CN106489996A - A kind of preparation method of microbial bactericide - Google Patents
A kind of preparation method of microbial bactericide Download PDFInfo
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- CN106489996A CN106489996A CN201610019899.0A CN201610019899A CN106489996A CN 106489996 A CN106489996 A CN 106489996A CN 201610019899 A CN201610019899 A CN 201610019899A CN 106489996 A CN106489996 A CN 106489996A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, 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
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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Abstract
The present invention relates to agricultural microbial agent technical field, more particularly to a kind of preparation method of microbial bactericide, concretely comprise the following steps, step 1：Bacillus subtilis inclined-plane culture, step 2：Bacillus subtilis Shaking culture, step 3：Bacillus subtilis seed culture, step 4：Fermentation of bacillus subtilis culture, step 5：Before fermentation of bacillus subtilis tank puts tank, 2h～6h adds the magnesium sulfate after sterilizing, addition is 0.001%～0.002%, and the microbial bactericide gemma high conversion rate of the present invention, total bacteria count are more, for plant disease prevention and control, pathogen implantation can be suppressed, Multiple Classes of Antibiotics, protide antibacterial material is produced, induction plant produces resistance, there is broad spectrum antibiotic activity, strengthen plant stress-resistance ability, effectively prevent and treat crop pest, improve crop disease-resistant, drought resisting, cold tolerance.
Description
Technical field
A kind of the present invention relates to agricultural microbial agent technical field, more particularly to preparation method of microbial bactericide.
Background technology
Plant pest is the formidable enemy of agricultural production, is the 15%～20% of total output because plant is subjected to the underproduction average loss that disease causes every year according to FAO (Food and Agriculture Organization of the United Nation) (FAO) statistics.For a long time, the preparation for control of crop disease is typically chemical pesticide.Long term frequent uses chemical pesticide, causes the pathogenic bacteria resistance to the action of a drug to strengthen, is gradually lowered the effect of chemical pesticide.At present, main settling mode is to increase dosage, directly results in agricultural cost raising；The pathogenic bacteria resistance to the action of a drug is strengthened, and difficulty of prevention and cure is increased；Pollution environment, the ecological balance are destroyed；Harmful toxic matter is remained in agricultural byproducts, is accumulated, and endangers the healthy of numerous people.Therefore, finding wide spectrum, the efficient, biological pesticide of low toxicity has become the common recognition of scientific research personnel and Pesticide use personnel.
The bioactivator with suppression plant disease that microbial bactericide refers to microorganism and its metabolite and processed by them.Microbial bactericide mainly suppresses pathogen to produce, disturb its biosynthesis and destroy its eucaryotic cell structure.Absorbability is strong, toxicity is low, and played the role of has stimulation plant growth concurrently.Current microbial bactericide mainly has three classes：1st, farm antibiotics.Farm antibiotics is pest and disease damage, crop smothering and the regulating crop growth development that can be suppressed or be killed crop by the secondary metabolite produced in fermentation process in low concentration.The kind that has developed and utilized has jinggangmeisu, qingfengmeisu, polyoxin etc..2nd, fungi microbe bactericide.Fungi microbe bactericide research and most widely used be Trichoderma, next to that being stained with the mould class of broom, Nematophagous fungi etc..Such preparation commercialization is little.3rd, bacterial micro-organism bactericide.Bacterial micro-organism bactericide has bacillus subtilis, bacillus licheniformis, Bacillus cercus, bacillus megaterium etc. based on bacillus, mainly.Which has suppression plant disease, and non-pathogenic bacteria, to person poultry harmless, the features such as free from environmental pollution.
Existing microbial bactericide in actual application range of application more single, its fungicidal spectrum is narrower.Additionally, many deficiencies are there is also in the aspect existing product such as industrial applications and environmental suitability, resistance, limit extensive application of the microorganism formulation in crops sterilization.
Content of the invention
The purpose of the present invention is by strain improvement and fermentation technology optimization, design a kind of industrialized preparing process of microbial bactericide, i.e. by the use of bacillus subtilis as production bacterial strain, process program and the culture medium prescription for optimizing designed by the application present invention, strengthen the bactericidal property of product, and extend its practical ranges.
