CN110408546B - Solid fermentation process of trichoderma viride - Google Patents
Solid fermentation process of trichoderma viride Download PDFInfo
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- CN110408546B CN110408546B CN201910605388.0A CN201910605388A CN110408546B CN 110408546 B CN110408546 B CN 110408546B CN 201910605388 A CN201910605388 A CN 201910605388A CN 110408546 B CN110408546 B CN 110408546B
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- C—CHEMISTRY; METALLURGY
- 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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- C—CHEMISTRY; METALLURGY
- 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 provides a trichoderma viride solid fermentation process, which comprises a seeding tank culture stage and a fermentation tank culture stage; the culture stage of the seeding tank comprises: controlling the pH value of the culture medium to be 5.0-5.2, the culture pressure to be 0.05-0.06MPa, and the culture temperature to be: 30 plus or minus 0.5 ℃. The culture stage of the seeding tank comprises: the ventilation volume is 1: 1; stirring speed: 180-; culturing time: 23-24 hours. The fermentation process of the invention has high spore yield, and the number of the prepared and dried trichoderma viride spores is more than or equal to 1.5 multiplied by 10 10 Per gram.
Description
Technical Field
The invention relates to a trichoderma viride solid fermentation process, and belongs to the technical field of microbial culture.
Background
Trichoderma viride is a kind of trichoderma, widely distributed in nature, and often saprophytic on wood, seeds and plant residues. The trichoderma viride can generate a plurality of enzyme systems with biological activity, the produced cellulase has one of strains with the highest activity, the produced cellulase has a degradation effect on crops, the effect is very good, and the trichoderma viride is also an antagonistic microorganism with rich resources and has an important effect in the prevention and treatment of plant pathological organisms.
CN201710426936.4 discloses a Trichoderma viride and a culture method thereof. The Trichoderma viride strain is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 5612. The method for culturing the trichoderma viride mainly comprises the steps of trichoderma viride strain activation, liquid strain preparation, solid fermentation and drying, wherein the solid fermentation adopts an edible fungus bag fermentation mode; the method adopts the traditional edible fungus production technology to produce the trichoderma viride spore powder, has reasonable process, low cost and easy implementation, and has good economic benefit, environmental benefit and social benefit.
CN201310372776.1 discloses a method for producing trichoderma viride by liquid-solid two-phase fermentation. The method for culturing trichoderma viride provided by the invention comprises the following steps: (1) performing liquid fermentation on trichoderma viride to obtain a liquid fermentation product; (2) and carrying out solid fermentation on the liquid fermentation product. The method for culturing the trichoderma viride has the advantages of economical and practical production process, high production efficiency, small investment, small occupied area, realization of large-scale production, no three-waste problem, suitability for popularization and application in large scale and huge economic benefit.
The prior art trichoderma viride fermentation process also has the following defects: low spore yield, long spore production time and high mixed bacterium rate.
Disclosure of Invention
The invention provides a trichoderma viride solid fermentation process, which aims to achieve the following aims: the yield of the trichoderma viride spores is improved, the spore production time is shortened, and the rate of mixed bacteria is reduced.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a trichoderma viride solid fermentation process comprises a seeding tank culture stage and a fermentation tank culture stage; the culture stage of the seeding tank comprises: controlling the pH value of the culture medium to be 5.0-5.2, the culture pressure to be 0.05-0.06MPa, and the culture temperature to be: 30 plus or minus 0.5 ℃.
The culture stage of the seeding tank comprises: the ventilation volume is 1: 1; stirring speed: 180-; culturing time: 23-24 hours.
The fermentation tank culture stage: for solid fermentation, the culture medium comprises solid materials and inorganic salt solution, and the material-liquid ratio is 3: 1.
the fermentation tank culture stage: the inoculation method comprises sterilizing the inoculation pipeline and the sterile air pipe with steam in advance for 1 hr, reducing pipeline steam pressure to 0.2Mpa and temperature to 30-32 deg.C, and pressing the strain into solid fermentation tank via pipeline; after inoculation, stirring is started for 30-32 minutes to uniformly mix the materials and the strains, and meanwhile, sterile air is introduced.
