CN103614323B - A kind of substratum of bacillus amyloliquefaciens and application - Google Patents
A kind of substratum of bacillus amyloliquefaciens and application Download PDFInfo
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- CN103614323B CN103614323B CN201310607909.9A CN201310607909A CN103614323B CN 103614323 B CN103614323 B CN 103614323B CN 201310607909 A CN201310607909 A CN 201310607909A CN 103614323 B CN103614323 B CN 103614323B
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Abstract
The invention provides substratum and the application of bacillus amyloliquefaciens, relate to microorganism field.Described fermention medium contains 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.Adopt this culture medium culturing solution starch bacillus amyloliquefaciens (bacillus amyloliquefaciens) K-8, control culture condition as follows: 0h ~ 8h: vapour-liquid ratio is (0.4 ~ 0.6): 1, temperature 25 DEG C ~ 28 DEG C; 8h ~ 24h: vapour-liquid ratio is (0.9 ~ 1.1): 1, temperature 28 DEG C ~ 30 DEG C; After 24h: vapour-liquid ratio is (0.7 ~ 0.9): 1, temperature 30 DEG C ~ 32 DEG C.Adopt substratum of the present invention and cultural method, be conducive to bacillus amyloliquefaciens (bacillus amyloliquefaciens) K-8 amount reproduction in the short period of time, gemma is formed in the short period of time in a large number.
Description
Technical field
The present invention relates to microorganism field, more particularly relate to a kind of bacillus amyloliquefaciens (
bacillus amyloliquefaciens) substratum of K-8 and application thereof.
Background technology
Bacillus amyloliquefaciens is the biocontrol microorganisms that a class research is a lot, antimicrobial spectrum is very wide.The investigator of countries in the world expands a large amount of research work to it in biological control, growth promotion, fertilizer etc.
Application number be disclose in the patent of invention of 201310070624.6 bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8, various crop pathogenic bacteria can be suppressed, 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) K-8 substratum in containing carbon source, nitrogenous source and inorganic salt; Carbon source is glucose, starch, one or both the mixture of cracking 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 DEG C of-32 DEG C of conditions, cultivate 24h-72h, obtain liquid microbial inoculum.But contriver finds in research process, adopt application number to be the liquid microbial inoculum that in 201310070624.6 prepared by open method, 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 the microbial preparations such as follow-up absorption, drying, there is the problems such as thalline mortality ratio is high, product viable bacteria amount is low, quality is unstable, shelf-lives is short, thus 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, gemma also can be made to be formed in a large number in the short period of time.
The present invention also provide cultivate separate starch bacillus amyloliquefaciens (
bacillus amyloliquefaciens) method of K-8, can amount reproduction thalline in the short period of time, gemma also can be made to be formed in a large number 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, 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 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 control as follows: 0h ~ 8h: vapour-liquid ratio is (0.4 ~ 0.6): 1, temperature 25 DEG C ~ 28 DEG C; 8h ~ 24h: vapour-liquid ratio is (0.9 ~ 1.1): 1, temperature 28 DEG C ~ 30 DEG C; After 24h: vapour-liquid ratio is (0.7 ~ 0.9): 1, temperature 30 DEG C ~ 32 DEG C.
In preferred technical scheme, in culturing process, air flow and temperature control as follows: 0h ~ 8h: vapour-liquid ratio is 0.5:1, temperature 28 DEG C; 8h ~ 24h: vapour-liquid ratio is 1:1, temperature 30 DEG C; After 24h: vapour-liquid ratio is 0.8:1, temperature 32 DEG C.
Bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8 be preserved on 08 28th, 2012 China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, 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, a large amount of thalline can be bred in shorter incubation time, form a large amount of gemma within a short period of time.Because gemma has very strong resistance to high temperature, ultraviolet, drying, ionizing rays and much poisonous chemical substance, therefore in fermented liquid, spore 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, shorten the production time, improve 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 uses 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) K-8 lyophilize pipe in, with aseptic inoculation pin picking part bacterium powder to (250mL shaking flask, LB liquid nutrient medium liquid amount 50ml) in LB shaking flask, 30 DEG C, cultivate 24h under 150r/min after, LB substratum becomes muddy.Get streak culture 24h-36h in bacterium liquid to LB solid medium, bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8 is good at LB cultured on solid medium, bacteria colony white or canescence, surface imperfection, opaque.
The preparation of primary seed solution: the single colony inoculation on picking LB solid medium is in first order seed shaking flask, and Flask volume is the liquid amount 50ml of 250mL, LB liquid culture medium.By first order seed shaking flask 30 DEG C, cultivate 24h under 150r/min, obtain primary seed solution.
