CN111073837B - Fermentation method for promoting Paenibacillus polymyxa to produce spores - Google Patents
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Abstract
The invention belongs to the field of biotechnology and bioengineering, and particularly relates to a fermentation method for promoting paenibacillus polymyxa to produce spores. The method comprises the following steps: activating Paenibacillus polymyxa in an activation medium; inoculating Paenibacillus polymyxa into a seed culture medium for seed culture; inoculating the seed culture solution to a fermentation culture solution for fermentation culture, sequentially raising the temperature by one stage, and regulating the pH value for continuous culture; raising the temperature in the second stage and regulating the pH value to continue culturing; when fermentation is finished, the pH is regulated; the invention can shorten the spore budding time in the fermentation process of Paenibacillus polymyxa, improve the spore quantity and budding rate, and the spore budding rate reaches more than 91.4%, thereby being beneficial to reducing the production cost and improving the product quality.
Description
Technical Field
The invention belongs to the field of biotechnology and bioengineering, and particularly relates to a fermentation method for promoting paenibacillus polymyxa to produce spores.
Background
The energy plant, namely jerusalem artichoke, is a perennial herb plant commonly known as jerusalem artichoke and rhizoma dioscoreae, and has the advantages of salt and alkali resistance, high yield and the like. The jerusalem artichoke contains inulin (also called inulin) which is polyfructose and can account for 15-20% of wet weight and about 80% of dry weight, and the biomass of the beach waste land can reach 3-6 tons/mu. Some Paenibacillus polymyxa produce powdery enzymes by itself, and inulin can be used as a fermentation carbon source.
Paenibacillus polymyxa (Paenibacillus polymyxa, previously called Bacillus polymyxa) is non-pathogenic to human beings or animals and plants, is a microorganism which can directly or indirectly promote plant growth, biocontrol effect and enhance plant resistance to adversity stress, and has important value in the development and production processes of biopesticides and fertilizers, and is living in plant rhizosphere soil or attached to plant roots. Recent research results are very different day by day, and understanding and cognition of the recent research results tend to be systematic. Because of its attractive application prospect, the U.S. Environmental Protection Agency (EPA) lists it as one of the commercially available microorganisms, and the agricultural community in China also lists it as a primary strain that is free of safety certification.
The use of Paenibacillus polymyxa HY96-2 strain to create microbial pesticides (0.1 hundred million CFU/g fine granule, trade name: kangdi Lei) has been reported, but the use of such factors as high cost of solid microbial inoculum, low strain number and poor use effect has been recently seen in the market. The research and development of the Paenibacillus polymyxa liquid spore type biological fertilizer microbial inoculum can avoid the defects, can increase the number and the use effect of living bacteria of the microbial fertilizer in soil, and has low production cost and multiple purposes. The increase of the spore amount of Paenibacillus polymyxa in the bacterial manure is one of key technologies for product development, and is beneficial to the transportation and long-term storage of the product.
Disclosure of Invention
Aiming at the problems of long spore formation time, low germination rate, low spore quantity and the like in the prior art, the invention provides a fermentation method for promoting paenibacillus polymyxa to produce spores.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
a fermentation method for promoting paenibacillus polymyxa to produce spores, comprising the following steps:
(1) Activating Paenibacillus polymyxa in an activation medium;
(2) Inoculating Paenibacillus polymyxa into a seed culture medium for seed culture;
(3) Inoculating the seed culture solution to a fermentation culture solution for fermentation culture, sequentially raising the temperature by one stage, and regulating the pH value for continuous culture; raising the temperature in the second stage and regulating the pH value to continue culturing; when fermentation is finished, the pH is regulated;
the fermentation culture and viable bacteria quantity improvement culture conditions are as follows: 30 ℃,120rpm, ph7.0, 24h;
the conditions of the first-stage temperature rise and pH value adjustment and continuous culture are as follows: 40-44 ℃,120rpm, pH7.5-8.0, 12h;
the secondary temperature rise and pH value adjustment continuous culture conditions are as follows: 45-50 ℃,120rpm, pH8.0-8.5, 12 h.
Preferably, the activation condition in the step (1) is 30 ℃, and the activation medium comprises the following components: (NH) 4 )HPO 4 1.0g/L，KCl 0.20g/L，MgSO 4 .7H 2 0.20g/L of O, 2.0g/L of yeast extract, 5.0g/L of glucose, 20.0g/L of agar and pH7.0, and 48. 48 h.
Preferably, the seed culture condition in the step (2) is 30 ℃,120rpm, and the seed culture medium comprises the following components: (NH) 4 )HPO4 1.0g/L，KCl 0.20g/L，MgSO 4 7H2O 0.20g/L, yeast extract 5.0g/L, glucose 10g/L, pH7.0, culture 12 h.