The fermentation tank culture stage: pot for storing foodThe pressure is 0.02-0.025Mpa, the culture temperature is 30 + -1 deg.C, and the air volume is controlled at 20 + -1 m 3 H, adjusting the air volume to 40 +/-1 m after heat production 3 /h。
The fermentation tank culture stage: after 16 hours of fermentation, stirring was started every 8 hours for 10 minutes.
The fermentation tank culture stage: after fermenting for 36 hours, transferring the solid material containing the seeds to a tray, continuing fermenting at 27-29 ℃ to produce spores, culturing for 2-3 days to find that the surface of the material is completely green spores, discharging, boiling, drying and drying.
The seeding tank culture stage: the adopted culture medium of the seeding tank comprises 18 +/-0.5 g/L of yeast extract, 25 +/-0.5 g/L of glucose, 2 +/-0.1 g/L of ammonium sulfate, 1.8 +/-0.1 g/L of shiitake extract, 0.6 +/-0.1 g/L of malt extract and 0.2 +/-0.05 g/L of cysteine.
The fermentation tank culture stage: the adopted culture medium raw materials comprise solid materials and inorganic salt solution; the solid material comprises corncob powder, seaweed meal, cottonseed cake meal, lentinan, cysteine, glutamic acid and vitamin C.
The fermentation tank culture stage: the inorganic salt solution is a mixed solution of ammonium sulfate and potassium dihydrogen phosphate.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
(1) the fermentation process of the invention has high spore yield, and the number of the prepared and dried trichoderma viride spores is more than or equal to 1.5 multiplied by 10 10 Per gram;
(2) the fermentation process of the invention has fast spore production, and the spore production time is 2-3 days;
(3) the fermentation process has low mixed bacteria rate, and the mixed bacteria rate is zero;
(4) the trichoderma viride spores prepared by the fermentation process have good quality stability.
Detailed Description
Example 1 solid fermentation Process of Trichoderma viride
A solid fermentation process of Trichoderma viride comprises a seeding tank culture stage and a fermentation tank culture stage.
First, seed tank culture
A 100 liter seeding tank is adopted; the inoculation amount is 1000ml
1. After the seed tank is checked to be correct, all the raw materials are put into the seed tank, the volume (50L) is determined, the temperature is raised to 121-122 ℃, the material is sterilized for 30 minutes, the volume after sterilization is 70L, and the pH value after sterilization is controlled to be 5.0.
2. Inoculation of
After the actual digestion is finished, the seeding tank is kept at the pressure and cooled, and the inoculation is prepared when the temperature is reduced to the culture temperature of 30 +/-0.5 ℃.
Alcohol cotton, forceps, tweezers and inoculating loop are prepared before inoculation. The cotton is evenly placed around the inoculating loop, the inoculating loop is placed around the inoculating opening and then ignited, and flame is required to seal the periphery of the inoculating opening.
And closing a valve between the inoculation bottle and the seed tank, and slowly opening the inoculation port cover, wherein the inoculation port cover cannot leave the flame closed range after being opened.
Burning the mouth of the seed bottle in flame, opening a cotton plug within the flame closed range, and quickly pouring the seed liquid into the inoculation bottle. And (3) in the flame range, plugging the cotton plug of the seed bottle into the bottle opening, then removing the inoculation flame, and after the inoculation is finished, closing the inoculation opening cover.
Opening the valve between the inoculating bottle and the seeding tank to make the seeds flow into the seeding tank naturally, extinguishing the flame and removing the inoculating loop.
Ventilating, turning on the stirring, and culturing normally.
Ventilation quantity: 1: 1; the culture temperature is as follows: 30 plus or minus 0.5 ℃; stirring speed: 180 revolutions per minute; culturing time: 24 hours;
and (5) detecting the sterility of the seed bottle.
3. Solid fermentation
1) Fermentation tank treatment: sterilizing and thoroughly cleaning the solid fermentation tube to ensure that no solid material is left in the solid fermentation tube.
And (3) air sterilizing the primary tank body at 125 ℃ for about 40 minutes, boiling the tank with alkaline water if the last time is infected with bacteria, and fumigating the room with formaldehyde before filling the tank.
Preparing materials: all solid materials are uniformly mixed, inorganic salt solution is added and mixed, and the material-liquid ratio is 3: 1.
introducing the material into a fermentation tank;
stirring, heating to 60 deg.C with jacket, sterilizing with steam at 121 deg.C for 40 min, and sterilizing with jacket.
