CN103409474A - Method for producing abscisic acid by solid state fermentation of fungi - Google Patents
Method for producing abscisic acid by solid state fermentation of fungi Download PDFInfo
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- CN103409474A CN103409474A CN2013102888840A CN201310288884A CN103409474A CN 103409474 A CN103409474 A CN 103409474A CN 2013102888840 A CN2013102888840 A CN 2013102888840A CN 201310288884 A CN201310288884 A CN 201310288884A CN 103409474 A CN103409474 A CN 103409474A
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- dormin
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Abstract
The invention discloses a method for producing abscisic acid by solid state fermentation of fungi, which specifically comprises the following steps: screening the mutagenic botrytis cinerea as a production strain, and performing activation and amplification to form a plate solid-state strain for use; preparing a fermentation medium; grinding the prepared plate solid-state strain, and uniformly dispersing into a sterilized culture tray for periodic fermentation culture; and purifying the fermentation product to obtain natural abscisic acid. Compared with a liquid-state fermentation method, the method disclosed by the invention has the advantages of high fermentation level, small investment, low contamination rate, no organic waste liquid and the like; and the average fermentation level of the abscisic acid can reach 2,934mg/kg, namely that each kilogram of medium contains 2.934g of abscisic acid, which is the highest fermentation level under the existing known industrial level.
Description
Technical field
The present invention relates to a kind of production method of dormin, particularly a kind of fungi solid state fermentation is produced the method for dormin.Background technology
Dormin (Abscisic Acid, be abbreviated as ABA) is one of plant five large spontaneous growth conditioning agents.The production cost of simple natural active dormin (+)-ABA is high.Due to expensive price and the difference on activity, dormin is not widely used in the middle of agriculture production always.
Existing dormin production method comprises: (1) plant extraction method (2) chemical synthesis (3) fungi liquid fermentation method.
Plant extraction method is that dormin is found the early stage method of purification of using.By series of steps such as enrichment, concentrated, extractions, extract the dormin in plant.Because the dormin that in plant, itself contains is extremely rare, cause the method to be applied in the middle of industrial production.
There are two kinds of optical configuration in dormin: (+)-ABA, (-)-ABA.In existing plant, only there is (+)-ABA, and only have (+)-ABA just to have physiologically active.The dormin of producing by chemical synthesis comprises two kinds of optical configuration, is the racemize dormin.With simple PBI 58, compare active low 1/3-1/2.
The fungi liquid fermentation method is popular PBI 58 production method of present stage.The method adopts Botrytis cinerea to carry out liquid state fermentation production.Botrytis cinerea is filamentous fungus, and its spontaneous growth environment is not liquid cultivation form.Therefore, liquid fermentation method can not be brought into play the highest fermentation capacity of this series fungi.Secondly, there are the problems such as huge, the easy microbiological contamination of investment, high expensive, discharging any waste liquor pollution in liquid fermentation method, and this is unfavorable for development and the application of whole dormin production industry.Particularly efflux high concentrated organic waste liquid, not only can cause enterprise to bear very large environmental protection pressure, the more important thing is ecotope is damaged.
Summary of the invention
The object of the present invention is to provide a kind of fungi solid state fermentation to produce the method for dormin.The present invention, by solid state fermentation production PBI 58, compares with liquid fermentation method, has that fermentation level is high, investment is little, the microbiological contamination rate is low, without advantages such as organic liquid wastes.In addition, utilize the method production PBI 58, very simple on purifying technique, can pass through conventional organism extracting method, can obtain the PBI 58 crystallization of ultra-high purity.This can advance industrialization and its application in agriculture production of whole PBI 58 industry to a great extent.
For achieving the above object, the invention provides following technical scheme:
A kind of fungi solid state fermentation is produced the method for dormin, and concrete steps are as follows:
(1) bacterial classification preparation
Screen the Botrytis cinerea of positive mutagenesis for producing bacterial classification, then, by activation and enlarged culturing, form the plate solid state bacterial, standby;
(2) fermention medium preparation
Fermention medium according to the raw material of weight percent is: millet 15-25%, rice 15-25%, glucose 0.1-2%, sucrose 0.1-4%, peptone 1-4%, CaCO
30.5-1.5%, Na
2HPO
412H
2O 0.1-0.5%, Ca (HO)
20.5-1.5%, MgSO
40.01-0.05%, purification residue 0.5-1%, other compositions are water; Wherein, the purification residue refers in the tunning leaching process and separates the concentrated non-dormin material obtained;
The making method of fermention medium: all raw materials are mixed, and poach, be placed in steam-cooking cabinet and carry out thermal pretreatment; Preheated substratum is broken up, and weighing divides to install to cultivates in tray, and every dish dress is got the 400-800g substratum; To cultivate tray and be placed in sterilizing cabinet, carry out autoclaving, standby;
(3) produce inoculation
Plate solid state bacterial prepared by step (1) pulverizes, and evenly spreads in the middle of the cultivation tray of step (2) after sterilizing; Each is cultivated tray and uses 2-4 plate bacterial classification; Inoculation weight is the 6-10% of substratum;
(4) fermentation culture
Vaccinated cultivation tray is moved into to sweathouse, be placed in the fermentation culture device and cultivate; Training mode is as follows:
The medial temperature of cultivating 0-48 hour be 26.5-28.5 ℃, average ventilation be the 5-10L/100 dish/day;
The medial temperature of cultivating 48-96 hour be 26-28 ℃, average ventilation be the 15-20L/100 dish/day;
The medial temperature of cultivating 96-216 hour be 22-24.5 ℃, average ventilation be the 40-50L/100 dish/day;
The medial temperature of cultivating 216-288 hour be 25.5-26.5 ℃, average ventilation be the 10-20L/100 dish/day;
(5) PBI 58 is purified: the tunning of step (4) is purified and obtained PBI 58.