Preferably, the fermentation medium in step (3) comprises the following components: 30-40g/L of inulin-containing jerusalem artichoke inulin crude extract, 4.0g/L of ammonium sulfate, 1.0g/L of calcium carbonate, 1.0g/L of magnesium sulfate, 3.0g/L of dipotassium hydrogen phosphate, 1.0g/L of corn steep liquor and 5.0g/L of yeast powder; and pH7.0, to 24 h.
Preferably, the strain of the Paenibacillus polymyxa is Paenibacillus polymyxa capable of producing inulasepaenibacillus polymyxa) The preservation number of the strain is CGMCC No. 3044.
Preferably, the preparation method of the Jerusalem artichoke inulin crude extract containing 30-40g/L inulin comprises the following steps: uniformly stirring jerusalem artichoke dry powder and water according to a mass ratio of 1:8, carrying out water bath heat preservation for 2 hours at 70 ℃, cooling to room temperature, regulating pH to 10, carrying out water bath heat preservation for 1 hour at 80 ℃, filtering to remove residues, and collecting and diluting 1 time to obtain 30-40g/L jerusalem artichoke inulin crude extract containing inulin.
Compared with the prior art, the invention has the following beneficial effects: the invention can shorten the spore budding time in the fermentation process of Paenibacillus polymyxa, improve the spore quantity and budding rate, and the spore budding rate reaches more than 91.4%, thereby being beneficial to reducing the production cost and improving the product quality.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The specific techniques or conditions are not identified in the examples and are set forth in the specification in accordance with the techniques or conditions described in the literature in this field. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the present invention, unless otherwise indicated, the pH of the medium refers to the initial pH, i.e., the pH before sterilization, and the other components are the same and are used in the preparation (before sterilization); CFU as used refers to colony forming units as determined by plate counting.
The microbial strain bred and preserved in the laboratory of the present invention is Paenibacillus polymyxa capable of producing inulasepaenibacillus polymyxa) CGMCC No.3044, the strain is preserved in China general microbiological culture Collection center (CGMCC) at present, and the preservation unit address is: beijing city dynastyThe registration number of the university Tunne way of the Yangregion, the microbiological institute of China academy of sciences is CGMCC No.3044, and the preservation date is: 2009, 4 months and 29 days. The strain is used as a fermentation strain.
The CGMCC No.3044 strain has the following properties:
1. colony morphology characterization:
white microcolonies (phi=0.6-1.2 mm) appear on nutrient agar after culturing at 30 ℃ for 48h, and the colonies are round, smooth in surface, sticky, convex in the center and transparent. With proper prolonged culture, colonies are enlarged and the size and shape of cells are changed little.
2. Physiological and biochemical characteristics:
a. culture temperature: 28-39 ℃ and the optimal temperature is 30 ℃;
b. growing in the pH range of 5.0-7.5;
c. pigment production: pigment is not produced;
d. NaCl resistant concentration: can grow at 5% concentration.
The present invention will be described in detail by examples.
Example 1
Activating Paenibacillus polymyxa in an activation medium under the activation condition of 30 ℃, wherein an activation plate contains (g/L): (NH) 4 )HPO 4 1.0，KCl 0.20，MgSO 4 .7H 2 O0.20, yeast extract 2.0, glucose 5.0, agar 20.0, and pH7.0, culture 48h; inoculating Paenibacillus polymyxa into a seed culture medium for culture, wherein the seed culture condition is 30 ℃,120rpm, and the seed culture medium contains (g/L): (NH) 4 )HPO4 1.0，KCl 0.20，MgSO 4 7H2O 0.20, yeast extract 5.0, glucose 10, pH7.0, culture 12h; inoculating the seed culture solution into a fermentation culture solution, wherein the culture condition is 30 ℃,120rpm, and the fermentation culture solution comprises: jerusalem artichoke inulin crude extract (containing inulin 32.4/g), ammonium sulfate 4.0g/L, calcium carbonate 1.0g/L, magnesium sulfate 1.0g/L, dipotassium hydrogen phosphate 3.0g/L, corn steep liquor 1.0g/L, and yeast powder 5.0g/L; and pH7.0, fermentation to 24h; heating the fermentation broth, regulating pH value, continuously culturing at 40deg.C, 120rpm, pH7.5,12h, performing H; then the fermentation broth was subjected to a second temperature increase and pH adjustment to continue the culture at 45℃and 120rpm at pH8.0, 12. 12 h. When fermentation is finished, pH7.0 is regulated, the spore yield in the fermentation liquid is 83.6%, and the spore amount reaches 5.88 Í 10 9 cfu/mL。
