms center preservation on April 21st, 2003, culture presevation CGMCC No.0924), it is a kind of gram positive bacterium of seeking saprogenesis, in soil, water body, plant surface and other physical environment, extensively exist, be easy to obtain, endogenous spore, high-output stress-resistance, reproduction speed is fast, nutritional requirement is simple, to farm crop and environmental safety, can produce antibacterial peptide, many fungies are all had stronger restraining effect.
Specific embodiments of the present invention is that the bacillus firmus that employing produces prompt peace peptide element carries out second order fermentation and cultivates in the liquid medium within, select different substratum for use at every grade of fermentation stage.In the fermentation of the second stage, to be suitable for the liquid nutrient medium of second stage fermentation, for example substratum B hereinafter described, as fermention medium, cultivate for some time under the suitable temperature, for example, at 26 ℃-32 ℃ after fermentation culture 10-30 hour, beginning liquid nutrient medium flow feeding.
Second stage liquid nutrient medium (for example substratum B) flow feeding mode can adopt continuously (at the uniform velocity or at the uniform velocity non-) stream to add and/or the intermittent type fed-batch mode, and the Continuous Flow add mode is preferred.
Described continuously (at the uniform velocity or at the uniform velocity non-) fed-batch mode, be to add in the fermentor tank of the second stage, until stopping preceding about 12 hours of fermentation (following jar) with liquid nutrient medium (for example substratum B) Continuous Flow that certain stream rate of acceleration (at the uniform velocity or at the uniform velocity non-) will be suitable for second stage fermentation.
Described intermittent type fed-batch mode is the mode that adds a defective material with certain interval of time, and liquid nutrient medium (for example substratum B) stream that intermittent type will be suitable for second stage fermentation adds in the fermentor tank of the second stage.Intermittent time is preferably every feed supplement in 3-24 hour 1-5 time, more preferably about feed supplement in 6 hours at interval 1 time.Those skilled in the art also can adopt other suitable timed interval feed supplement as required.
Fermentation condition: 26 ℃-32 ℃ of temperature, PH:6-8
Fermentation time: 3-5 days
Adopt technology of the present invention, when fermenting in the second stage, a liquid nutrient medium flow feeding and without discharging.By regulating (raising or reduction) fermented liquid carbon-nitrogen ratio and dissolved oxygen concentration, controlled temperature, foam etc. reduce the metabolism of bacterium, improve prompt peace peptide element productive rate.
After the fermentation ends, remove thalline and other solid substance, the pH value of transferring fermented liquid with sulfuric acid or hydrochloric acid is perhaps saltoutd at the ammonium sulfate of 40%-70% with saturation ratio to 2-5.Collecting precipitation, with the precipitation that the lower alcohol dissolving obtains, precipitation is 1: 20 to 1: 40 with the ratio of lower alcohol solvent; This lysate carries out silica gel column chromatography after concentrating, with ethanol, methyl alcohol and trichloromethane as eluting solvent, collection contains the elutriant of prompt peace peptide element, through concentrate the back leaving standstill with stirring state under, cooling gradually, the prompt peptide element of pacifying is separated out with solid form, through collecting, washing, drying, the i.e. plain product of De Jiean peptide.
