CN103333851B - Method of producing conidiospore by solid fermentation of trichoderma harzianum - Google Patents
Method of producing conidiospore by solid fermentation of trichoderma harzianum Download PDFInfo
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Abstract
The invention provides a method of producing conidiospore by solid fermentation of trichoderma harzianum. Trichoderma harzianum used in the method is purchased from Agricultural Culture Collection of China, and the number is ACCC30371. The method of producing conidiospore comprises following steps: trichoderma harzianum is subjected to culture activation, slant culture, solid seed culture, and solid fermentation so as to obtain conidiospore. A solid phase fermentation medium of the solid fermentation is composed of inorganic salts, wheat bran, and soybean meal or corn flour. According to the solid fermentation of trichoderma harzianum, a large amount of conidiospore is produced, the largest amount of matrix conidiospore is 4.89*10<9>cfu/g, the fermentation unit is high, no waste is produced during producing processes, and no environment pollution is caused. A mixture of conidiospore-medium obtained by the solid fermentation can be used directly for production after drying, and the obtained conidiospore can be used for bio-control of crops and decomposition of straw.
Description
Technical field
The present invention relates to a kind of conidial production method, specifically, relate to a kind of trichoderma harziarum solid fermentation and produce conidial method.
Background technology
Trichoderma (Trichoderma spp.) belongs to Mycophyta, Deuteromycotina, hyphomycetes, Moniliales, and sticky spore mushroom, being the ubiquitous fungi of a class, being common in soil, is the important group of soil microorganisms.Fungus Trichoderma is distributed widely in nature, is extensively present in rotten wood, seed, plant residue, air, therefrom can be separated and obtains.
Trichoderma has important economic implications, is widely applied to all respects of agricultural, as controlling plant diseases, Promoting plant growth, biologically rehabilitating soil and environment, is applied to biological organic fertilizer etc.In recent years, the research and apply of Trichoderma obtained and developed fast.In conjunction with research methods such as genetics, molecular biology, biological chemistry and ecology, Trichoderma and meta-bolites thereof are at Promoting plant growth, have additional nutrients the absorption rate of material, improves the output of farm crop, and strengthen and certainly will have more wide application prospect to the defensive aspect of disease.Particularly under the overall background advocating green agriculture, the large scale application of Trichoderma has become a part indispensable in structure modern times and efficient biological pest control system.
Along with the progress of society, people more and more profoundly recognize that the long-term a large amount of chemical pesticide that uses is to the harm of ecotope and human health.Biological control can overcome these drawbacks effectively, and thus biological pesticide is more and more subject to people's attention.The wherein application of antagonistic microbe in disease flocking biocontrol extremely people's concern.In the antagonistic microbe to have found for 2013, it is active that Trichoderma has good biological and ecological methods to prevent plant disease, pests, and erosion.
Applying maximum in Trichoderma is trichoderma harziarum (Trichoderma harzianum).In trichoderma harziarum production process, easily occur that mycelial growth is too vigorous, and the problem that conidium is underproduce.Make microbial inoculum with trichoderma harziarum and be that this microbial inoculum is containing described conidial quantity for the key of agriculture aspect, quantity is high, microbial inoculum shelf-lives will be long, the microbial inoculum that this ectoconidium quantity is high can be sprouted rapidly in field and surely grow, form dominant colony, and then generation meta-bolites, act on the growth microenvironment of plant and plant, thus play biological and ecological methods to prevent plant disease, pests, and erosion and promote plant strain growth effect.Therefore, the quality of described conidial output and microbial inoculum is closely related, thus needs to research and develop a kind of high conidiiferous method of trichoderma harziarum.
Summary of the invention
The object of this invention is to provide a kind of trichoderma harziarum high conidiiferous method in solid medium.
In order to realize the object of the invention, first the present invention provides a kind of fermention medium, and described substratum is made up of the material of following weight part: wheat bran 3-4.5 part, bean cake powder 0.75-1.5 part, inorganic ion solution 4-6.25 part; Wherein, the Ingredient percent of described inorganic ion solution is: ammonium sulfate 0.5% ~ 2%, SODIUM PHOSPHATE, MONOBASIC 0.05% ~ 0.8%, Sodium phosphate dibasic 0.05% ~ 0.8%, dipotassium hydrogen phosphate 0.05% ~ 0.5%, magnesium sulfate 0.1% ~ 1%, manganous sulfate 0.1% ~ 1%, sucrose 0.5% ~ 2.5%, glucose 0.05% ~ 2%, and surplus is water.
Preferably, described substratum is made up of the material of following weight part: wheat bran 3.8-4.2 part, bean cake powder 0.8-1.2 part, inorganic ion solution 4.6-5.6 part; Wherein, the Ingredient percent of described inorganic ion solution is: ammonium sulfate 0.8% ~ 1.4%, SODIUM PHOSPHATE, MONOBASIC 0.1% ~ 0.5%, Sodium phosphate dibasic 0.1% ~ 0.5%, dipotassium hydrogen phosphate 0.1% ~ 0.4%, magnesium sulfate 0.2% ~ 0.8%, manganous sulfate 0.2% ~ 0.8%, sucrose 1% ~ 1.8%, glucose 0.1% ~ 1.2%, and surplus is water.
