CN103614323A - Culture medium of bacillus amyloliquefaciens and application - Google Patents
Culture medium of bacillus amyloliquefaciens and application Download PDFInfo
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Abstract
The invention provides a culture medium of bacillus amyloliquefaciens and application, relating to the field of microorganisms. The fermentation medium contains 5-10g/L of glucose, 10-30g/L of molasses, 5-10g/L of silkworm chrysalis meal, 5-20g/L of corn steep liquor, 10-20g/L of sodium chloride, 1-10g/L of calcium carbonate, 0.1-1g/L of magnesium sulfate, 0.01-0.1g/L of manganese sulfate and 0.02-0.08g/L of ferrous chloride, and the pH value is 7.0-7.5. The bacillus amyloliquefaciens K-8 is cultured by the culture medium, and the culture conditions are controlled as follows: from the 0th-8th hour, the gas-liquid ratio is (0.4-0.6):1, and the temperature is 25-28 DEG C; from the 8th-24th hour, the gas-liquid ratio is (0.9-1.1):1, and the temperature is 28-30 DEG C; after the 24th hour, the gas-liquid ratio is (0.7-0.9):1, and the temperature is 30-32 DEG C. By adopting the culture medium and culture method provided by the invention, mass propagation of the bacillus amyloliquefaciens K-8 in relatively short time is facilitated so that a great quantity of spores are formed in relatively short time.
Description
Technical field
The present invention relates to microorganism field, more particularly relate to a kind of bacillus amyloliquefaciens (
bacillus amyloliquefaciens) substratum and the application thereof of K-8.
Background technology
Bacillus amyloliquefaciens is that a class research is a lot, the very wide biocontrol microorganisms of antimicrobial spectrum.The investigator of countries in the world has launched a large amount of research work to it at aspects such as biological control, growth promotion, fertilizer.
Application number be in 201310070624.6 patent of invention, disclose bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8, can suppress various crop pathogenic bacteria, as fungal diseases such as the bacterial diseases such as bacterial wilt of tomato pathogenic bacteria, angular leaf spot of cucumber pathogenic bacteria, Prospect on Kiwifruit Bacterial Canker pathogenic bacteria, soft rot of Chinese cabbage pathogenic bacteria and watermelon blight pathogenic bacteria, rice sheath blight disease pathogenic bacteria and canker of apple fruit pathogenic bacterias simultaneously.Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) contain carbon source, nitrogenous source and inorganic salt in the substratum of K-8; One or both the mixture that carbon source is glucose, starch, crack rice in powder and molasses; Nitrogenous source is one or both the mixture in groundnut meal, soyflour, yeast powder, protein powder and dried silkworm chrysalis meal; Inorganic salt are one or both the mixture in sodium-chlor, dipotassium hydrogen phosphate, potassium primary phosphate, ammonium sulfate, manganous sulfate, magnesium sulfate, calcium carbonate.Under 28 ℃ of-32 ℃ of conditions, cultivate 24h-72h, obtain liquid microbial inoculum.But contriver finds in research process, adopting application number is the liquid microbial inoculum that in 201310070624.6 prepared by open method, and spore forming rate is not high, sporulation overlong time.If spore forming rate is low in liquid microbial inoculum, in the course of processing of follow-up absorption, the microbial preparation such as dry, there is the problems such as thalline mortality ratio is high, product viable bacteria amount is low, quality is unstable, shelf-lives is short, thereby have a strong impact on the result of use of preparation.
Summary of the invention
The object of this invention is to provide a kind of bacillus amyloliquefaciens (
bacillus amyloliquefaciens) substratum of K-8, adopt this substratum be conducive to bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8 amount reproduction in the short period of time, also can make gemma form in a large number in the short period of time.
The present invention also provide cultivate to separate starch bacillus amyloliquefaciens (
bacillus amyloliquefaciens) method of K-8, amount reproduction thalline, also can make gemma form in a large number in the short period of time in the short period of time.
Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) fermention medium of K-8, contain: glucose 5～10g/L, molasses 10～30g/L, dried silkworm chrysalis meal 5～10g/L, corn steep liquor 5～20g/L, sodium-chlor 10～20g/L, calcium carbonate 1～10g/L, magnesium sulfate 0.1～1g/L, manganous sulfate 0.01～0.1g/L, iron protochloride 0.02～0.08g/L, pH7.0～7.5.
In preferred technical scheme, bacillus amyloliquefaciens (
bacillus amyloliquefaciens) fermention medium of K-8, contain glucose 5g/L, molasses 20g/L, dried silkworm chrysalis meal 5g/L, corn steep liquor 15g/L, sodium-chlor 15g/L, calcium carbonate 5g/L, magnesium sulfate 0.5g/L, manganous sulfate 0.05g/L, iron protochloride 0.04g/L, pH7.0～7.5.
A kind of fermentation solution starch bacillus amyloliquefaciens (
bacillus amyloliquefaciens) method of K-8, adopt described culture medium culturing solution starch bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8, in culturing process, air flow and temperature are controlled as follows: 0h～8h: vapour-liquid ratio is (0.4～0.6): 1, and 25 ℃～28 ℃ of temperature; 8h～24h: vapour-liquid ratio is (0.9～1.1): 1,28 ℃～30 ℃ of temperature; After 24h: vapour-liquid ratio is (0.7～0.9): 1,30 ℃～32 ℃ of temperature.
In preferred technical scheme, in culturing process, air flow and temperature are controlled as follows: 0h～8h: vapour-liquid ratio is 0.5:1,28 ℃ of temperature; 8h～24h: vapour-liquid ratio is 1:1,30 ℃ of temperature; After 24h: vapour-liquid ratio is 0.8:1,32 ℃ of temperature.
Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 08 28th, 2012, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), its preserving number is CGMCC No.6486.
Beneficial effect:
Adopt the present invention be exclusively used in bacillus amyloliquefaciens (
bacillus amyloliquefaciens) substratum of K-8, can in shorter incubation time, breed a large amount of thalline, form a large amount of gemma within a short period of time.Because gemma has very strong resistance to high temperature, ultraviolet ray, dry, ionizing rays and a lot of poisonous chemical substance, therefore in fermented liquid, gemma content is high, in the course of processing, thalline survival rate is high, and the preparation living spores content of acquisition is high, shelf-lives long, constant product quality.Due to bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8 can form a large amount of gemma within a short period of time in substratum of the present invention, shortened the production time, improved production efficiency, saved production cost, for solid basis is established in the industrialization of bacillus amyloliquefaciens.
Embodiment
The solvent that in the present invention, substratum is used is water.Vapour-liquid ratio refers to per minute air flow (m
3) and fermentating liquid volume (m
3) ratio.
embodiment 1 bacillus amyloliquefaciens (
bacillus amyloliquefaciens) cultivation of K-8
(1) bacillus amyloliquefaciens (
bacillus amyloliquefaciens) activation of K-8 and the preparation of seed liquor
Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) activation of K-8: from bacillus amyloliquefaciens (
bacillus amyloliquefaciens) in the lyophilize pipe of K-8, with aseptic inoculation pin picking part bacterium powder (250mL shaking flask, LB liquid nutrient medium liquid amount 50ml) to LB shaking flask, under 30 ℃, 150r/min, cultivate after 24h, it is muddy that LB substratum becomes.Get bacterium liquid to streak culture 24h-36h in LB solid medium, bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8 well-grown on LB solid medium, bacteria colony white or canescence, surface imperfection is opaque.
The preparation of primary seed solution: the single colony inoculation on picking LB solid medium is in first order seed shaking flask, and shaking flask volume is 250mL, the liquid amount 50ml of LB liquid culture medium.First order seed shaking flask is cultivated to 24h under 30 ℃, 150r/min, obtain primary seed solution.
