CN114437939A - Liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia - Google Patents
Liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia Download PDFInfo
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- CN114437939A CN114437939A CN202210068022.6A CN202210068022A CN114437939A CN 114437939 A CN114437939 A CN 114437939A CN 202210068022 A CN202210068022 A CN 202210068022A CN 114437939 A CN114437939 A CN 114437939A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
Abstract
The invention is applicable to the technical field of microbial fermentation engineering, and provides a liquid fermentation method for promoting trichoderma longibrachiatum to produce conidium, which comprises the following steps: preparing a liquid culture medium of trichoderma according to the formula of the culture medium; II, secondly: adjusting the pH value of the liquid culture medium to 7 by using a sodium hydroxide solution, and then sterilizing at high temperature and high pressure to obtain a liquid fermentation culture medium of trichoderma; thirdly, the method comprises the following steps: inoculating the preserved trichoderma longibrachiatum strain on a potato glucose agar culture medium, and performing activated culture to obtain a fungus cake of the trichoderma longibrachiatum strain; fourthly, the method comprises the following steps: inoculating a fungus cake of the trichoderma longibrachiatum strain in a triangular flask filled with a culture medium; fifthly: carrying out shake culture on the inoculated culture medium at 25-28 ℃ and 200rpm/min for 4-6 days to obtain a seed culture solution; sixthly, the method comprises the following steps: inoculating the seed culture solution into a fermentation tank filled with liquid culture medium according to the inoculation amount of 1%, and culturing at 28-Fermenting at 30 deg.C with ventilation of 0.8-1.0L/L for 48h to produce conidia of 1 × 109cfu/mL, low cost of the culture medium formula, less impurities in the culture solution and convenient processing of subsequent dosage forms.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation engineering, and particularly relates to a liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia.
Background
Trichoderma spp has multiple biocontrol functions such as competition, antibiotic, heavy parasitism, induced resistance and the like as an important broad-spectrum antagonistic biocontrol bacterium, and has an inhibiting effect on various pathogenic fungi, such as Rhizoctonia solani, Pythium spp, Fusarium spp and the like. The trichoderma biocontrol agent mainly adopts a conidium preparation, and the trichoderma viride (T.viride) and trichoderma harzianum (T.harzianum) are mainly produced in a large amount of commercial production at present, so that the optimization of fermentation conditions and the increase of conidium yield of strains are important research directions for developing microbial agents.
Trichoderma longibrachiatum (t. longibrachiatum) is an important biocontrol bacterium, and the fermentation methods currently used for the bacterium are mainly solid fermentation and liquid fermentation. The solid fermentation has the defects of too long fermentation time, more impurities and unsuitability for aerial spraying by an unmanned aerial vehicle, and the liquid fermentation has the problem of low conidium yield. Wangyitong et al (Wangyitong, Zhaoyang, Huangya Li, etc., application of Trichoderma longibrachiatum JG9-31 in preventing and treating gray mold 2021, CN113249228A) propose a liquid fermentation method for producing chlamydospore by Trichoderma longibrachiatum, but the chlamydospore production conditions are stricter than those of conidiospore, and the conidiospore is more widely used in practical application. In order to determine the disease control effect of trichoderma longibrachiatum, a large amount of conidia are needed, the concentration of the conidia generated by liquid fermentation is low at present, a conidia suspension with higher concentration than that of conventional spraying is needed when an unmanned aerial vehicle is used for aviation spraying, and the prior art cannot meet the test requirement. Therefore, the liquid fermentation method for producing conidia with high yield by trichoderma longibrachiatum is urgently needed to be developed, and the liquid fermentation method has important practical application value for developing airplane control by using an unmanned aerial vehicle in the future.
Disclosure of Invention
The invention provides a liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia, and aims to solve the problems that the conidia of the trichoderma longibrachiatum is low in conidia yield and cannot meet the requirements of general tests under the condition of the prior art, and the development of application basic research is limited.