More preferred, described substratum is made up of the material of following weight part: 4 parts, wheat bran, bean cake powder 1 part, inorganic ion solution 5 parts; Wherein, the Ingredient percent of described inorganic ion solution is: ammonium sulfate 1.25%, SODIUM PHOSPHATE, MONOBASIC 0.4%, Sodium phosphate dibasic 0.4%, dipotassium hydrogen phosphate 0.25%, magnesium sulfate 0.55%, manganous sulfate 0.55%, sucrose 1.5%, glucose 1%, and surplus is water.
Present invention also offers the application of aforesaid fermentation substratum in trichoderma harziarum fermentative production conidium.
Present invention also offers a kind of trichoderma harziarum solid fermentation and produce conidial method, comprise the preparation of slant strains, the preparation of ferment-seeded and the preparation of fermention medium and fermentating controling process step.
Wherein fermentation step with aforementioned fermention medium for substratum, be placed in fermentation culture case and cultivate under fluorescent lamp irradiates, culture temperature is 22 DEG C ~ 29 DEG C, incubation time is 6 ~ 9 days, sterile air is passed between incubation period, air flow is 2.5L/min, ensures that in fermenting case, relative humidity, more than 80%, stirred a subculture every 2 ~ 3 days during the fermentation.
When temperature is lower than 22 DEG C, trichoderma harziarum poor growth; When temperature is higher than 29 DEG C, the substratum in fermenting case can degenerate fouling, causes fermenting unsuccessfully.Fermentation relative humidity lower than 80% time, substratum easily becomes dry, and substratum exsiccation secondary fermentation is only produced mycelia and do not produced spore.When fermentation time was lower than 6 days, trichoderma harziarum not yet all produces conidium; Fermentation time 6 ~ 9 days is the conidiiferous active period of trichoderma harziarum; When fermentation time was more than 9 days, substratum no longer produces conidium.
Present invention also offers and utilize aforesaid method to produce the trichoderma harziarum conidium obtained.
Present invention also offers a kind of containing above-mentioned conidial bacteria agent.
This microbial inoculum is mainly used in becoming thoroughly decomposed of stalk.Major microorganisms is trichoderma harziarum, subtilis.Contents of Main Components is wherein: trichoderma harziarum 1.05 hundred million/g, aspergillus niger 2.0 hundred million/g, aspergillus oryzae 2.0 hundred million/g, subtilis 5.0 hundred million/g, Bacillus licheniformis 5.0 hundred million/g.
The present invention, to the fermentation process of trichoderma harziarum, can produce a large amount of conidiums, and matrix spore amount reaches as high as 4.89*10
9cfu/g, fermentation unit is high, and production process does not produce waste material, whole process zero release, environment friendly and pollution-free.Spore-the substrate mixture obtained that ferments can be directly used in production after drying, and the conidium of producing can be used for the biological and ecological methods to prevent plant disease, pests, and erosion of farm crop and becoming thoroughly decomposed of stalk.
Accompanying drawing explanation
Fig. 1 is the trichoderma harziarum that fermention medium of the present invention grows;
Fig. 2 is that the method for the invention produces the conidial powder obtained;
Fig. 3 is that the method for the invention produces the conidium microscopy figure obtained;
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment 1 fermentative production conidium
1, the making method of liquid seeds
Be that ACCC31707 bacterial classification activates on PDA test tube slant by the deposit number purchased from Chinese agriculture Microbiological Culture Collection administrative center, be then forwarded to after activated in the eggplant-shape bottle of PDA and cultivate, culture temperature 28 DEG C, incubation time 4 days.
Preparation trichoderma harziarum inclined-plane seed liquid activation medium, medium component is by massfraction: ammonium sulphate content is 1.5%, glucose content is 1.2%, tween 80 content is 0.2%, surplus is water.Join substratum 250mL according to the above ratio, 121 DEG C of sterilizings 15 minutes, scrape cultured eggplant bottle inclined-plane lawn after cooling and pour in this liquid nutrient medium in Bechtop, and cultivate 18 hours in constant-temperature table, culture temperature is 28 DEG C.
Detecting suspension colony number is 1.62*10
8cfu/mL.Detection method is for adopting gradient dilution method, and detect with coating method, detection substratum is PDA substratum.
2, triangular flask solid seed method of making
Press wheat bran 4.8Kg, bean cake powder 1.2Kg, the proportions solid seed culture medium of inorganic ion solution 4Kg.The composition of inorganic ion solution is: ammonium sulfate 8g, potassium primary phosphate 8g, crystalline sulfuric acid magnesium 16g, manganous sulfate 0.8g, ferrous sulfate 2.4g, glucose 40g, surplus is water.Divide after preparing substratum and be filled to triangular flask, the loading amount of each triangular flask is 90g solid seed culture medium.Be stoppered parchment with tampon and tie up sterilising treatment after openning, temperature 121 DEG C, sterilizing 25 minutes.
After the cooling of solid seed culture medium, the liquid seeds that inoculation culture is good in Bechtop, inoculum size is 10mL, puts into constant incubator and cultivate after vibration mixes, and fluorescent lamp irradiates lower cultivation, and culture temperature is 24 DEG C.Incubation time is 4 days
3, solid fermentation produces spore
Press wheat bran 4Kg, bean cake powder 1Kg, the good solid fermentation substratum of proportional arrangement of inorganic ion solution 5Kg.The composition of inorganic ion solution is ammonium sulfate 62.5g, SODIUM PHOSPHATE, MONOBASIC 20g, Sodium phosphate dibasic 20g, potassium primary phosphate 12.5g, magnesium sulfate 27.5g, manganous sulfate 27.5g, sucrose 75g, glucose 50g, and surplus is water.Divide after preparing and be filled to flour bag sterilizing, the loading amount of every bag is about 3.3Kg, and sterilising conditions is 121 DEG C, sterilization time 30 minutes.