The preparation of secondary seed solution: the culture medium prescription in seeding tank (g/L) is: glucose 25, sucrose 25, protein powder 10, yeast powder 15, potassium primary phosphate 0.5, manganous sulfate 0.05, solvent is water.Primary seed solution is accessed in secondary seed tank, and inoculum size is 1%-5%(V/V), culture condition is: ventilation ratio 1:1, mixing speed is 200r/min, 30 ℃ of culture temperature, tank pressure 0.01MPa, incubation time is 20h.Cultivate while finishing and obtain secondary seed solution, its viable bacteria content is 1-3 * 10
8cfu/mL, now thalline is energetic.
(2) fermentation culture
By bacillus amyloliquefaciens (
bacillus amyloliquefaciens) secondary seed solution of K-8, according to inoculum size, being 2%(V/V) in access fermentor tank, the substratum and the culture condition that in each test, use are as follows respectively.
Test 1:
Adopt substratum 1: glucose 5g/L, molasses 10g/L, dried silkworm chrysalis meal 5g/L, corn steep liquor 5g/L, sodium-chlor 10g/L, calcium carbonate 1g/L, magnesium sulfate 0.1g/L, manganous sulfate 0.01g/L, iron protochloride 0.02g/L, pH7.0.
Culture condition: tank pressure 0.01MPa; 0h-8h: vapour-liquid ratio is 0.5:1,25 ℃ of temperature; 8h-24h: vapour-liquid ratio is 1:1,28 ℃ of temperature; After 24h: vapour-liquid ratio is 0.8:1,30 ℃ of temperature.Mixing speed: 180r/min.
Test 2:
Adopt substratum 2: glucose 5g/L, molasses 20g/L, dried silkworm chrysalis meal 5g/L, corn steep liquor 15g/L, sodium-chlor 15g/L, calcium carbonate 5g/L, magnesium sulfate 0.5g/L, manganous sulfate 0.05g/L, iron protochloride 0.04g/L, pH7.3.
Culture condition: tank pressure 0.01MPa; 0h-8h: vapour-liquid ratio is 0.5:1,28 ℃ of temperature; 8h-24h: vapour-liquid ratio is 1:1,30 ℃ of temperature; After 24h: vapour-liquid ratio is 0.8:1,32 ℃ of temperature.Mixing speed: 150r/min.
Test 3:
Adopt substratum 3: glucose 10g/L, molasses 30g/L, dried silkworm chrysalis meal 10g/L, corn steep liquor 20g/L, sodium-chlor 20g/L, calcium carbonate 10g/L, magnesium sulfate 1g/L, manganous sulfate 0.1g/L, iron protochloride 0.08g/L, pH7.5.
Culture condition: tank pressure 0.01MPa; 0h-8h: vapour-liquid ratio is 0.5:1,28 ℃ of temperature; 8h-24h: vapour-liquid ratio is 1:1,30 ℃ of temperature; After 24h: vapour-liquid ratio is 0.8:1,32 ℃ of temperature.Mixing speed: 200r/min.
Test 4:
Adopt substratum 4: glucose 50g/L, starch 50g/L, protein powder 10g/L, yeast powder 20g/L, potassium primary phosphate 0.5g/L, manganous sulfate 0.05g/L, pH7.3.Contriver is through test of many times discovery, and when the pH of substratum 4 is 7.0-7.5, in fermenting process, viable bacteria content and gemma content are similar, when pH is greater than 7.5 or while being less than 7.0, can reduce viable bacteria content and gemma, therefore, control substratum 4 herein for pH7.3, carry out simultaneous test.
Culture condition: ventilation ratio is that 1:1, mixing speed are that 150r/min, tank pressure 0.01MPa, 30 ℃ of culture temperature, incubation time are 48h.
Result is as shown in table 1 below.