Example 2
Activating Paenibacillus polymyxa in an activation medium under the activation condition of 30 ℃, wherein an activation plate contains (g/L): (NH) 4 )HPO 4 1.0，KCl 0.20，MgSO 4 .7H 2 O0.20, yeast extract 2.0, glucose 5.0, agar 20.0, and pH 6.0, culture 48h; inoculating Paenibacillus polymyxa into a seed culture medium for culture, wherein the seed culture condition is 30 ℃,120rpm, and the seed culture medium contains (g/L): (NH) 4 )HPO4 1.0，KCl 0.20，MgSO 4 7H2O 0.20, yeast extract 5.0, glucose 10, pH7.0, culture 12h; inoculating the seed culture solution into a fermentation culture solution, wherein the culture condition is 30 ℃,120rpm, and the fermentation culture solution comprises: jerusalem artichoke inulin crude extract (containing inulin 35.2 g/L), ammonium sulfate 4.0g/L, calcium carbonate 1.0g/L, magnesium sulfate 1.0g/L, dipotassium hydrogen phosphate 3.0g/L, corn steep liquor 1.0g/L, and yeast powder 5.0g/L; and pH7.0, to 24 h. Heating the fermentation broth at first stage, regulating pH value, continuously culturing at 42 deg.C and 120rpm, and regulating pH to 8.0 and 12. 12h; then the fermentation broth was subjected to a second temperature increase and pH adjustment to continue the culture at 48℃and 120rpm at pH 8.5, 12. 12 h. When fermentation is finished, pH7.0 is regulated, the spore yield in the fermentation liquid is more than 91.4%, and the spore amount reaches 7.09 Í 10 9 cfu/mL。
Example 3
Activating Paenibacillus polymyxa in an activation medium under the activation condition of 30 ℃, wherein an activation plate contains (g/L): (NH) 4 )HPO 4 1.0，KCl 0.20，MgSO 4 .7H 2 O0.20, yeast extract 2.0, glucose 5.0, agar 20.0, and pH7.0, culture 48h; inoculating Paenibacillus polymyxa into a seed culture medium for culture, wherein the seed culture condition is 30 ℃,120rpm, and the seed culture medium contains (g/L): (NH) 4 )HPO4 1.0，KCl 0.20，MgSO 4 7H2O 0.20, yeast extract 5.0, glucose 10, andpH7.0, culture 12h; inoculating the seed culture solution into a fermentation culture solution, wherein the culture condition is 30 ℃,120rpm, and the fermentation culture solution comprises: crude extract of Jerusalem artichoke inulin (containing inulin 33.9 g/L), ammonium sulfate 4.0g/L, calcium carbonate 1.0g/L, magnesium sulfate 1.0g/L, dipotassium hydrogen phosphate 3.0g/L, corn steep liquor 1.0g/L, and yeast powder 5.0g/L; and pH7.0, fermentation 24 h. Heating the fermentation broth at first stage, regulating pH value, continuously culturing at 44deg.C, 120rpm, and pH of 7.5, 12 and h; then the fermentation broth was subjected to a second temperature increase and pH adjustment to continue the culture at 50℃and 120rpm at pH 8.5, 12. 12 h. When fermentation is finished, pH7.0 is regulated, the spore yield in the fermentation liquid is more than 87.5%, and the spore amount reaches 6.15 Í 10 9 cfu/mL。
Comparative example 1
Activating Paenibacillus polymyxa in an activation medium under the activation condition of 30 ℃, wherein an activation plate contains (g/L): (NH) 4 )HPO 4 1.0，KCl 0.20，MgSO 4 .7H 2 O0.20, yeast extract 2.0, glucose 5.0, agar 20.0, and pH7.0, culture 48h; inoculating Paenibacillus polymyxa into a seed culture medium for culture, wherein the seed culture condition is 30 ℃,120rpm, and the seed culture medium contains (g/L): (NH) 4 )HPO4 1.0，KCl 0.20，MgSO 4 7H2O 0.20, yeast extract 5.0, glucose 10, pH7.0, culture 12h; inoculating the seed culture solution into a fermentation culture solution, wherein the culture condition is 30 ℃,120rpm, and the fermentation culture solution comprises: crude extract of Jerusalem artichoke inulin (containing inulin 33.9 g/L), ammonium sulfate 4.0g/L, calcium carbonate 1.0g/L, magnesium sulfate 1.0g/L, dipotassium hydrogen phosphate 3.0g/L, corn steep liquor 1.0g/L, and yeast powder 5.0g/L; and pH7.0, fermentation 48 and h, the germination rate in the fermentation liquid is lower than 0.1%, and the spore amount is about 6.0 Í 10 4 cfu/mL; fermenting 72 h, wherein the spore yield in the fermentation liquid is lower than 0.1%, and the spore quantity is about 1.06 Í 10 6 cfu/mL。
While the foregoing is directed to embodiments of the present invention, other and further details of the invention may be had by the present invention, it should be understood that the foregoing description is merely illustrative of the present invention and that no limitations are intended to the scope of the invention, except insofar as modifications, equivalents, improvements or modifications are within the spirit and principles of the invention.