Air filter, air line, fermenting case are carried out vapor sterilization, fermentation stainless steel plate pressure kettle sterilising treatment, sterilising conditions is 121 DEG C, sterilization time 15 minutes.And by Peracetic Acid to environment disinfection.
The solid seed that inoculation culture is good in Bechtop, stainless steel plate dress substratum 1Kg/ dish, the solid seed 100g that inoculation culture is good, puts into fermentation culture case after seed and solid fermentation substratum being mixed cultivate with scoop, and fluorescent lamp irradiates lower cultivation, culture temperature is 25 DEG C, pass into sterile air, intake is 2.5L/min, and in adjustment fermenting case, relative humidity is more than 80%, stirred a subculture every 2 ~ 3 days during the fermentation, incubation time is 7 days.
4, aftertreatment
Cultivation terminates fermented substrate air blast heat-circulation oven dry, and drying temperature is 35 DEG C, and namely obtain trichoderma harziarum conidial powder (as shown in Figure 1) after drying and crushing, carry out microscopy to conidium, microscopy figure as shown in Figure 2.Detect trichoderma harziarum conidium amount and reach 4.89*10
9cfu/g.Detection method is coating method, and detection substratum used is PDA substratum.
Embodiment 2 fermentative production conidium
1, the making method of liquid seeds
Be that ACCC31707 bacterial classification activates on PDA test tube slant by the deposit number purchased from Chinese agriculture Microbiological Culture Collection administrative center, be then forwarded to after activated in the eggplant-shape bottle of PDA and cultivate, culture temperature 28 DEG C, incubation time 4 days.
Preparation trichoderma harziarum inclined-plane seed liquid activation medium, medium component is by massfraction: ammonium sulphate content is 1.5%, glucose content is 1%, tween 80 content is 0.1%, surplus is water.Join substratum 250mL according to the above ratio, 121 DEG C of sterilizings 15 minutes, scrape cultured eggplant bottle inclined-plane lawn after cooling and pour in this liquid nutrient medium in Bechtop, and cultivate 18 hours in constant-temperature table, culture temperature is 24 DEG C.
Detecting suspension colony number is 1.2*10
8cfu/mL.Detection method is for adopting gradient dilution method, and detect with coating method, detection substratum is PDA substratum.
2, triangular flask solid seed method of making
Press wheat bran 4.8Kg, bean cake powder 1.2Kg, the proportions solid seed culture medium of inorganic ion solution 4Kg.The composition of inorganic ion solution is: ammonium sulfate 8g, potassium primary phosphate 8g, magnesium sulfate 16g, manganous sulfate 0.8g, ferrous sulfate 2.4g, glucose 40g, and surplus is water.Divide after preparing substratum and be filled to triangular flask, the loading amount of each triangular flask is 90g solid seed culture medium.Be stoppered parchment with tampon and tie up sterilising treatment after openning, temperature 121 DEG C, sterilizing 25 minutes.
After the cooling of solid seed culture medium, the liquid seeds that inoculation culture is good in Bechtop, inoculum size is 8mL, puts into constant incubator and cultivate after vibration mixes, and fluorescent lamp irradiates lower cultivation, and culture temperature is 24 DEG C.Incubation time is 4 days, and be advisable just to have overgrowed with white hypha in triangular flask, mycelial growth should not be excessively of a specified duration, crosses and then can produce spore for a long time, be unfavorable for trichoderma harziarum quick growth and breeding in tray.
3, solid fermentation produces spore
Press wheat bran 3Kg, bean cake powder 0.75Kg, the good solid fermentation substratum of proportional arrangement of inorganic ion solution 6.25Kg.The composition of inorganic ion solution is ammonium sulfate 31.25g, SODIUM PHOSPHATE, MONOBASIC 3.12g, Sodium phosphate dibasic 3.12g, potassium primary phosphate 3.12g, magnesium sulfate 6.25g, manganous sulfate 6.25g, sucrose 31.25g, glucose 3.12g, and surplus is water.Divide after preparing and be filled to flour bag sterilizing, the loading amount of every bag is about 3.3Kg, and sterilising conditions is 121 DEG C, sterilization time 30 minutes.
Air filter, air line, fermenting case are carried out vapor sterilization, fermentation stainless steel plate pressure kettle sterilising treatment, sterilising conditions is 121 DEG C, sterilization time 15 minutes.And by Peracetic Acid to environment disinfection.
The solid seed that inoculation culture is good in Bechtop, stainless steel plate dress substratum 1Kg/ dish, the solid seed 50g that inoculation culture is good, puts into fermentation culture case after seed and solid fermentation substratum being mixed cultivate with scoop, and fluorescent lamp irradiates lower cultivation, culture temperature is 24 DEG C, pass into sterile air, intake is 2.5L/min, and in adjustment fermenting case, relative humidity is more than 80%, stirred a subculture every 2 ~ 3 days during the fermentation, incubation time is 7 days.