The impact of the different culture condition of table 1 on result
The medium component of testing 1,2,3 three group is identical, and just the content of nutrient media components and culture process are different.Test 1 is compared with test 2, its highest viable bacteria content (52.9 * 10
8cfu/mL) lower than the highest viable bacteria content (75.8 * 10 of testing 2
8cfu/mL).In this simultaneous test, it is 32h that the 1 microscopy gemma rate of testing reaches time of 100%, and viable bacteria content is now 47.3 * 10
8cfu/mL.And the 2 microscopy gemma rates of testing reach time of 100%, be 28h, viable bacteria content is now 70.5 * 10
8cfu/mL.Test 2 result and be better than test 1.
Test 3 is compared with test 2, and medium component is identical with culture process, and just the content of nutrient media components is different.In this simultaneous test, it is 28h that the 3 microscopy gemma rates of testing reach time of 100%, and viable bacteria content is now 60.7 * 10
8cfu/mL, lower than 70.5 * 10 of test 2
8cfu/mL.Test 2 result and be better than test 3.
Test 4 for application number be the preferred cultural method of disclosed bacillus amyloliquefaciens in 201310070624.6 applications for a patent for invention.From table 1 data, its viable bacteria content reaches the highest by (71.7 * 10 when 32h
8cfu/mL), but because the nutritive ingredient in substratum is too abundant, therefore, up to 44h, in fermented liquid, microscopy is just found gemma, even if 48h is arrived in fermentation, its gemma rate is 30% left and right, and viable bacteria content is now 38.4 * 10
8cfu/mL.
Consider viable bacteria content and these three factors of gemma rate in fermentation time, fermented liquid, testing 2 is best combinations.
the synergy of embodiment 2 inorganic salt
In order to investigate the impact of part inorganic salt on thalline fermentation and sporulation in fermented liquid, ad hoc this simultaneous test of having counted.
The basic medium using in simultaneous test contains: glucose 5g/L, molasses 20g/L, dried silkworm chrysalis meal 5g/L, corn steep liquor 15g/L, sodium-chlor 15g/L, calcium carbonate 5g/L, pH7.0～7.5.
Culture condition is also identical:
Tank pressure 0.01MPa;
0h-8h: vapour-liquid ratio is 0.5:1,28 ℃ of temperature;
8h-24h: vapour-liquid ratio is 1:1,30 ℃ of temperature;
After 24h: vapour-liquid ratio is 0.8:1,32 ℃ of temperature.
Other composition and the content that in each test, add are as follows:
test 5:magnesium sulfate 0.5g/L, manganous sulfate 0.05g/L, iron protochloride 0.04g/L.
test 6:magnesium sulfate 0.5g/L, manganous sulfate 0.05g/L.
test 7:iron protochloride 0.04g/L.
Result is as shown in table 2:
The impact of table 2 inorganic salt on sporulation time and content
As seen from the results in Table 2, test 5 when 28h, microscopy gemma rate reaches 100%, and viable bacteria content is now 73.6 * 10
8cfu/mL, it is 32h that the 6 microscopy gemma rates of testing reach time of 100%, but now viable bacteria content is 65.4 * 10
8cfu/mL, it is 32h that the 7 microscopy gemma rates of testing reach time of 100%, viable bacteria content is now only 62.1 * 10
8cfu/mL.From this test, can find out, only in basic fermention medium, add magnesium sulfate, manganous sulfate and iron protochloride simultaneously, can shift to an earlier date 4h and make gemma rate reach 100%, thereby shorten fermentation time, cost-saving, make microbial inoculum be easy to processing, constant product quality.
Claims (4)
- Bacillus amyloliquefaciens ( bacillus amyloliquefaciens) fermention medium of K-8, it is characterized in that containing: glucose 5～10g/L, molasses 10～30g/L, dried silkworm chrysalis meal 5～10g/L, corn steep liquor 5～20g/L, sodium-chlor 10～20g/L, calcium carbonate 1～10g/L, magnesium sulfate 0.1～1g/L, manganous sulfate 0.01～0.1g/L, iron protochloride 0.02～0.08g/L, pH7.0～7.5.