The invention is realized in such a way that a liquid fermentation method for promoting trichoderma longibrachiatum to produce conidium comprises the following steps:
the method comprises the following steps: preparing a liquid culture medium of trichoderma according to the formula of the culture medium;
step two: adjusting the pH value of the liquid culture medium to 7 by using a sodium hydroxide solution, dividing the culture medium into a plurality of parts, filling the parts in triangular flasks, and sterilizing at high temperature and high pressure to obtain a liquid fermentation culture medium for trichoderma;
step three: inoculating the preserved trichoderma longibrachiatum strain on a potato glucose agar culture medium, and performing activated culture to obtain a fungus cake of the trichoderma longibrachiatum strain;
step four: inoculating a fungus cake of the trichoderma longibrachiatum strain in a triangular flask filled with a culture medium;
step five: and carrying out shake culture on the inoculated culture medium at 25-28 ℃ and 200rpm/min for 4-6 days to obtain a seed culture solution.
Step six: inoculating the seed culture solution into a fermentation tank filled with liquid culture medium according to the inoculation amount of 1%, and fermenting for 48h under the conditions of temperature of 28-30 ℃ and ventilation capacity of 0.8-1.0L/L to obtain fermentation liquor containing a large amount of conidia.
The method can obtain a large amount of conidia, and has the advantages of low cost, less impurities in the fermentation culture solution, difficult blockage of the spray head and convenient subsequent formulation processing.
Preferably, in the first step, the components in the culture medium formula comprise 5-6g/L of yeast extract powder, 20g/L of glucose, 7g/L of sodium nitrate, 0.1g/L of ferrous sulfate, 1.25g/L of magnesium sulfate and 4g/L of potassium dihydrogen phosphate.
Preferably, in step two, the prepared liquid culture medium is adjusted to pH 7 by using 1mol/L sodium hydroxide solution.
Preferably, the sterilization under high temperature and high pressure in the second step is performed at 121 ℃ and 105KPa for 20 min.
Preferably, in the third step, the preserved trichoderma longibrachiatum strain is inoculated on a potato glucose agar culture medium for activation culture for 5-7 days to obtain the trichoderma longibrachiatum cake.
Preferably, in step four, the liquid volume of the culture medium in a 500mL triangular flask is 70-200mL, and 2-5 fungus cakes with the diameter of 6mm are inoculated.
Preferably, the liquid loading volume of the 500mL triangular flask is 70mL, and 2 fungus cakes with the diameter of 6mm are inoculated.
Compared with the prior art, the invention has the beneficial effects that: the invention relates to a liquid fermentation method for promoting trichoderma longibrachiatum to produce conidium, which comprises the steps of inoculating the trichoderma conidium to a specific culture medium, carrying out shake culture for 4-6 days at 25-28 ℃ under the condition of 200rpm/min, wherein the specific culture medium comprises 5-6g of yeast extract powder, 20g of glucose, 7g of sodium nitrate, 0.1g of ferrous sulfate, 1.25g of magnesium sulfate, 4g of potassium dihydrogen phosphate and 1L of distilled water, and the pH value is adjusted to 7.
Drawings
FIG. 1 is a schematic flow diagram of the process of the present invention;
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Referring to fig. 1, the present invention provides a technical solution:
a liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia comprises the following steps:
the method comprises the following steps: and preparing a liquid culture medium of the trichoderma according to the formula of the culture medium. The components in the culture medium formula comprise 5-6g/L of yeast extract powder, 20g/L of glucose, 7g/L of sodium nitrate, 0.1g/L of ferrous sulfate, 1.25g/L of magnesium sulfate and 4g/L of potassium dihydrogen phosphate.
Step two: adjusting pH of the liquid culture medium to 7 with 1mol/L sodium hydroxide solution, dividing the culture medium into multiple parts, placing in triangular flasks, and sterilizing at 121 deg.C and 105KPa for 20min to obtain liquid fermentation culture medium of Trichoderma.
Step three: and inoculating the preserved trichoderma longibrachiatum strain on a potato glucose agar culture medium for activation culture for 5-7d to obtain the trichoderma longibrachiatum cake.
Step four: inoculating the fungus cake of the Trichoderma longibrachiatum strain in a triangular flask filled with the culture medium. The liquid loading of the culture medium in a 500mL triangular flask is 70-200mL, and 2-5 fungus cakes with the diameter of 6mm are inoculated. Preferably, the liquid loading volume of the 500mL triangular flask is 70mL, and 2 fungus cakes with the diameter of 6mm are inoculated.