The culture medium A that second order fermentation of the present invention adopted, B, it is specifically composed as follows:
Culture medium A: glucose 0.5%-5.0% peptone 0.3%-5.0%
Extractum carnis 0.1%-2.0% sal epsom 0.05%-1.0%
Potassium primary phosphate 0.1%-1.0%
Substratum B:
| Composition | General range | Preferable range | Most preferred range |
| Starch | ??0.1％-5.0％ | ??0.3％-3.0％ | ??0.5％-2.0％ |
| Dextrin | ??0.1％-3.0％ | ??0.5％-3.0％ | ??0.5％-2.0％ |
| Lactose | ??0.1％-3.0％ | ??0.3％-3.0％ | ??0.5％-2.0％ |
| Sucrose | ??0.1％-4.0％ | ??0.3％-3.0％ | ??0.5％-2.0％ |
| Glycerine | ??0.1％-2.0％ | ??0.5％-2.0％ | ??0.5％-1.0％ |
| Semen Maydis powder | ??0.1％-5.0％ | ??0.5％-3.0％ | ??0.5％-2.0％ |
| Waste molasses | ??0.1％-8.0％ | ??0.5％-5.0％ | ??0.5％-3.0％ |
| Glucose | ??0.1％-10.0％ | ??0.5％-6.0％ | ??1.0％-3.0％ |
| Peptone | ??0.3％-10.0％ | ??0.5％-8.0％ | ??1.0％-5.0％ |
| Ammonium nitrate | ??0.1％-8.0％ | ??0.5％-5.0％ | ??0.5％-3.0％ |
| Ammonium sulfate | ??0.1％-7.0％ | ??0.5％-5.0％ | ??0.5％-3.0％ |
| Groundnut meal | ??0.1％-15.0％ | ??0.5％-10.0％ | ??0.5％-5.0％ |
| Soybean cake powder | ??0.1％-15.0％ | ??0.5％-10.0％ | ??0.5％-5.0％ |
| Biological nitrogen | ??0.5％-15.0％ | ??0.5％-12.0％ | ??1.0％-8.0％ |
| Potassium primary phosphate | ??0.01％-5.0％ | ??0.05％-3.0％ | ??0.05％-1.0％ |
| Zinc sulfate | ??0.01％-5.0％ | ??0.05％-3.0％ | ??0.05％-1.0％ |
| Sodium-chlor | ??0.01％-5.0％ | ??0.05％-3.0％ | ??0.05％-1.0％ |
| Ferric sulfate | ??0.01％-5.0％ | ??0.05％-3.0％ | ??0.05％-1.0％ |
The victory peace peptide element that the present invention produced is a kind of antibacterial peptide, and its amino acid is formed peculiar, does not newly see the antibacterial peptide of being made up of the similar amino acid of report through looking into.
The victory peace peptide element that the present invention produced, its chemical constitution, promptly amino acid consists of (content meter by weight percentage):
Aspartic acid 40.1% Serine 8.1% L-glutamic acid 14.6% glycine 0.1%
Isoleucine 0.7% Gelucystine 0.8% methionine(Met) 0.9% leucine 0.2%
Phenylalanine 0.3% tyrosine 18.0% Methionin 0.2% proline(Pro) 9.6%
Xie Ansuan 0.9% ammonia 5.5%
The zymotechnique whole process of the preferred embodiment of the invention is:
In culture medium A, solid culture or be loaded in the triangular flask 26 ℃-32 ℃ after shake-flask culture 10-24 hour is inoculated in the inoculum size of 5%-30% and carries out fermentative production in the fermentor tank that is added with sterilising medium B with the bacterial classification inoculation after the activation.Bacillus firmus is inoculated in behind the fermentor tank in 26 ℃-32 ℃, and preferred 28 ℃-30 ℃ after fermentation culture 8-15 hour, beginning substratum B flow feeding.Substratum B flow feeding mode has two kinds, a kind ofly is continuously that (at the uniform velocity or at the uniform velocity non-) stream adds, and a kind of is that intermittent type stream adds, and the Continuous Flow add mode is preferred.
Before adopting during a kind of mode, with the speed of 0.01-5.0L/h (at the uniform velocity or at the uniform velocity non-) fed-batch medium B continuously, until stopping fermentation (following jar) precontract 12 hours.
When adopting a kind of mode in back, i.e. intermittent type fed-batch medium B, the intermittent time can be every feed supplement in 3-24 hour 1-5 time, is preferably about interval feed supplement in 6 hours 1 time, and each feed supplement amount is the 0.01%-1.0% of fermented liquid cumulative volume, preferably 0.1%-0.5%.
Fermentation condition: 26 ℃-32 ℃ of temperature, PH:6-8
Fermentation time: 3-5 days
Accompanying drawing 1 is seen in full technical process of the present invention.