For realizing above-mentioned goal of the invention, technical scheme is as follows：
A kind of preparation method of microbial bactericide, concretely comprises the following steps：
Step 1：Bacillus subtilis inclined-plane culture, the temperature of the bacillus subtilis inclined-plane culture are 35 DEG C～42 DEG C, and the time is 42h～52h, and the bacillus subtilis inclined-plane culture culture medium prescription is：Sodium chloride 0.2%～0.8%, beef extract 0.5%～2%, peptone 0.2%～0.8%, agar powder 1%～2%, remaining are water, pH value are adjusted to 7.0～8.5, contra bevel after sterilizing, inoculated and cultured；
Step 2：Bacillus subtilis Shaking culture, the temperature of the bacillus subtilis Shaking culture is 35 DEG C～42 DEG C, and the time is 30h～36h, and speed of agitator is 200rpm, and the bacillus subtilis Shaking culture culture medium prescription is：Corn protein powder 0.2%～0.8%, peptone 0.5%～2%, starch 1%～2%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.05%～0.15%, remaining are water, and pH value is adjusted to 7.0～8.5, inoculated and cultured after the cooling that sterilizes；
Step 3：Bacillus subtilis seed culture, the temperature of the bacillus subtilis seed culture is 35 DEG C～42 DEG C, time is 24h～32h, inoculum concentration is 2%～4%, and initial speed of agitator is 400rpm, and before 6h, ventilation ratio is 0.6,1.3 are adjusted to afterwards, culture transferring is carried out when the numerical value of tail carbon peaks to be begun to decline, pH adjustment is not carried out during the bacillus subtilis seed culture, and the bacillus subtilis seed culture culture medium prescription is：Corn protein powder 0.2%～1%, peptone 0.5%～2%, starch 1%～3%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.02%～0.35%；Polyether antifoam agent 0.01%, remaining are water, and pH value is adjusted to 7.0～8.5, and coefficient is 0.6, ventilate after sterilizing pressurize, when being cooled to 35 DEG C～42 DEG C, inoculated and cultured；
Step 4：Fermentation of bacillus subtilis culture, the temperature of the fermentation of bacillus subtilis culture is 35 DEG C～42 DEG C, time is 15h～26h, and initial speed of agitator is 400rpm, and inoculum concentration is 5%～10%, before 6h, ventilation ratio is 0.8, stepped up to 1.5, when pH is less than 6.0 in sweat according to zymotic fluid dissolved oxygen afterwards, in zymotic fluid, add ammoniacal liquor to carry out pH value adjustment, pH is more than 6.0 for control, and the culture medium prescription of the fermentation of bacillus subtilis culture is：Corn protein powder 0.5%～2%, cottonseed meal 0.2%～1%, starch 1%～2%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.05%, polyether antifoam agent 0.01%, remaining are water, and pH value is adjusted to 7.0～8.5 before sterilizing, coefficient is 0.6, ventilation pressurize after sterilizing, when being cooled to 35 DEG C～42 DEG C, inoculated and cultured；
Step 5：Before fermentation of bacillus subtilis tank puts tank, 2h～6h adds the magnesium sulfate after sterilizing, and addition is 0.001%～0.002%, promotes the formation of gemma, increases the active ingredient in zymotic fluid, treat bacterial content >=5*10 in zymotic fluid9Cfu/ml, and gemma ratio more than 90% when, fermentor liquid is discharged, finished product is carried out.
Beneficial effects of the present invention are：
The microbial bactericide gemma high conversion rate of the present invention, total bacteria count is more, for plant disease prevention and control, pathogen implantation can be suppressed, Multiple Classes of Antibiotics, protide antibacterial material is produced, induction plant produces resistance, there is broad spectrum antibiotic activity, strengthen plant stress-resistance ability, effectively prevent and treat crop pest, improve crop disease-resistant, drought resisting, cold tolerance.There is efficient capacity of decomposition simultaneously, it is residual to decompose root toxin, fertile residual, medicine, produces various plants desired nutritional composition and growth factor, so as to stimulate crop growth.
Specific embodiment
The present invention is further elaborated with reference to embodiments.