Step five: and carrying out shake culture on the inoculated culture medium at 25-28 ℃ and 200rpm/min for 4-6 days to obtain a seed culture solution.
Step six: inoculating the seed culture solution into a fermentation tank filled with liquid culture medium according to the inoculation amount of 1%, and fermenting for 48h under the conditions of temperature of 28-30 ℃ and ventilation capacity of 0.8-1.0L/L to obtain fermentation liquor containing a large amount of conidia.
The liquid fermentation method provided by the invention can obtain a large amount of conidia within 2 days, the formula cost of the culture medium is low, impurities in the culture solution are less, the subsequent preparation processing is convenient, and the industrial production is realized.
The Trichoderma longibrachiatum strains used in the present embodiment are all strains isolated from Pinus massoniana by the applicant.
Example 1
A liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia comprises the following steps:
1) preparing a liquid culture medium of trichoderma according to a culture medium formula, adding 2.5g of yeast extract powder, 10g of glucose, 3.5g of sodium nitrate, 0.05g of ferrous sulfate, 0.625g of magnesium sulfate and 2g of monopotassium phosphate into 500mL of pure water;
2) adjusting pH of the solution to 7 with 1mol/L sodium hydroxide solution, subpackaging the culture medium into 500mL triangular flasks at a liquid loading of 70mL, and sterilizing at 121 deg.C and 105KPa for 20 min.
3) Inoculating the preserved Trichoderma longibrachiatum strain to potato glucose agar culture medium, and activating and culturing for 5-7d to obtain fungus cake;
4) inoculating 2 fungus cakes with the diameter of 6mm in a triangular flask filled with a culture medium;
5) the inoculated culture medium was cultured under shaking at 28 ℃ and 200rpm/min for 5 days, and the spore yield was observed.
Example 2
A liquid fermentation method for promoting trichoderma longibrachiatum to produce conidium comprises the following steps:
1) preparing a liquid culture medium of trichoderma according to a culture medium formula, adding 2.5g of yeast extract powder, 10g of glucose, 3.5g of sodium nitrate, 0.05g of ferrous sulfate, 0.625g of magnesium sulfate and 2g of monopotassium phosphate into 500mL of pure water;
2) adjusting pH of the solution to 7 with 1mol/L sodium hydroxide solution, subpackaging the culture medium into 500mL triangular flasks at a liquid loading of 100mL, and sterilizing at 121 deg.C and 105KPa for 20 min.
3) Inoculating the preserved Trichoderma longibrachiatum strain to potato glucose agar culture medium, and activating and culturing for 5-7d to obtain fungus cake;
4) inoculating 3 fungus cakes with the diameter of 6mm in a triangular flask filled with a culture medium;
5) the inoculated culture medium was cultured under shaking at 28 ℃ and 200rpm/min for 5 days, and the spore yield was observed.
Example 3
A liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia comprises the following steps:
1) preparing a liquid culture medium of trichoderma according to a culture medium formula, adding 3g of yeast extract powder, 10g of glucose, 3.5g of sodium nitrate, 0.05g of ferrous sulfate, 0.625g of magnesium sulfate and 2g of monopotassium phosphate into 500mL of pure water;
2) adjusting pH of the solution to 7 with 1mol/L sodium hydroxide solution, subpackaging the culture medium into 500mL triangular flasks at a liquid loading of 200mL, and sterilizing at 121 deg.C and 105KPa for 20 min.
3) Inoculating the preserved Trichoderma longibrachiatum strain to potato glucose agar culture medium, and activating and culturing for 5-7d to obtain fungus cake;
4) inoculating 3 fungus cakes with the diameter of 6mm in a triangular flask filled with a culture medium;
5) the inoculated culture medium was cultured under shaking at 28 ℃ and 200rpm/min for 5 days, and the spore yield was observed.
Example 4
A liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia comprises the following steps:
steps 1-5 are the same as in example 3.
(6) Inoculating the seed culture solution into a fermentation tank filled with liquid culture medium at an inoculation amount of 1%, fermenting at 30 deg.C with ventilation of 0.8L/L for 48h, and recording spore yield.
Example 5
A liquid fermentation method for promoting trichoderma longibrachiatum to produce conidia comprises the following steps:
steps 1-5 are the same as in example 3.