After sterilization, the temperature is reduced to 30 ℃ for inoculation.
2) Inoculation of
Sterilizing the inoculation pipeline and the sterile air pipe with steam for 1 hr, reducing the pipeline steam pressure to 0.2MPa and 30 deg.C, and pressing the strain into the solid fermentation tank via pipeline.
After inoculation, stirring is started for 30 minutes to uniformly mix the materials and the strains, and meanwhile, sterile air is introduced.
3) Fermenting in a fermentation tank
After inoculation, the pot pressure was maintained at 0.02 MPa.
The culture temperature is 30 +/-1 ℃.
After inoculation, the air volume was controlled at 20m 3 H, regulating the air volume to 40 m after heat production 3 /h。
After 16 hours stirring was switched on every 8 hours for 10 minutes.
After 36 hours, transferring the solid material containing the seeds to a tray, continuing fermenting at 27 ℃ to produce spores, after culturing for 2 days, finding that the surface of the material is completely green spores, discharging, boiling, drying and drying.
The detection shows that the number of the trichoderma viride spores after drying is more than or equal to 1.5 multiplied by 10 10 Number of bacteria per gram is zero.
Example 2 solid fermentation Process of Trichoderma viride
A solid fermentation process of Trichoderma viride comprises a seeding tank culture stage and a fermentation tank culture stage.
The strain preservation number of the trichoderma viride is CICC 13027.
Seeding tank culture
A 100 liter seed tank was used: the inoculation amount is 1000 ml;
1) seeding tank culture medium preparation
The adopted culture medium of the seeding tank comprises 18g/L of yeast extract, 25g/L of glucose, 2g/L of ammonium sulfate, 1.8g/L of shiitake extract, 0.6g/L of malt extract and 0.2g/L of cysteine;
the mushroom extract comprises the following components: purchased from West Anwan Biotechnology Ltd, cat # WF-5002;
the malt extract is prepared by the following steps: purchased from Quancao Biotech, Inc., Guangzhou, cat # JG-167.
After the seed tank is checked to be correct, all the culture medium raw materials of the seed tank are put into the seed tank, the volume is 50L, the temperature is increased to 121-.
The volume after elimination is as follows: i.e., the volume of medium after sterilization.
2) Inoculating and culturing
After the actual digestion is finished, maintaining the pressure in the seeding tank and cooling, and preparing for inoculation when the temperature is reduced to the culture temperature of 30 +/-0.5 ℃; maintaining the pressure in the seeding tank at 0.05 MPa.
Alcohol cotton, forceps, tweezers and inoculating loop are prepared before inoculation. The cotton is evenly placed around the inoculating loop, the inoculating loop is placed around the inoculating opening and then ignited, and flame is required to seal the periphery of the inoculating opening.
And closing a valve between the inoculation bottle and the seed tank, and slowly opening the inoculation port cover, wherein the inoculation port cover cannot leave the flame closed range after being opened.
Burning the mouth of the seed bottle in flame, opening a cotton plug within the flame closed range, and quickly pouring the seed liquid into the inoculation bottle. And (3) in the flame range, plugging the cotton plug of the seed bottle into the bottle opening, then removing the inoculation flame, and after the inoculation is finished, closing the inoculation opening cover.
Opening the valve between the inoculating bottle and the seeding tank to make the seeds flow into the seeding tank naturally, extinguishing the flame and removing the inoculating loop.
Ventilating, opening stirring, and normally culturing; controlling ventilation quantity in the culture process: 1: 1; the culture temperature is as follows: 30 plus or minus 0.5 ℃; stirring speed: 180 revolutions per minute; culturing time: 24 hours;
and (5) detecting the sterility of the seed bottle.
2. Solid fermentation
1) Fermenter treatment
Sterilizing and thoroughly cleaning the solid fermentation tube to ensure that no solid material is left in the solid fermentation tube.
Emptying the primary tank body at the temperature of 125 ℃ and the pressure of 103.4kPa for about 40 minutes; if the last time of contamination, the pot is boiled with alkaline water, and the room is fumigated with formaldehyde and then can be filled.
Preparing a culture medium: all solid materials are uniformly mixed, inorganic salt solution is added and mixed, and the material-liquid ratio is 3: 1.
the solid material is as follows: 40% of corn cob meal, 30% of seaweed meal, 20% of cottonseed cake meal, 6% of lentinan, 2% of cysteine, 1.5% of glutamic acid and 0.5% of vitamin C.