4, aftertreatment
Cultivation terminates fermented substrate air blast heat-circulation oven dry, and drying temperature is 35 DEG C, namely obtains trichoderma harziarum conidial powder after drying and crushing, detects trichoderma harziarum conidium amount and reaches 2.65*10
9cfu/g.Detection method is coating method, and detection substratum used is PDA substratum.Embodiment 3 fermentative production conidium
1, the making method of liquid seeds
Be that ACCC31707 bacterial classification activates on PDA test tube slant by the deposit number purchased from Chinese agriculture Microbiological Culture Collection administrative center, be then forwarded to after activated in the eggplant-shape bottle of PDA and cultivate, culture temperature 28 DEG C, incubation time 4 days.
Preparation trichoderma harziarum inclined-plane seed liquid activation medium, medium component is by massfraction: ammonium sulphate content is 1.5%, glucose content is 1.2%, tween 80 content is 0.2%, surplus is water.Join substratum 250mL according to the above ratio, 121 DEG C of sterilizings 15 minutes, scrape cultured eggplant bottle inclined-plane lawn after cooling and pour in this liquid nutrient medium in Bechtop, and cultivate 18 hours in constant-temperature table, culture temperature is 28 DEG C.
Detecting suspension colony number is 1.62*10
8cfu/mL.Detection method is for adopting gradient dilution method, and detect with coating method, detection substratum is PDA substratum.
2, triangular flask solid seed method of making
Press wheat bran 4.8Kg, bean cake powder 1.2Kg, the proportions solid seed culture medium of inorganic ion solution 4Kg.The composition of inorganic ion solution is: ammonium sulfate 8g, potassium primary phosphate 8g, crystalline sulfuric acid magnesium 16g, manganous sulfate 0.8g, ferrous sulfate 2.4g, glucose 40g, surplus is water.Divide after preparing substratum and be filled to triangular flask, the loading amount of each triangular flask is 90g solid seed culture medium.Be stoppered parchment with tampon and tie up sterilising treatment after openning, temperature 121 DEG C, sterilizing 25 minutes.
After the cooling of solid seed culture medium, the liquid seeds that inoculation culture is good in Bechtop, inoculum size is 10mL, puts into constant incubator and cultivate after vibration mixes, and fluorescent lamp irradiates lower cultivation, and culture temperature is 24 DEG C.Incubation time is 4 days
3, solid fermentation produces spore
Press wheat bran 4.5Kg, bean cake powder 1.5Kg, the good solid fermentation substratum of proportions of inorganic ion solution 4Kg.The composition of inorganic ion solution is ammonium sulfate 80g, SODIUM PHOSPHATE, MONOBASIC 32g, Sodium phosphate dibasic 32g, potassium primary phosphate 20g, magnesium sulfate 40g, manganous sulfate 40g, sucrose 100g, glucose 80g, and surplus is water.Divide after preparing and be filled to flour bag sterilizing, the loading amount of every bag is about 3.3Kg, and sterilising conditions is 121 DEG C, sterilization time 30 minutes.
Air filter, air line, fermenting case are carried out vapor sterilization, fermentation stainless steel plate pressure kettle sterilising treatment, sterilising conditions is 121 DEG C, sterilization time 15 minutes.And by Peracetic Acid to environment disinfection.
The solid seed that inoculation culture is good in Bechtop, stainless steel plate dress substratum 1Kg/ dish, the solid seed 100g that inoculation culture is good, put into fermentation culture case after seed and solid fermentation substratum being mixed with scoop to cultivate, daylight etc. irradiate lower cultivation, and culture temperature is 25 DEG C, pass into sterile air 2.5L/min, in adjustment fermenting case, relative humidity is more than 80%, and stirred a subculture every 2 ~ 3 days during the fermentation, incubation time is 8 days.
4, aftertreatment
Cultivation terminates fermented substrate air blast heat-circulation oven dry, and drying temperature is 35 DEG C, namely obtains trichoderma harziarum conidial powder after drying and crushing, detects trichoderma harziarum conidium amount and reaches 1.83*10
9cfu/g.Detection method is coating method, and detection substratum used is PDA substratum.Embodiment 4 fermentative production conidium
1, the making method of liquid seeds
Be that ACCC31707 bacterial classification activates on PDA test tube slant by the deposit number purchased from Chinese agriculture Microbiological Culture Collection administrative center, be then forwarded to after activated in the eggplant-shape bottle of PDA and cultivate, culture temperature 28 DEG C, incubation time 4 days.
Preparation trichoderma harziarum inclined-plane seed liquid activation medium, medium component is by massfraction: ammonium sulphate content is 1.5%, glucose content is 1.2%, tween 80 content is 0.2%, surplus is water.Join substratum 250mL according to the above ratio, 121 DEG C of sterilizings 15 minutes, scrape cultured eggplant bottle inclined-plane lawn after cooling and pour in this liquid nutrient medium in Bechtop, and cultivate 18 hours in constant-temperature table, culture temperature is 28 DEG C.
Detecting suspension colony number is 1.62*10
8cfu/mL.Detection method is for adopting gradient dilution method, and detect with coating method, detection substratum is PDA substratum.
2, triangular flask solid seed method of making
Press wheat bran 4.8Kg, bean cake powder 1.2Kg, the proportions solid seed culture medium of inorganic ion solution 4Kg.The composition of inorganic ion solution is: ammonium sulfate 8g, potassium primary phosphate 8g, crystalline sulfuric acid magnesium 16g, manganous sulfate 0.8g, ferrous sulfate 2.4g, glucose 40g, surplus is water.Divide after preparing substratum and be filled to triangular flask, the loading amount of each triangular flask is 90g solid seed culture medium.Be stoppered parchment with tampon and tie up sterilising treatment after openning, temperature 121 DEG C, sterilizing 25 minutes.