A kind of preparation method of microbial bactericide, concretely comprises the following steps：
Step 1：Bacillus subtilis inclined-plane culture, the temperature of the bacillus subtilis inclined-plane culture are 35 DEG C～42 DEG C, and the time is 42h～52h, and the bacillus subtilis inclined-plane culture culture medium prescription is：Sodium chloride 0.2%～0.8%, beef extract 0.5%～2%, peptone 0.2%～0.8%, agar powder 1%～2%, remaining are water, pH value are adjusted to 7.0～8.5, contra bevel after sterilizing, inoculated and cultured；
Step 2：Bacillus subtilis Shaking culture, the temperature of the bacillus subtilis Shaking culture is 35 DEG C～42 DEG C, and the time is 30h～36h, and speed of agitator is 200rpm, and the bacillus subtilis Shaking culture culture medium prescription is：Corn protein powder 0.2%～0.8%, peptone 0.5%～2%, starch 1%～2%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.05%～0.15%, remaining are water, and pH value is adjusted to 7.0～8.5, inoculated and cultured after the cooling that sterilizes；
Step 3：Bacillus subtilis seed culture：The temperature of the bacillus subtilis seed culture is 35 DEG C～42 DEG C, time is 24h～32h, inoculum concentration is 2%～4%, initial speed of agitator is 400rpm, before 6h, ventilation ratio is 0.6, is adjusted to 1.3 afterwards, carries out culture transferring when the numerical value of tail carbon peaks to be begun to decline, pH adjustment is not carried out during the bacillus subtilis seed culture, and the bacillus subtilis seed culture culture medium prescription is：Corn protein powder 0.2%～1%, peptone 0.5%～2%, starch 1%～3%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.02%～0.35%；Polyether antifoam agent 0.01%, remaining are water, and pH value is adjusted to 7.0～8.5, and coefficient is 0.6, ventilate after sterilizing pressurize, when being cooled to 35 DEG C～42 DEG C, inoculated and cultured；
Step 4：Fermentation of bacillus subtilis culture, the temperature of the fermentation of bacillus subtilis culture is 35 DEG C～42 DEG C, time is 15h～26h, and initial speed of agitator is 400rpm, and inoculum concentration is 5%～10%, before 6h, ventilation ratio is 0.8, stepped up to 1.5, when pH is less than 6.0 in sweat according to zymotic fluid dissolved oxygen afterwards, in zymotic fluid, add ammoniacal liquor to carry out pH value adjustment, pH is more than 6.0 for control, and the culture medium prescription of the fermentation of bacillus subtilis culture is：Corn protein powder 0.5%～2%, cottonseed meal 0.2%～1%, starch 1%～2%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.05%, polyether antifoam agent 0.01%, remaining are water, and pH value is adjusted to 7.0～8.5 before sterilizing, coefficient is 0.6, ventilation pressurize after sterilizing, when being cooled to 35 DEG C～42 DEG C, inoculated and cultured；
Step 5：Before fermentation of bacillus subtilis tank puts tank, 2h～6h adds the magnesium sulfate after sterilizing, and addition is 0.001%～0.002%, promotes the formation of gemma, increases the active ingredient in zymotic fluid, treat bacterial content >=5*10 in zymotic fluid9Cfu/ml, and gemma ratio more than 90% when, fermentor liquid is discharged, finished product is carried out.
Above the enforcement of the present invention is illustrated, but the content is only presently preferred embodiments of the present invention, it is impossible to be considered as limiting protection scope of the present invention.All impartial changes that is made according to the present patent application scope and improvement, all should belong within the patent covering scope of the present invention.