The corncob powder: the fineness is 500 meshes;
the seaweed powder comprises the following components: is spirulina powder, purchased from Jinan Riyue Ying chemical Co Ltd, ground to 200 mesh fineness;
the inorganic salt solution is a mixed solution of ammonium sulfate and potassium dihydrogen phosphate; the contents are as follows: 1.2g/L of ammonium sulfate and 0.8 g/L of monopotassium phosphate;
introducing the material into a fermentation tank;
stirring, heating to 60 deg.C, sterilizing with steam at 123 deg.C for 40 min.
After sterilization, the temperature is reduced to 30 ℃ for inoculation.
2) Inoculation of
Sterilizing the inoculation pipeline and the sterile air pipe with steam for 1 hr, reducing the pipeline steam pressure to 0.2MPa and 30 deg.C, and pressing the strain into the solid fermentation tank via pipeline.
After inoculation, stirring is started for 30 minutes to uniformly mix the materials and the strains, and meanwhile, sterile air is introduced.
3) Fermenting in a fermentation tank
After inoculation, the pot pressure was maintained at 0.02 MPa.
The culture temperature is 30 +/-1 ℃.
After inoculation, the air volume was controlled at 20m 3 H, regulating the air volume to 40 m after heat production 3 /h。
After 16 hours stirring was switched on every 8 hours for 10 minutes.
After 36 hours, transferring the solid material containing the seeds to a tray, continuing fermentation at 29 ℃ to produce spores, after culturing for 3 days, finding that the surface of the material is completely green spores, discharging, boiling, drying and drying.
The detection shows that the number of the trichoderma viride spores after drying is more than or equal to 3.2 multiplied by 10 11 Number of bacteria per gram is zero.
In addition, the trichoderma viride spores prepared by the method have good stability and long quality guarantee period which is up to 3 years.
Except for special description, the percentages are mass percentages, and the ratios are mass ratios.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (1)
1. A trichoderma viride solid fermentation process is characterized by comprising a seeding tank culture stage and a fermentation tank culture stage;
the strain preservation number of the trichoderma viride is CICC 13027;
the method comprises the following steps: seeding tank culture
A 100 liter seed tank was used: the inoculation amount is 1000 ml;
1) seeding tank culture medium preparation
The adopted culture medium of the seeding tank comprises 18g/L of yeast extract, 25g/L of glucose, 2g/L of ammonium sulfate, 1.8g/L of shiitake extract, 0.6g/L of malt extract and 0.2g/L of cysteine;
the mushroom extract comprises the following components: purchased from West Anwan Biotechnology Ltd, cat # WF-5002; the malt extract is prepared by the following steps: purchased from the grass Biotech Ltd, Guangzhou, cat # JG-167;
after the seed tank is checked to be correct, all the culture medium raw materials of the seed tank are put into the seed tank, the volume is 50L, the temperature is increased to 121-;
the volume after elimination is as follows: i.e. the volume of the culture medium after sterilization;
2) inoculating and culturing
After the actual digestion is finished, maintaining the pressure in the seeding tank and cooling, and preparing for inoculation when the temperature is reduced to the culture temperature of 30 +/-0.5 ℃; maintaining the pressure of the seeding tank at 0.05 Mpa; preparing alcohol cotton, forceps, tweezers and inoculating loops before inoculation; uniformly placing cotton around an inoculating loop, igniting after the inoculating loop is placed on an inoculating opening, and requiring flame to seal the periphery of the inoculating opening; closing a valve between the inoculation bottle and the seed tank, slowly opening an inoculation port cover, and preventing the inoculation port cover from leaving the flame closed range after opening; burning the mouth of the seed bottle in flame, opening a cotton plug within the flame closed range, and quickly pouring the seed liquid into an inoculation bottle; the cotton plug of the seed bottle is plugged into the bottle mouth in the flame range, then the inoculation flame is removed, and after the inoculation is finished, the inoculation opening cover is closed; opening a valve between the inoculation bottle and the seed tank, putting out flame after the seeds naturally flow into the seed tank, and removing the inoculating loop; ventilating, opening stirring, and normally culturing; controlling ventilation rate in the culture process: 1: 1; the culture temperature is as follows: 30 plus or minus 0.5 ℃; stirring speed: 180 revolutions per minute; culturing time: 24 hours; detecting the sterility of the seed bottle;