After the cooling of solid seed culture medium, the liquid seeds that inoculation culture is good in Bechtop, inoculum size is 10mL, puts into constant incubator and cultivate after vibration mixes, and fluorescent lamp irradiates lower cultivation, and culture temperature is 24 DEG C.Incubation time is 4 days
3, solid fermentation produces spore
Press wheat bran 4.2Kg, bean cake powder 1.2Kg, the proportions solid seed culture medium of inorganic ion solution 5.6Kg.The composition of inorganic ion solution is: the composition of inorganic ion solution is ammonium sulfate 78.4g, SODIUM PHOSPHATE, MONOBASIC 28g, Sodium phosphate dibasic 28g, potassium primary phosphate 22.4g, magnesium sulfate 44.8g, manganous sulfate 44.8g, sucrose 100.8g, glucose 67.2g, and surplus is water.Divide after preparing and be filled to flour bag sterilizing, the loading amount of every bag is about 3.3Kg, and sterilising conditions is 121 DEG C, sterilization time 30 minutes.
Air filter, air line, fermenting case are carried out vapor sterilization, fermentation stainless steel plate pressure kettle sterilising treatment, sterilising conditions is 121 DEG C, sterilization time 15 minutes.And by Peracetic Acid to environment disinfection.
The solid seed that inoculation culture is good in Bechtop, stainless steel plate dress substratum 1Kg/ dish, the solid seed 100g that inoculation culture is good, puts into fermentation culture case after seed and solid fermentation substratum being mixed cultivate with scoop, and fluorescent lamp irradiates lower cultivation, culture temperature is 22 DEG C, pass into sterile air, intake is 2.5L/min, and in adjustment fermenting case, relative humidity is more than 80%, stirred a subculture every 2 ~ 3 days during the fermentation, incubation time is 9 days.
4, aftertreatment
Cultivation terminates fermented substrate air blast heat-circulation oven dry, and drying temperature is 35 DEG C, namely obtains trichoderma harziarum conidial powder after drying and crushing, detects trichoderma harziarum conidium amount and reaches 3.5*10
9cfu/g.Detection method is coating method, and detection substratum used is PDA substratum.
Embodiment 5 fermentative production conidium
1, the making method of liquid seeds
Be that ACCC31707 bacterial classification activates on PDA test tube slant by the deposit number purchased from Chinese agriculture Microbiological Culture Collection administrative center, be then forwarded to after activated in the eggplant-shape bottle of PDA and cultivate, culture temperature 28 DEG C, incubation time 4 days.
Preparation trichoderma harziarum inclined-plane seed liquid activation medium, medium component is by massfraction: ammonium sulphate content is 1.5%, glucose content is 1.2%, tween 80 content is 0.2%, surplus is water.Join substratum 250mL according to the above ratio, 121 DEG C of sterilizings 15 minutes, scrape cultured eggplant bottle inclined-plane lawn after cooling and pour in this liquid nutrient medium in Bechtop, and cultivate 18 hours in constant-temperature table, culture temperature is 28 DEG C.
Detecting suspension colony number is 1.62*10
8cfu/mL.Detection method is for adopting gradient dilution method, and detect with coating method, detection substratum is PDA substratum.
2, triangular flask solid seed method of making
Press wheat bran 4.8Kg, bean cake powder 1.2Kg, the proportions solid seed culture medium of inorganic ion solution 4Kg.The composition of inorganic ion solution is: ammonium sulfate 8g, potassium primary phosphate 8g, crystalline sulfuric acid magnesium 16g, manganous sulfate 0.8g, ferrous sulfate 2.4g, glucose 40g, surplus is water.Divide after preparing substratum and be filled to triangular flask, the loading amount of each triangular flask is 90g solid seed culture medium.Be stoppered parchment with tampon and tie up sterilising treatment after openning, temperature 121 DEG C, sterilizing 25 minutes.
After the cooling of solid seed culture medium, the liquid seeds that inoculation culture is good in Bechtop, inoculum size is 10mL, puts into constant incubator and cultivate after vibration mixes, and fluorescent lamp irradiates lower cultivation, and culture temperature is 24 DEG C.Incubation time is 4 days
3, solid fermentation produces spore
Press wheat bran 3.8Kg, bean cake powder 0.8Kg, the good solid fermentation substratum of proportions of inorganic ion solution 4.6Kg.The composition of inorganic ion solution is ammonium sulfate 36.8g, SODIUM PHOSPHATE, MONOBASIC 4.6g, Sodium phosphate dibasic 4.6g, potassium primary phosphate 4.6g, magnesium sulfate 9.2g, manganous sulfate 9.2g, sucrose 46g, glucose 4.6g, and surplus is water.Divide after preparing and be filled to flour bag sterilizing, the loading amount of every bag is about 3.3Kg, and sterilising conditions is 121 DEG C, sterilization time 30 minutes.
Air filter, air line, fermenting case are carried out vapor sterilization, fermentation stainless steel plate pressure kettle sterilising treatment, sterilising conditions is 121 DEG C, sterilization time 15 minutes.And by Peracetic Acid to environment disinfection.