Claims (1)
1. a kind of preparation method of microbial bactericide, it is characterised in that concretely comprise the following steps：
Step 1：Bacillus subtilis inclined-plane culture, the temperature of the bacillus subtilis inclined-plane culture are 35 DEG C～42 DEG C, and the time is 42h～52h, and the bacillus subtilis inclined-plane culture culture medium prescription is：Sodium chloride 0.2%～0.8%, beef extract 0.5%～2%, peptone 0.2%～0.8%, agar powder 1%～2%, remaining are water, pH value are adjusted to 7.0～8.5, contra bevel after sterilizing, inoculated and cultured；
Step 2：Bacillus subtilis Shaking culture, the temperature of the bacillus subtilis Shaking culture is 35 DEG C～42 DEG C, and the time is 30h～36h, and speed of agitator is 200rpm, and the bacillus subtilis Shaking culture culture medium prescription is：Corn protein powder 0.2%～0.8%, peptone 0.5%～2%, starch 1%～2%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.05%～0.15%, remaining are water, and pH value is adjusted to 7.0～8.5, inoculated and cultured after the cooling that sterilizes；
Step 3：Bacillus subtilis seed culture, the temperature of the bacillus subtilis seed culture is 35 DEG C～42 DEG C, time is 24h～32h, inoculum concentration is 2%～4%, and initial speed of agitator is 400rpm, and before 6h, ventilation ratio is 0.6,1.3 are adjusted to afterwards, culture transferring is carried out when the numerical value of tail carbon peaks to be begun to decline, pH adjustment is not carried out during the bacillus subtilis seed culture, and the bacillus subtilis seed culture culture medium prescription is：Corn protein powder 0.2%～1%, peptone 0.5%～2%, starch 1%～3%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.02%～0.35%；Polyether antifoam agent 0.01%, remaining are water, and pH value is adjusted to 7.0～8.5, and coefficient is 0.6, ventilate after sterilizing pressurize, when being cooled to 35 DEG C～42 DEG C, inoculated and cultured；
Step 4：Fermentation of bacillus subtilis culture, the temperature of the fermentation of bacillus subtilis culture is 35 DEG C～42 DEG C, time is 15h～26h, and initial speed of agitator is 400rpm, and inoculum concentration is 5%～10%, before 6h, ventilation ratio is 0.8, stepped up to 1.5, when pH is less than 6.0 in sweat according to zymotic fluid dissolved oxygen afterwards, in zymotic fluid, add ammoniacal liquor to carry out pH value adjustment, pH is more than 6.0 for control, and the culture medium prescription of the fermentation of bacillus subtilis culture is：Corn protein powder 0.5%～2%, cottonseed meal 0.2%～1%, starch 1%～2%, glucose 0.5%～2%, potassium dihydrogen phosphate 0.05%, polyether antifoam agent 0.01%, remaining are water, and pH value is adjusted to 7.0～8.5 before sterilizing, coefficient is 0.6, ventilation pressurize after sterilizing, when being cooled to 35 DEG C～42 DEG C, inoculated and cultured；
Step 5：Before fermentation of bacillus subtilis tank puts tank, 2h～6h adds the magnesium sulfate after sterilizing, and addition is 0.001%～0.002%, promotes the formation of gemma, increases the active ingredient in zymotic fluid, treat bacterial content >=5*10 in zymotic fluid9Cfu/ml, and gemma ratio more than 90% when, fermentor liquid is discharged, finished product is carried out.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113016593A (en) * | 2021-03-04 | 2021-06-25 | 云南泓庆农业科技有限公司 | Cultivation method of soybean sprouts |
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| CN105132312A (en) * | 2015-08-17 | 2015-12-09 | 河南科技学院 | Bacillus subtilis and application thereof as well as microbial fungicide containing bacillus subtilis and preparation method of microbial fungicide |
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- 2016-01-13 CN CN201610019899.0A patent/CN106489996A/en active Pending
Patent Citations (7)
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| CN1432284A (en) * | 2002-01-18 | 2003-07-30 | 福建泰禾生化科技股份有限公司 | High-efficiency biological germicide and its prepn process |
| CN1506455A (en) * | 2002-12-07 | 2004-06-23 | 福建农林大学 | Endophytic Bacillus subtilis and its prepn |
| CN102433283A (en) * | 2011-12-19 | 2012-05-02 | 湖南省微生物研究所 | High-density production process for forage bacillus subtilis, microbial inoculum prepared by using forage bacillus subtilis and application of microbial inoculum |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113016593A (en) * | 2021-03-04 | 2021-06-25 | 云南泓庆农业科技有限公司 | Cultivation method of soybean sprouts |
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Application publication date: 20170315 |