The preparation of secondary seed solution: the culture medium prescription (g/L) in seeding tank is: glucose 25, sucrose 25, protein powder 10, yeast powder 15, potassium primary phosphate 0.5, manganous sulfate 0.05, and solvent is water.Primary seed solution accessed in secondary seed tank, inoculum size is 1%-5%(V/V), culture condition is: ventilation ratio 1:1, and mixing speed is 200r/min, culture temperature 30 DEG C, tank pressure 0.01MPa, and incubation time is 20h.Obtain secondary seed solution at the end of cultivation, 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, be 2%(V/V according to inoculum size) access in fermentor tank, the substratum used in each test and culture condition 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, temperature 25 DEG C; 8h-24h: vapour-liquid ratio is 1:1, temperature 28 DEG C; After 24h: vapour-liquid ratio is 0.8:1, temperature 30 DEG C.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, temperature 28 DEG C; 8h-24h: vapour-liquid ratio is 1:1, temperature 30 DEG C; After 24h: vapour-liquid ratio is 0.8:1, temperature 32 DEG C.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, temperature 28 DEG C; 8h-24h: vapour-liquid ratio is 1:1, temperature 30 DEG C; After 24h: vapour-liquid ratio is 0.8:1, temperature 32 DEG C.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 finds through test of many times, when the pH of substratum 4 is 7.0-7.5, in fermenting process viable bacteria content and spore content similar, when pH be greater than 7.5 or be less than 7.0 time, viable bacteria content and gemma can be reduced, therefore, controlling substratum 4 is herein pH7.3, carries out simultaneous test.
Culture condition: ventilation ratio is 1:1, mixing speed is 150r/min, tank pressure 0.01MPa, culture temperature 30 DEG C, incubation time are 48h.
Result is as shown in table 1 below.
The different culture condition of table 1 is on the impact of result
The medium component testing 1,2,3 three group is identical, and the content and the culture process that are nutrient media components are different.Test 1 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 test 2
8cfu/mL).In this simultaneous test, the time that 1 microscopy gemma rate of testing reaches 100% is 32h, and viable bacteria content is now 47.3 × 10
8cfu/mL.And the time that 2 microscopy gemma rates of testing reach 100% is 28h, viable bacteria content is now 70.5 × 10
8cfu/mL.Namely the result testing 2 is 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, the time that 3 microscopy gemma rates of testing reach 100% is 28h, and viable bacteria content is now 60.7 × 10
8cfu/mL, lower than 70.5 × 10 of test 2
8cfu/mL.Namely the result testing 2 is better than test 3.
Test 4 for application number be the preferred cultural method of bacillus amyloliquefaciens disclosed 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 due to the nutritive ingredient in substratum too abundant, therefore until 44h, in fermented liquid, microscopy just finds gemma, even if fermentation is to 48h, its gemma rate ability about 30%, viable bacteria content is now 38.4 × 10
8cfu/mL.
Consider the viable bacteria content in fermentation time, fermented liquid and these three factors of gemma rate, test 2 is best combinations.
the synergy of embodiment 2 inorganic salt
In order to investigate the impact on thalline fermentation and sporulation of part inorganic salt in fermented liquid, spy devises this simultaneous test.
The basic medium used 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, temperature 28 DEG C;
8h-24h: vapour-liquid ratio is 1:1, temperature 30 DEG C;
After 24h: vapour-liquid ratio is 0.8:1, temperature 32 DEG C.
Other composition added in each test and content 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:
Table 2 inorganic salt are on the impact of sporulation time and content
As seen from the results in Table 2, test 5 is when 28h, and namely microscopy gemma rate reaches 100%, and viable bacteria content is now 73.6 × 10
8cfu/mL, the time that 6 microscopy gemma rates of testing reach 100% is 32h, but now viable bacteria content is 65.4 × 10
8cfu/mL, the time that 7 microscopy gemma rates of testing reach 100% is 32h, and viable bacteria content is now only 62.1 × 10
8cfu/mL.As can be seen from this test, only in basal fermentation medium, add magnesium sulfate, manganous sulfate and iron protochloride simultaneously, 4h can be shifted to an earlier date and make gemma rate reach 100%, thus shorten fermentation time, cost-saving, make microbial inoculum be easy to processing, constant product quality.
Claims (3)
1. one kind fermentation bacillus amyloliquefaciens (
bacillus amyloliquefaciens) method of K-8, it is characterized in that adopting fermention medium cultivate bacillus amyloliquefaciens (
bacillus amyloliquefaciens) K-8, in culturing process, air flow and temperature control as follows: 0h ~ 8h: vapour-liquid ratio is (0.4 ~ 0.6): 1, temperature 25 DEG C ~ 28 DEG C; 8h ~ 24h: vapour-liquid ratio is (0.9 ~ 1.1): 1, temperature 28 DEG C ~ 30 DEG C; After 24h: vapour-liquid ratio is (0.7 ~ 0.9): 1, temperature 30 DEG C ~ 32 DEG C; Described fermention medium contains: 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.
2. ferment according to claim 1 bacillus amyloliquefaciens (
bacillus amyloliquefaciens) method of K-8, it is characterized in that described fermention medium 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, magnesium sulfate 0.5g/L, manganous sulfate 0.05g/L, iron protochloride 0.04g/L, pH7.0 ~ 7.5.
3. ferment according to claim 2 bacillus amyloliquefaciens (
bacillus amyloliquefaciens) method of K-8, it is characterized in that in culturing process, air flow and temperature control as follows: 0h ~ 8h: vapour-liquid ratio is 0.5:1, temperature 28 DEG C; 8h ~ 24h: vapour-liquid ratio is 1:1, temperature 30 DEG C; After 24h: vapour-liquid ratio is 0.8:1, temperature 32 DEG C.
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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 |
| 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 |
| CN112280712A (en) * | 2020-11-02 | 2021-01-29 | 南京农业大学 | High-density liquid fermentation medium for bacteria and fermentation method thereof |
| CN118479915A (en) * | 2024-05-14 | 2024-08-13 | 云南省农业科学院农业环境资源研究所 | Biofertilizer for improving yield of peppers and preparation method thereof |
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