Adopt technology of the present invention, when fermenting in the second stage, a liquid nutrient medium flow feeding and without discharging.By regulating (raising or reduction) fermented liquid carbon-nitrogen ratio and dissolved oxygen concentration, controlled temperature, foam etc. make bacterial strain produce prompt peace peptide element with higher substrate conversion efficiency and product synthesis rate, and prompt output of pacifying the peptide element can reach 5000 units.
After the fermentation ends, remove thalline and other solid substance, the pH value of transferring fermented liquid with sulfuric acid or hydrochloric acid is perhaps saltoutd at the ammonium sulfate of 40%-70% with saturation ratio to 2-5.Collecting precipitation, with the precipitation that the lower alcohol dissolving obtains, precipitation is 1: 20 to 1: 40 with the ratio of lower alcohol solvent; This lysate carries out silica gel column chromatography after concentrating, with ethanol, methyl alcohol and trichloromethane as eluting solvent, collection contains the elutriant of prompt peace peptide element, through concentrate the back leaving standstill with stirring state under, cooling gradually, the prompt peptide element of pacifying is separated out with solid form, through collecting, washing, drying, the i.e. plain pure product of De Jiean peptide.
The present invention has the following advantages:
One, bacterial classification of the present invention produces the prompt peace of unique antibacterial substance peptide element, and the fermentation production rate height is effective.
Two, the plain output height of the prompt peace of secondary fed batch fermentation explained hereafter peptide, substratum utilization ratio height, the living contaminants rate is low;
Three, fermentation, extraction process flow process are simple, and rate of recovery height is easy to operate.
Four, description of drawings
Accompanying drawing is the schema of the plain fermentative production of prompt peace peptide.
By finding out clearly among the figure that carrying out second order fermentation with the plain production of prompt peace peptide bacterial classification according to required condition earlier cultivates, in the fermentation culture process of the second stage, carry out flow feeding again, then tunning is carried out the process that aftertreatment obtains prompt peace peptide element.
Five, embodiment
Embodiment 1
With 10 of 1000mL triangular flasks, every bottled 300mL culture medium A (glucose 2.0% peptone 3.0% extractum carnis 0.5% sal epsom 0.1% potassium primary phosphate 0.2%.) in 120 ℃ of sterilizations, the plain bacterium bacillus firmus bacterium liquid that produces of the victory peace peptide after the inoculation activation of cooling back places under 30 ℃ of temperature shake-flask culture 18 hours.Dress 50L sterilising medium B (starch 1.0% dextrin 2.0% lactose 1.0% sucrose 2.0% glycerine 0.5% Semen Maydis powder 1.0% waste molasses 3.0% glucose 2.0% peptone 5.0% ammonium nitrate 1.0% ammonium sulfate 0.5% groundnut meal 2.0% soybean cake powder 0.5% biological nitrogen 1.0% potassium primary phosphate 0.5% zinc sulfate 0.5% sodium-chlor 0.5% ferric sulfate 0.1% in cultured seed liquid is inoculated in by the inoculum size of 5%-10%.) the 100L fermentor tank in, under 28 ℃ of-30 ℃ of temperature, aeration-agitation fermentation is after 8-15 hour, with the continuous fed-batch medium B of the speed of 0.1-1.0L/h, until stopping fermentation (following jar) precontract 12 hours.Fermentation PH 6-8, fermentation period 5 days.
After the fermentation ends, remove thalline and other solid substance, the pH value of transferring fermented liquid with sulfuric acid or hydrochloric acid is perhaps saltoutd at the ammonium sulfate of 40%-70% with saturation ratio to 2-5.Collecting precipitation, with the precipitation that the lower alcohol dissolving obtains, precipitation is 1: 20 to 1: 40 with the ratio of lower alcohol solvent; This lysate carries out silica gel column chromatography after concentrating, with ethanol, methyl alcohol and trichloromethane as eluting solvent, collection contains the elutriant of prompt peace peptide element, through concentrate the back leaving standstill with stirring state under, cooling gradually, the prompt peptide element of pacifying is separated out with solid form, through collecting, washing, drying, the i.e. plain product of De Jiean peptide.