step two: solid fermentation
1) Fermenter treatment
Sterilizing and thoroughly cleaning the solid fermentation tube to ensure that no solid materials remain in the solid fermentation tube; emptying the primary tank body at the temperature of 125 ℃ and the pressure of 103.4kPa for about 40 minutes; if the last time of contamination, the pot is boiled with alkaline water, and the room is fumigated by formaldehyde and then can be filled; preparing a culture medium: all solid materials are uniformly mixed, inorganic salt solution is added and mixed, and the material-liquid ratio is 3: 1;
the solid material is as follows: 40% of corn cob meal, 30% of seaweed meal, 20% of cottonseed cake meal, 6% of lentinan, 2% of cysteine, 1.5% of glutamic acid and 0.5% of vitamin C;
the corncob powder: the fineness is 500 meshes; the seaweed powder comprises the following components: is spirulina powder, purchased from Jinan Riyue Ying chemical Co Ltd, ground to 200 mesh fineness; the inorganic salt solution is a mixed solution of ammonium sulfate and potassium dihydrogen phosphate; the contents are as follows: 1.2g/L of ammonium sulfate and 0.8 g/L of monopotassium phosphate;
introducing the material into a fermentation tank;
stirring, uniformly mixing, heating by using a jacket, stopping heating by using the jacket when the temperature is raised to 60 ℃, starting steam sterilization in a tank, wherein the sterilization temperature is 123 ℃, and the time is 40 minutes; cooling to 30 ℃ for inoculation after sterilization;
2) inoculation of
Sterilizing the inoculation pipeline and the sterile air pipe with steam for 1 hour in advance, wherein the steam pressure of the pipeline is more than or equal to 0.2Mpa, the temperature is reduced to 30 ℃, and the strain is pressed into a solid fermentation tank through the pipeline; stirring for 30 minutes after inoculation is finished, uniformly mixing the materials and the strains, and simultaneously introducing sterile air;
3) fermenting in a fermentation tank
After inoculation, the tank pressure is kept at 0.02 MPa; the culture temperature is 30 +/-1 ℃; after inoculation, the air volume was controlled at 20m 3 H, regulating the air volume to 40 m after heat generation 3 H; starting stirring once every 8 hours after 16 hours, wherein the starting time is 10 minutes; after 36 hours, transferring the solid material containing the seeds to a tray, continuing fermentation at 29 ℃ to produce spores, after culturing for 3 days, finding that the surface of the material is completely green spores, discharging, boiling, drying and drying.
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| CN103131642A (en) * | 2013-03-18 | 2013-06-05 | 济南巨微生物技术有限公司 | Trichoderma viride and culture method and application thereof |
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| CN106676017A (en) * | 2016-12-30 | 2017-05-17 | 沂源康源生物科技有限公司 | Trichoderma viride and large-scale preparation method thereof |
| CN107118971A (en) * | 2017-06-08 | 2017-09-01 | 黄河三角洲京博化工研究院有限公司 | A kind of Trichoderma viride and its cultural method |
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| CN103131642A (en) * | 2013-03-18 | 2013-06-05 | 济南巨微生物技术有限公司 | Trichoderma viride and culture method and application thereof |
| CN103525748A (en) * | 2013-10-31 | 2014-01-22 | 湖南泰谷生物科技股份有限公司 | Production method of Trichoderma harzianum chlamydospore |
| CN106676017A (en) * | 2016-12-30 | 2017-05-17 | 沂源康源生物科技有限公司 | Trichoderma viride and large-scale preparation method thereof |
| CN107118971A (en) * | 2017-06-08 | 2017-09-01 | 黄河三角洲京博化工研究院有限公司 | A kind of Trichoderma viride and its cultural method |
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Denomination of invention: A Solid State Fermentation Process of Trichoderma viride Effective date of registration: 20230316 Granted publication date: 20220816 Pledgee: Weifang rural commercial bank Limited by Share Ltd. hi tech sub branch Pledgor: SHANDONG SUKAHAN BIO-TECHNOLOGY Co.,Ltd. Registration number: Y2023980034955 |