The solid seed that inoculation culture is good in Bechtop, stainless steel plate dress substratum 1Kg/ dish, the solid seed 100g that inoculation culture is good, puts into fermentation culture case after seed and solid fermentation substratum being mixed cultivate with scoop, and fluorescent lamp irradiates lower cultivation, culture temperature is 29 DEG C, pass into sterile air, intake is 2.5L/min, and in adjustment fermenting case, relative humidity is more than 80%, stirred a subculture every 2 ~ 3 days during the fermentation, incubation time is 6 days.
4, aftertreatment
Cultivation terminates fermented substrate air blast heat-circulation oven dry, and drying temperature is 35 DEG C, namely obtains trichoderma harziarum conidial powder after drying and crushing, detects trichoderma harziarum conidium amount and reaches 3.8*10
9cfu/g.Detection method is coating method, and detection substratum used is PDA substratum.
Embodiment 6 fermentative production conidium (contrast experiment)
For advantage of the present invention is described, select domesticly to publish more advanced method and compare explanation.The present embodiment be current domestic announcement be suitable for the conidial fermentation process of Workshop Production trichoderma harziarum, China Patent Publication No. is CN101748070A.
Concrete steps are as follows:
1, slant strains activation
Adopt solid PDA medium, ACCC30317 bacterial strain two generation seed is seeded on test-tube culture medium, cultivate 4 days for 28 DEG C.
2, the preparation of seed fermentation liquid
The composition of liquid fermentation and culture liquid is: whole wheat flour 2%, glucose 0.5%, yeast extract paste 0.2%, CaCO
31.0%, KH
2pO
40.4%, MgSO
47H
2o, pH7.0.Adopt the bottled nutrient solution of triangle of 500mL, liquid amount is 200mL.Adopt High Temperature High Pressure 121 DEG C, the sterilizing of 20min condition.Aseptically inoculate trichoderma harziarum inclined-plane seed to shaking flask.Be placed in double-deck low capacity shaking table to cultivate, culture temperature is 26 DEG C, 200rmp, and open shaking table fluorescent lamp, incubation time is 5 days.
3, solid fermentation is cultivated
In compound sugarcane sheet slag: Semen Maydis powder: soybean cake powder ratio is the proportions solid medium 4kg of 93:6:2, and add water 6kg, mixing.Point be filled to flour bag sterilizing after preparing, the loading amount of every bag is about 3.3Kg, sterilising conditions be 0.13 ~ 0.17MPa high pressure, 121 DEG C, sterilization time 100 minutes.
Air filter, air line, fermenting case are carried out vapor sterilization, fermentation stainless steel plate pressure kettle sterilising treatment, sterilising conditions is 121 DEG C, sterilization time 15 minutes.And by Peracetic Acid to environment disinfection.
The liquid seeds that inoculation culture is good in Bechtop, stainless steel plate dress substratum 1Kg/ dish, the liquid seeds 100g that inoculation culture is good, put into fermentation culture case after liquid seeds and solid fermentation substratum being mixed with scoop to cultivate, cultivate under sun exposure, culture temperature is 25 DEG C, pass into sterile air 2.5L/min, in adjustment fermenting case, relative humidity is more than 80%, and stirred a subculture every 2 ~ 3 days during the fermentation, incubation time is 8 days.
4, aftertreatment
Cultivation terminates, by fermented substrate natural air drying, namely to obtain trichoderma harziarum conidial powder after drying and crushing, detects trichoderma harziarum conidium amount and reaches 8.6*10
8cfu/g.Detection method is coating method, and detection substratum used is PDA substratum.
Relatively can be found out by embodiment, the method of the invention improves an order of magnitude than the conidium amount quantity that disclosed trichoderma harziarum fermentation product conidium method is cultivated at present, be more suitable for scale operation trichoderma harziarum conidium, can economies of scale be produced.
Embodiment 7 is containing the preparation of conidium microbial inoculum
Present invention also offers a kind of containing above-mentioned conidial a kind of biotechnological formulation.This biotechnological formulation is soluble straw decomposing agent, its, and this biotechnological formulation is constructed as follows by weight: composite fungus agent 20 parts, nutritive ingredient 50 parts, plant-sourced emulsification permeate agent 5 parts, soluble carrier 25 parts.Described composite fungus agent is constructed as follows: trichoderma harziarum 1.05 hundred million/g, aspergillus niger 2.0 hundred million/g, aspergillus oryzae 2.0 hundred million/g, subtilis 5.0 hundred million/g and Bacillus licheniformis 5.0 hundred million/g.
Described composite fungus agent is obtained by following steps:
The preparation of bacterium mixing spore powder:
1) slant culture: by subtilis and Bacillus licheniformis, original strain is aseptically inoculated on slant medium respectively, cultivates 48 hours under 28 ± 2 DEG C of conditions;
2) shaking table is cultivated: by above-mentioned steps 1) bacterial classification cultivated aseptically is inoculated in liquid nutrient medium respectively, and under pH6.5, temperature are 30 DEG C of conditions, 160r/min shaking table cultivates 36 hours;
3) fermentor cultivation: by above-mentioned steps 2) bacterial classification cultivated aseptically is inoculated in fermentation tank culture medium respectively, pH8.0, tank pressure 0.5kg, temperature be 30 DEG C, under ventilation 1:0.8-1.1 condition, cultivate after 56 hours, bacterium number is greater than 1.0 × 10
10cfu/mL, 80% thalline changes into gemma tank at present, obtains fermented liquid;
4) by the fermented liquid that obtains in step 3) by etc. weight mixing, be prepared into bacterium mixing spore powder through concentrate drying;
In the preparation of above-mentioned bacterium mixing spore powder, the formula of the slant medium selected in step 1) is as follows: glucose 15g, fish peptone 5g, yeast extract paste 5g, water 1000mL, agar 15g.Above-mentioned steps 2) in liquid culture based formulas as follows: glucose 10g, extractum carnis 5g, yeast powder 5g, starch 10g, soybean cake powder 5g, K
2hPO
40.5g, MgSO
40.2g, water 1000mL.Above-mentioned steps 3) in fermentor cultivation based formulas as follows: Semen Maydis powder 26kg, soybean cake powder 16kg, ammonium sulfate 4kg, glucose 8kg, yeast powder 2.5kg, peptone 1.7kg, add water to 600kg.