- According to claim 1 bacillus amyloliquefaciens ( bacillus amyloliquefaciens) fermention medium of K-8, it is characterized in that containing glucose 5g/L, molasses 20g/L, dried silkworm chrysalis meal 5g/L, corn steep liquor 15g/L, sodium-chlor 15g/L, calcium carbonate 5g/L, magnesium sulfate 0.5g/L, manganous sulfate 0.05g/L, iron protochloride 0.04g/L, pH7.0～7.5.
- A fermentation separate starch bacillus amyloliquefaciens ( bacillus amyloliquefaciens) method of K-8, it is characterized in that adopting culture medium culturing solution starch bacillus amyloliquefaciens described in claim 1 or 2 ( bacillus amyloliquefaciens) K-8, in culturing process, air flow and temperature are controlled as follows: 0h～8h: vapour-liquid ratio is (0.4～0.6): 1, and 25 ℃～28 ℃ of temperature; 8h～24h: vapour-liquid ratio is (0.9～1.1): 1,28 ℃～30 ℃ of temperature; After 24h: vapour-liquid ratio is (0.7～0.9): 1,30 ℃～32 ℃ of temperature.
- According to claim 3 preparation separate starch bacillus amyloliquefaciens ( bacillus amyloliquefaciens) method of K-8, it is characterized in that in culturing process that air flow and temperature are controlled as follows: 0h～8h: vapour-liquid ratio is 0.5:1,28 ℃ of temperature; 8h～24h: vapour-liquid ratio is 1:1,30 ℃ of temperature; After 24h: vapour-liquid ratio is 0.8:1,32 ℃ of temperature.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104232549A (en) * | 2014-09-29 | 2014-12-24 | 浙江汇能动物药品有限公司 | Silkworm chrysalis extract powder-containing microbiological culture medium and application thereof |
| CN106434418A (en) * | 2016-08-05 | 2017-02-22 | 四川农业大学 | Culture medium for bacillus amyloliquefaciens culture medium for producing bacteriostatic active substances |
| CN108753760A (en) * | 2018-05-25 | 2018-11-06 | 广州市天旭食品添加剂有限公司 | A kind of bacillus amyloliquefaciens producing lab ferment fermentation process |
| CN110447659A (en) * | 2019-08-15 | 2019-11-15 | 江苏苏滨生物农化有限公司 | A kind of bacillus amyloliquefaciens suspending agent and preparation method thereof |
| CN110447659B (en) * | 2019-08-15 | 2020-08-21 | 江苏苏滨生物农化有限公司 | Bacillus amyloliquefaciens suspending agent and preparation method thereof |
| CN110878274A (en) * | 2019-12-30 | 2020-03-13 | 中化农业生态科技(湖北)有限公司 | Bacillus amyloliquefaciens culture process method |
| CN113293119A (en) * | 2020-11-02 | 2021-08-24 | 南京农业大学 | High-density liquid fermentation medium for bacteria and fermentation method thereof |
| CN113293119B (en) * | 2020-11-02 | 2023-10-13 | 南京农业大学 | Bacterial high-density liquid fermentation medium and fermentation method thereof |
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Effective date of registration: 20220928 Address after: No. 1, West Ring Road, Low Carbon Industrial Park, Wuhai High-tech Industrial Development Zone, Hainan District, Wuhai City, Inner Mongolia Autonomous Region, 016000 Patentee after: Inner Mongolia Yongtai Chemical Co.,Ltd. Address before: 224500 Coastal Industrial Park, Binhai County Economic Development Zone, Yancheng City, Jiangsu Province Patentee before: JIANGSU SUBIN AGROCHEMICAL Co.,Ltd. |