Claims (1)
1. A fermentation method for promoting paenibacillus polymyxa to produce spores, which is characterized by comprising the following steps:
(1) Activating Paenibacillus polymyxa in an activation medium;
the activation condition is 30 ℃, and the activation culture medium comprises the following components: (NH) 4 )HPO 4 1.0g/L，KCl 0.20g/L，MgSO 4 ·7H 2 0.20g/L of O, 2.0g/L of yeast extract, 5.0g/L of glucose, 20.0g/L of agar and pH7.0, and culturing for 48h;
the strain of the Paenibacillus polymyxa is Paenibacillus polymyxa (paenibacillus polymyxa) capable of producing inulase, and the preservation number of the strain is CGMCC No.3044;
(2) Inoculating Paenibacillus polymyxa into a seed culture medium for seed culture;
the seed culture conditions were 30 ℃,120rpm, and the seed culture medium included the following components: (NH) 4 )HPO 4 1.0g/L，KCl 0.20g/L，MgSO 4 ·7H 2 0.20g/L of O, 5.0g/L of yeast extract, 10g/L of glucose and pH7.0, and culturing for 12h;
(3) Inoculating the seed culture solution to a fermentation culture solution for fermentation culture, sequentially raising the temperature by one stage, and regulating the pH value for continuous culture; raising the temperature in the second stage and regulating the pH value to continue culturing; when fermentation is finished, the pH is regulated; the fermentation culture and viable bacteria quantity improvement culture conditions are as follows: 30 ℃,120rpm, pH7.0, 24h;
the conditions of the first-stage temperature rise and pH value adjustment and continuous culture are as follows: 40-44 ℃,120rpm, pH7.5-8.0, 12h;
the secondary temperature rise and pH value adjustment continuous culture conditions are as follows: 45-50 ℃,120rpm, pH8.0-8.5, 12h;
the fermentation medium comprises the following components: 30-40g/L of inulin-containing jerusalem artichoke inulin crude extract, 4.0g/L of ammonium sulfate, 1.0g/L of calcium carbonate, 1.0g/L of magnesium sulfate, 3.0g/L of dipotassium hydrogen phosphate, 1.0g/L of corn steep liquor and 5.0g/L of yeast powder; and pH7.0, fermenting for 24h;
the preparation method of the Jerusalem artichoke inulin crude extract containing 30-40g/L inulin comprises the following steps: uniformly stirring jerusalem artichoke dry powder and water according to a mass ratio of 1:8, carrying out water bath heat preservation for 2 hours at 70 ℃, cooling to room temperature, regulating pH to 10, carrying out water bath heat preservation for 1 hour at 80 ℃, filtering to remove residues, and collecting and diluting 1 time to obtain 30-40g/L jerusalem artichoke inulin crude extract containing inulin.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101565683A (en) * | 2009-06-01 | 2009-10-28 | 盐城工学院 | Bacillus polymyxa and method for preparing optically-pure R, R type 2, 3-butanedio by utilizing same |
| CN106947715A (en) * | 2017-03-23 | 2017-07-14 | 中国科学院青岛生物能源与过程研究所 | A kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate |
| CN109251879A (en) * | 2018-11-22 | 2019-01-22 | 青岛中达农业科技有限公司 | A kind of Jie meter La series bacillus fermentation process in high density |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101565683A (en) * | 2009-06-01 | 2009-10-28 | 盐城工学院 | Bacillus polymyxa and method for preparing optically-pure R, R type 2, 3-butanedio by utilizing same |
| CN106947715A (en) * | 2017-03-23 | 2017-07-14 | 中国科学院青岛生物能源与过程研究所 | A kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate |
| CN109251879A (en) * | 2018-11-22 | 2019-01-22 | 青岛中达农业科技有限公司 | A kind of Jie meter La series bacillus fermentation process in high density |
Non-Patent Citations (1)
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| 多粘类芽孢杆菌SQR-21芽孢形成、萌发特征研究;霍振华;《中国博士学位论文全文数据库（电子期刊）》;20120615(第06期);第37-40、45-47页 * |
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