The preparation of fungi mixing spore powder:
1) aspergillus oryzae, aspergillus niger, original strain is aseptically inoculated on slant medium respectively, cultivates 48 hours under 28 ± 2 DEG C of conditions;
2) shaking table is cultivated: by above-mentioned steps 1) bacterial classification cultivated aseptically is inoculated in liquid nutrient medium respectively, and under pH6.6, temperature are 30 DEG C of conditions, 160r/min shaking table cultivates 24 hours;
3) fermentor cultivation: by above-mentioned steps 2) bacterial classification cultivated aseptically is inoculated in fermentation tank culture medium respectively, pH6.6, tank pressure 0.5kg, temperature be 28 DEG C, ventilation is 1:0.6, after cultivation 48h, mycelium stops fermentation when accounting for 20% of cumulative volume, carries out solid fermentation and produces spore;
4) solid fermentation produce spore: by above-mentioned steps 3) by fermentation tank cultivate after mycelium be inoculated on solid fermentation substratum, cultivate 48h, 90% produce spore;
5) production method of trichoderma harziarum is undertaken by method described in present patent application;
6) by above-mentioned steps 4), 5) in produce spore completely substratum be immersed in clear water respectively and make spore suspension, thus obtain trichoderma harziarum spore suspension, aspergillus oryzae spore suspension, aspergillus niger spore suspension;
7) by above-mentioned steps 5) in obtain trichoderma harziarum spore suspension, aspergillus niger spore suspension, aspergillus oryzae spore suspension respectively concentrate drying be prepared into fungi mixing spore powder; Then, fungi mixing spore powder is hybridly prepared into according to 1: 1: 1 ratio.
In the preparation of above-mentioned fungi mixing spore powder, the formula of the slant medium selected in step 1) is as follows: glucose 20g, murphy juice 200g, agar 20g, water 1000mL, pH nature.Above-mentioned steps 2) in liquid culture based formulas as follows: white sugar 20g, yeast extract paste 0.5g.Starch 20g, potassium primary phosphate 0.5g, magnesium sulfate 0.2g, sodium-chlor 0.2g, water 1000mL.Above-mentioned steps 3) in fermentor cultivation based formulas as follows: starch 12kg, soybean cake powder 1.2kg, Semen Maydis powder 3kg, white sugar 12kg, yeast extract paste 0.3kg, magnesium sulfate 0.12kg, sodium-chlor 0.12kg, add water to 600kg.Above-mentioned steps 4) in solid fermentation culture medium prescription as follows: solid material: cotton seed hulls 25kg, Semen Maydis powder 10kg, wheat bran 65kg.The weight ratio of solid material and water is 1:0.8.Above-mentioned obtained bacterium mixing spore powder and fungi mixing spore powder are mixed according to the ratio of 4:1, thus prepares composite fungus agent.
Described emulsifying agent is plant-sourced tensio-active agent, and described plant-sourced tensio-active agent is Oil-tea-cake extractive substance.
Described nutritive ingredient prepares by the following method: by white sugar, brown sugar (Saccharum Sinensis Roxb.), starch, protein powder, iron, boron, zinc, copper, manganese and molybdenum with amino acid or humic acid chelating, mix obtained according to the quality proportioning of 1:2.8:5:1:0.2.
Described amino acid and or the iron of humic acid chelating, boron, zinc, copper, manganese and molybdenum purchased from Chengdu chelating Bioisystech Co., Ltd.Name of product is chelated microelement.Wherein iron, boron, zinc, copper, manganese, molybdenum are amino-acid chelate.Chelated microelements total content (iron+boron+zinc+copper+manganese+molybdenum) >=8%.
Described soluble carrier is Zulkovsky starch.
Embodiment 8 is become thoroughly decomposed the effect experimental of stalk
1, test objective:
The effect that checking is degraded to straw decomposing to the Degradation of maize straw and trichoderma harziarum containing the conidial microbial inoculum of trichoderma harziarum.
2, test materials:
A kind of soluble straw decomposing agent, its prepared by embodiment 7;
By a kind of soluble straw decomposing agent, its not adding trichoderma harziarum prepared by embodiment 7 method;
Stalk: maize straw (pulverizing);
Urea: general application urea;
Water: tap water.
3, experimental design:
1) test site: greenhouse.Room temp controls: 22 DEG C-25 DEG C, medial humidity: 60%.