(6) Inoculating the seed culture solution into a fermentation tank filled with liquid culture medium at an inoculation amount of 1%, fermenting at 28 deg.C with ventilation of 1.0L/L for 48h, and recording spore yield.
Comparative example 1
A potato dextrose liquid culture medium is prepared, the liquid filling amount per bottle in a 500mL triangular flask is 100mL, a trichoderma cake is inoculated according to the method of the example 1, the culture is performed for 5 days under the conditions of 28 ℃ and 200rpm/min, and the sporulation amount is observed.
As a result of the experiments, the spore production amounts of 1X 10 spores in examples 1-3 were all8cfu/mL or more, and the spore yield of comparative example 1 was 1X 105cfu/mL or less, and hyphae grow vigorously. In example 4, the amount of conidia of Trichoderma produced was 1.45X 109cfu/mL, the spore yield of the Trichoderma conidia in example 5 was 1.2X 109cfu/mL。
The method for fermenting the trichoderma liquid provided by the invention has the advantages that the spore yield of conidia is obviously increased, and the spore yield can reach 1 multiplied by 10 in the expanded reproduction9cfu/mL or more.
Therefore, the liquid fermentation method provided by the invention can obtain a large amount of conidia within 2 days, the formula cost of the culture medium is low, impurities in the culture solution are less, the spray head is not easy to block, and the subsequent preparation formulation processing is facilitated.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. A liquid fermentation method for promoting trichoderma longibrachiatum to produce conidium is characterized in that: the method comprises the following steps:
the method comprises the following steps: preparing a liquid culture medium of trichoderma according to the formula of the culture medium;
step two: adjusting the pH value of the liquid culture medium to 7 by using a sodium hydroxide solution, dividing the culture medium into a plurality of parts, filling the parts in triangular flasks, and sterilizing at high temperature and high pressure to obtain a liquid fermentation culture medium for trichoderma;
step three: inoculating the preserved trichoderma longibrachiatum strain on a potato glucose agar culture medium, and performing activated culture to obtain a fungus cake of the trichoderma longibrachiatum strain;
step four: inoculating a fungus cake of the trichoderma longibrachiatum strain in a triangular flask filled with a culture medium;
step five: and carrying out shake culture on the inoculated culture medium at 25-28 ℃ and 200rpm/min for 4-6 days to obtain a seed culture solution.
Step six: inoculating the seed culture solution into a fermentation tank filled with liquid culture medium according to the inoculation amount of 1%, and fermenting for 48h under the conditions of temperature of 28-30 ℃ and ventilation capacity of 0.8-1.0L/L to obtain fermentation liquor containing a large amount of conidia.
2. The liquid fermentation method for promoting the production of conidia by trichoderma longibrachiatum according to claim 1, wherein: in the first step, the components in the culture medium formula comprise 5-6g/L of yeast extract powder, 20g/L of glucose, 7g/L of sodium nitrate, 0.1g/L of ferrous sulfate, 1.25g/L of magnesium sulfate and 4g/L of potassium dihydrogen phosphate.
3. The liquid fermentation method for promoting the production of conidia of trichoderma longibrachiatum according to claim 1, wherein: in the second step, the pH of the prepared liquid culture medium is adjusted to 7 by using 1mol/L sodium hydroxide solution.
4. The liquid fermentation method for promoting the production of conidia by trichoderma longibrachiatum according to claim 1, wherein: sterilizing at high temperature and high pressure in step two at 121 deg.C and 105KPa for 20 min.
5. The liquid fermentation method for promoting the production of conidia by trichoderma longibrachiatum according to claim 1, wherein: in the third step, the preserved trichoderma longibrachiatum strain is inoculated on a potato glucose agar culture medium for activation culture for 5-7d, and the trichoderma longibrachiatum cake is obtained.
6. The liquid fermentation method for promoting the production of conidia of trichoderma longibrachiatum according to claim 1, wherein: in the fourth step, the liquid loading of the culture medium in a 500mL triangular flask is 70-200mL, and 2-5 fungus cakes with the diameter of 6mm are inoculated.
7. The liquid fermentation method for promoting the production of conidia by trichoderma longibrachiatum according to claim 6, wherein: preferably, the liquid loading volume of the 500mL triangular flask is 70mL, and 2 fungus cakes with the diameter of 6mm are inoculated.
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