After testing, through fermentation in 5 days, the plain output of prompt peace peptide can reach more than 5000 units, and product recovery rate reaches more than 80%.
Embodiment 2
With 100 of 1000mL triangular flasks, every bottled 300mL culture medium A (glucose 3.0% peptone 2.0% extractum carnis 0.8% sal epsom 0.1% potassium primary phosphate 0.5%.) in 120 ℃ of sterilizations, the plain bacterium bacillus firmus bacterium liquid that produces of the victory peace peptide after the inoculation activation of cooling back places under 30 ℃ of temperature shake-flask culture 18 hours.Dress 250L sterilising medium B (starch 2.0% dextrin 2.0% lactose 1.0% sucrose 1.0% glycerine 0.5% Semen Maydis powder 0.5% waste molasses 3.0% glucose 1.0% peptone 1.0% ammonium nitrate 3.0% ammonium sulfate 2.0% groundnut meal 5.0% soybean cake powder 2.0% biological nitrogen 3.0% potassium primary phosphate 0.1% zinc sulfate 0.1% sodium-chlor 0.5% ferric sulfate 0.5% in cultured seed liquid is inoculated in by the inoculum size of 10%-15%.) the 500L fermentor tank in, under 28 ℃ of-30 ℃ of temperature, aeration-agitation fermentation is after 10-15 hour, with the continuous fed-batch medium B of the speed of 0.5-2.0L/h, until stopping fermentation (following jar) precontract 12 hours.Fermentation PH6-8, fermentation period 5 days.
After the fermentation ends, remove thalline and other solid substance, the pH value of transferring fermented liquid with sulfuric acid or hydrochloric acid is perhaps saltoutd at the ammonium sulfate of 40%-70% with saturation ratio to 2-5.Collecting precipitation, with the precipitation that the lower alcohol dissolving obtains, precipitation is 1: 20 to 1: 40 with the ratio of lower alcohol solvent; This lysate carries out silica gel column chromatography after concentrating, with ethanol, methyl alcohol and trichloromethane as eluting solvent, collection contains the elutriant of prompt peace peptide element, through concentrate the back leaving standstill with stirring state under, cooling gradually, the prompt peptide element of pacifying is separated out with solid form, through collecting, washing, drying, the i.e. plain product of De Jiean peptide.
After testing, through fermentation in 5 days, the plain output of prompt peace peptide can reach more than 5000 units, and product recovery rate reaches more than 80%.
Embodiment 3: with 100 of 1000mL triangular flasks, every bottled 300mL culture medium A (glucose 4.0% peptone 3.0% extractum carnis 0.8%8 sal epsom 0.3% potassium primary phosphate 0.4%.) in 120 ℃ of sterilizations, the plain bacterium bacillus firmus bacterium liquid that produces of the victory peace peptide after the inoculation activation of cooling back places under 30 ℃ of temperature shake-flask culture 18 hours.Dress 250L sterilising medium B (starch 2.0% dextrin 1.0% lactose 1.0% sucrose 2.0% glycerine 0.5% Semen Maydis powder 0.5 waste molasses 0.5% glucose 3.0% peptone 5.0% ammonium nitrate 0.5% ammonium sulfate 0.5% groundnut meal 1.0% soybean cake powder 2.0% biological nitrogen 5.0% potassium primary phosphate 1.0% zinc sulfate 0.1% sodium-chlor 0.1% ferric sulfate 0.5% in cultured seed liquid is inoculated in by the inoculum size of 10%-15%.) the 500L fermentor tank in, under 28 ℃ of-30 ℃ of temperature, aeration-agitation fermentation is after 10-15 hour, with intermittent type fed-batch medium B, the intermittent time be feed supplement in 6 hours approximately at interval 1 time, the feed supplement amount is the 0.1%-0.5% of fermented liquid cumulative volume at every turn.Fermentation PH6-8, fermentation period 5 days.