2) straw degradative test arranges 4 process:
Process 1: stalk 220g+ soluble straw decomposing agent, its+urea+water
Process 2: stalk 220g+ does not add a kind of soluble straw decomposing agent, its+urea+water of trichoderma harziarum
Process 3: stalk 220g+ urea+water
Process 4: stalk 220g+ water
4, test method:
1) bacterium liquid or solution is prepared:
Process 1: in recommendation ratio (a kind of soluble straw decomposing agent, its: water: urea=150: 45000: 4000) (weight ratio) prepares bacterium liquid.
Process 2: (do not add a kind of soluble straw decomposing agent, its of trichoderma harziarum: water: urea=150: 45000: 4000) (weight ratio) prepares bacterium liquid in recommendation ratio.
Process 3: do not add a kind of soluble straw decomposing agent, its, in recommendation ratio (water: urea=45000: 4000) (weight ratio) obtain solution.
Process 4: only add water.
2) maize straw is filled basin by each process 220g, calculate by recommendation amount, process 1 sprays bacterium liquid 16.5ml, and process 2 sprays urea soln 16.5ml, and process 3 sprays urea soln 16.5ml, and process 4 sprays water 16.5ml, is closed by stalk with soil.
3) spray water every day, keep stalk humidity, be beneficial to microorganism growth and straw decomposing, each Treating straw injection flow rate is basically identical, keeps water content to be 60%.
4) within every three days, observe a straw decomposition degree, project comprises: color, feel, stretching resistance.
5) each Treating straw is dried, take quality, calculate decomposition rate, compare the decomposition speed of each process.
5, test-results:
1) color, feel and stretching resistance change: the straw decomposition changing conditions comparing 3 process from color, feel and stretching resistance aspect, through a kind of straw decomposition Be very effective of soluble straw decomposing agent, its process after two weeks, stalk color blackening in process 1, grabbed rotten to the corn sense with hand, stretching resistance is on duty mutually.The decomposition speed of process 1 comparatively processes 2, process 3 and process 4 fast, check through on-the-spot naked eyes find that process 1 degree of becoming thoroughly decomposed comparatively processes 2, process 3 and process 4 large.
2) decomposition rate: decomposition rate is the ratio that the weight of rotten sample accounts for gross weight.Through calculating, within 15 days, aftertreatment 1 decomposition rate is 94.5%, is 66.2% than the process 2(rate of becoming thoroughly decomposed) exceed 28.3%, be 38.3% than process 3(decomposition rate) exceed 56.2%, be 33.8% than process 4(decomposition rate) exceed 60.7%.
6, conclusion:
A kind of soluble straw decomposing agent, its of this description of test has to maize straw effect of becoming thoroughly decomposed very significantly, illustrates that trichoderma harziarum plays important effect in this microbial inoculum simultaneously.Concrete reason is that maize straw forms primarily of Mierocrystalline cellulose, hemicellulose and xylogen.Mierocrystalline cellulose, hemicellulose, xylogen form a complicated structure, and wherein the encapsulation action of xylogen is sternly levied and hampered cellulase to cellulosic effect.And trichoderma harziarum can produce the materials such as laccase to the xylogen in stalk and decomposes, and then the cellulase that the cellulosic material be conducive to inside by stalk comes out directly and other microorganisms produce reacts, thus accelerates the progress of straw decomposing.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some amendments to it or improve, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (3)
1. a fermention medium, is characterized in that, described substratum is made up of the material of following weight part: 4 parts, wheat bran, bean cake powder 1 part, inorganic ion solution 5 parts; Wherein, the Ingredient percent of described inorganic ion solution is: ammonium sulfate 1.25%, SODIUM PHOSPHATE, MONOBASIC 0.4%, Sodium phosphate dibasic 0.4%, dipotassium hydrogen phosphate 0.25%, magnesium sulfate 0.55%, manganous sulfate 0.55%, sucrose 1.5%, glucose 1%, and surplus is water.
2. the application of fermention medium according to claim 1 in trichoderma harziarum fermentative production conidium.
3. a trichoderma harziarum solid fermentation produces conidial method, comprise the preparation of slant strains, the preparation of ferment-seeded and the preparation of fermention medium and fermentating controling process step, it is characterized in that: with fermention medium according to claim 1 for substratum, be placed in fermentation culture case and cultivate under fluorescent lamp irradiates, culture temperature is 22 DEG C ~ 29 DEG C, incubation time is 6 ~ 9 days, sterile air is passed between incubation period, air flow is 2.5L/min, ensures that in fermenting case, relative humidity is more than 80%.
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| CN105802863B (en) * | 2016-06-01 | 2020-01-10 | 东北农业大学 | Trichoderma harzianum fermentation process, wettable powder and application thereof |
| CN106635824B (en) * | 2016-11-01 | 2019-08-06 | 东北林业大学 | A kind of trichoderma harzianum process for solid state fermentation |
| CN110885761A (en) * | 2019-11-25 | 2020-03-17 | 中国林业科学研究院林产化学工业研究所 | Trichoderma harzianum solid state fermentation substrate and culture method thereof |
| CN114214210A (en) * | 2021-12-27 | 2022-03-22 | 中国农业大学 | Method for rapidly preparing maize puccinia polycephala bacterial liquid in batches |
| CN114395520A (en) * | 2022-02-14 | 2022-04-26 | 山东省农业科学院 | Method for efficiently producing conidia in large scale by solid state fermentation of trichoderma harzianum |
| CN118667743A (en) * | 2024-08-01 | 2024-09-20 | 深圳诺普信作物科学股份有限公司 | Production method of trichoderma harzianum spore powder |
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