After the fermentation ends, remove thalline and other solid substance, the pH value of transferring fermented liquid with sulfuric acid or hydrochloric acid is perhaps saltoutd at the ammonium sulfate of 40%-70% with saturation ratio to 2-5.Collecting precipitation, with the precipitation that the lower alcohol dissolving obtains, precipitation is 1: 20 to 1: 40 with the ratio of lower alcohol solvent; This lysate carries out silica gel column chromatography after concentrating, with ethanol, methyl alcohol and trichloromethane as eluting solvent, collection contains the elutriant of prompt peace peptide element, through concentrate the back leaving standstill with stirring state under, cooling gradually, the prompt peptide element of pacifying is separated out with solid form, through collecting, washing, drying, the i.e. plain product of De Jiean peptide.
After testing, through fermentation in 5 days, the plain output of prompt peace peptide can reach more than 5000 units, and product recovery rate reaches more than 80%.
The plain control of application example 1 prompt peace peptide vegetable fungi disease: 100 times-300 times of the plain aqua dilutions of victory peace peptide with 1% spray the control fungal diseases of plants as the field
Application example 2: the plain control of prompt peace peptide cereal fungal disease
100 times-300 times of the plain aqua dilutions of victory peace peptide with 1% spray the control fungal diseases of plants as the field.Spray with certain density ABA (dormin) if the peptide element is pacified in victory, can improve the long-lasting of prevention effect and control.
Claims (13)
1, a kind of preparation method of biological antibiotic agent, it is characterized in that: the bacillus firmus that can produce prompt peace peptide element is cultivated in the level liquid substratum, as primary seed solution, primary seed solution is inoculated in the liquid nutrient medium of the second stage, after cultivating one suitable period, begin to carry out second stage liquid nutrient medium fed batch fermentation and cultivate, after the fermentation ends, from above-mentioned fermentation culture, collect prompt peace peptide element.
2, the described bacillus firmus Bacillus firmus that can produce prompt peace peptide element of claim 1 handed over common micro-organisms center preservation, culture presevation CGMCC No.0924 on April 21st, 2003.
3, according to the preparation method of the described biological antibiotic agent of claim 1, it is characterized in that: first step liquid nutrient medium is specifically composed as follows:
Glucose 0.5%-5.0% peptone 0.3%-5.0% extractum carnis 0.1%-2.0%
Sal epsom 0.05%1.0% potassium primary phosphate 0.1%-1.0%.
4, the preparation method of biological antibiotic agent according to claim 3 is characterized in that: the-level liquid nutrient medium is preferably composed as follows:
Glucose 2.0%-4.0% peptone 1.0%-3.0% extractum carnis 0.5%-1.0%
Sal epsom 0.1%-0.5% potassium primary phosphate 0.1%-0.5%.
5, according to the preparation method of the described biological antibiotic agent of claim 1, it is characterized in that: second stage liquid nutrient medium is specifically composed as follows:
Starch 0.1%-5.0% dextrin 0.1%-3.0% lactose 0.1%-3.0% sucrose 0.1%4.0%
Glycerine 0.1%-2.0% Semen Maydis powder 0.1%-5.0% waste molasses 0.1%-8.0% glucose 0.1%-10.0%
Peptone 0.3%-10.0% ammonium nitrate 0.1%-8.0% ammonium sulfate 0.1%-7.0% groundnut meal
0.1%-15.0% soybean cake powder 0.1%-15.0% biological nitrogen 0.5%-15.0% potassium primary phosphate
0.01%-5.0% zinc sulfate 0.01%-5.0% sodium-chlor 0.01%-5.0% ferric sulfate 0.01%-5.0%.
6, the preparation method of biological antibiotic agent according to claim 5 is characterized in that: second stage liquid nutrient medium is preferably composed as follows:
Starch 0.3%-3.0% dextrin 0.5%-3.0% lactose 0.3%-3.0% sucrose 0.3%-3.0%
Glycerine 0.5%-2.0% Semen Maydis powder 0.5%-3.0% waste molasses 0.5%-5.0% glucose 1.0%-6.0%
Peptone 1.0%-8.0% ammonium nitrate 0.5%-5.0% ammonium sulfate 0.5%-5.0% groundnut meal
0.5%-10.0% soybean cake powder 0.5%-10.0% biological nitrogen 0.5%-12.0% potassium primary phosphate
0.05%-3.0% zinc sulfate 0.05%-3.0% sodium-chlor 0.05%-3.0% ferric sulfate 0.05%-3.0%.
7, the preparation method of biological antibiotic agent according to claim 6 is characterized in that: second stage liquid nutrient medium is most preferably formed as follows:
Starch 0.5%-2.0% dextrin 0.5%-2.0% lactose 0.5%-2.0% sucrose 0.5%-2.0%
Glycerine 0.5%-1.0% Semen Maydis powder 0.5%-2.0% waste molasses 0.5%-3.0% glucose 1.0%-3.0%
Peptone 1.0%-5.0% ammonium nitrate 0.5%-3.0% ammonium sulfate 0.5%-3.0% groundnut meal
0.5%-5.0% soybean cake powder 0.5%-5.0% biological nitrogen 1.0%-8.0% potassium primary phosphate
0.05%-1.0% zinc sulfate 0.05%-1.0% sodium-chlor 0.05%-1.0% ferric sulfate 0.05%-1.0%.
8, according to the preparation method of the described biological antibiotic agent of claim 1, it is characterized in that: flow feeding can be taked the Continuous Flow add mode, the speed that Continuous Flow adds can be at the uniform velocity also can right and wrong at the uniform velocity.
9, according to the preparation method of the described biological antibiotic agent of claim 1, it is characterized in that: flow feeding also can be taked the intermittent type fed-batch mode.
10, according to the preparation method of the described biological antibiotic agent of claim 1, it is characterized in that: first step fermentation inoculum size 5%-30%, 26 ℃ of-32 ℃ of shake-flask culture 10-24h.
11, according to the preparation method of the described biological antibiotic agent of claim 1, it is characterized in that: second stage fermentation condition: 26 ℃-32 ℃ of temperature, PH:6-8, fermentation time: 3-5 days.
12, according to the preparation method of the described biological antibiotic agent of claim 1, the extracting method of wherein prompt peace peptide element is: after the fermentation ends, remove thalline and other solid substance, the pH value of transferring fermented liquid with sulfuric acid or hydrochloric acid is perhaps saltoutd at the ammonium sulfate of 40%-70% with saturation ratio to 2-5.Collecting precipitation, with the precipitation that the lower alcohol dissolving obtains, precipitation is 1: 20 to 1: 40 with the ratio of lower alcohol solvent; This lysate carries out silica gel column chromatography after concentrating, with ethanol, methyl alcohol and trichloromethane as eluting solvent, collection contains the elutriant of prompt peace peptide element, through concentrate the back leaving standstill with stirring state under, cooling gradually, the prompt peptide element of pacifying is separated out with solid form, through collecting, washing, drying, the i.e. plain product of De Jiean peptide.
13, victory that the present invention produced peace peptide element, its chemical constitution, promptly amino acid is formed by weight percentage content and is counted:
Aspartic acid 40.1% Serine 8.1% L-glutamic acid 14.6% glycine 0.1%
Isoleucine 0.7% Gelucystine 0.8% methionine(Met) 0.9% leucine 0.2%
Phenylalanine 0.3% tyrosine 18.0% Methionin 0.2% proline(Pro) 9.6%
Xie Ansuan 0.9% ammonia 5.5%.
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| CN102876611A (en) * | 2012-10-12 | 2013-01-16 | 湖南师范大学 | Bacillus firmus for killing plant parasitic nematodes, and preparation method and application thereof |
| CN103651394A (en) * | 2013-12-17 | 2014-03-26 | 北京燕化永乐生物科技股份有限公司 | Sterilization composition containing Jiean-peptide |
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| CN103651395A (en) * | 2013-12-17 | 2014-03-26 | 北京燕化永乐生物科技股份有限公司 | Sterilization composition |
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| CN103651772A (en) * | 2013-12-18 | 2014-03-26 | 北京燕化永乐生物科技股份有限公司 | Sterilization composition |
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