As the further scheme of the present invention: in step (1) by natural screening, ultraviolet mutagenesis, ethyl sulfate mutagenesis, the laser radiation of YAG double frequency pulse and in producing dynamically screening obtain the Botrytis cinerea of positive mutagenesis.
As the present invention's scheme further: in described production, dynamically the concrete steps of screening are as follows:
1) bacterial classification collection: in daily production process, every batch of random 10-50 of selection coils the fermentation tray pollution-free, that growing way is good; From picking bacterial classification the fermentation tray, be placed in the PDA plate, 26-30 ℃ of static cultivation 5-7 days, make bacterial classification to be tested;
2) fermentation culture: cultivate in the middle of bacterial classification is inoculated in to the fermentation tray that contains substratum; Culture condition and daily production are consistent;
3) mutant strain detects: the ABA content in fermented product is detected, record the fermentation rate of bacterial classification to be tested; If the fermentation rate of bacterial classification to be tested, without considerable change, is given up it, corresponding tunning is incorporated to daily production, extracts wherein dormin; If positive mutant occurs, it is carried out to stability test;
4) positive mutant stability test: from choosing the 3-8 strain positive mutant, and measure its 3-8 strain fermentation rate in generation that goes down to posterity; Fermentation culture conditions and daily production are consistent; If bacterial classification, in obviously decline of interior appearance of 3-8 generation, is looked it and is passed through stability test;
5) positive mutant comes into operation: will put in production and application by the positive mutant of stability test, and continue above dynamically screening step.
As the further scheme of the present invention: step is carried out activation culture by culture medium A to Botrytis cinerea in (1), described culture medium A according to the raw material of weight percent is: potato 10-20%, agar 1-3%, glucose 0.1-1%, lactose 0.1-0.5%, sucrose 1-2%, Ca (NO3) 24H2O 0.01-0.05%, peptone 1-2%, Na2HPO412H2O 0.1-0.5%, other compositions are water.
As the further scheme of the present invention: in step (1), by substratum B, the botrytis sp bacterium of activation is carried out to enlarged culturing, substratum B is for solid-state inoculation; Described substratum B according to the raw material of weight percent is: millet 5-10%, rice 5-10%, glucose 0.1-1%, sucrose 1-2%, peptone 1-2%, Ca (NO
3)
24H
2O 0.01-0.05%, Na
2HPO
412H
2O 0.1-0.5%, agar 1-3%, other compositions are water; Described millet, rice need boiling, cross leaching juice.
As the further scheme of the present invention: the fermention medium described in step (2) according to the raw material of weight percent is: millet 20%, rice 20%, glucose 1%, sucrose 2%, peptone 2.5%, CaCO
31%, Na
2HPO
412H
2O 0.3%, Ca (HO)
21%, MgSO
40.03%, the purification residue 0.75%, and other compositions are water; In the making method of fermention medium, every dish dress is got the 600g substratum.
As the further scheme of the present invention: in step (3), each is cultivated tray and uses 3 plate bacterial classifications; Inoculation weight is 8% of substratum.
As the further scheme of the present invention: the training mode in step (4) is as follows:
The medial temperature of cultivating 0-48 hour be 27 ℃, average ventilation be the 7.5L/100 dish/day;
The medial temperature of cultivating 48-96 hour be 27 ℃, average ventilation be the 17.5L/100 dish/day;
The medial temperature of cultivating 96-216 hour be 23 ℃, average ventilation be the 45L/100 dish/day;
The medial temperature of cultivating 216-288 hour be 26 ℃, average ventilation be the 15L/100 dish/day.
As the present invention's scheme further: after cultivating 96 hours, will cultivate the tray left-hand thread, and can effectively reduce because air flow increases the substratum moisture loss brought, the water activity of assurance late stage of culture substratum.
Compared with prior art, the invention has the beneficial effects as follows:
1. liquid fermentation method is produced the known fermentation level of dormin and is up to 2.0g/L.And by the method, the average fermentation level of dormin can reach 2934mg/kg, namely in every kilogram of substratum, contains the 2.934g dormin.This is the highest fermentation level that present known industrial level is issued to.
2. utilize traditional organism method of purification, the rate of recovery of dormin can reach 90% in laboratory, can reach 83% in the middle of pilot scale.In pilot process, the PBI 58 yield of a whole set of technique is 2.4g/kg, and namely the per kilogram substratum can finally obtain PBI 58 (purity > 98.5%) 2.4g.
The accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, below will the accompanying drawing of required use in embodiment or description of the Prior Art be briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the process flow sheet that the fungi solid state fermentation is produced dormin.
Fig. 2 is the process flow sheet that PBI 58 is purified.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, rather than whole embodiment.Based on the embodiment in the present invention, those of ordinary skills, not making under the creative work prerequisite the every other embodiment obtained, belong to the scope of protection of the invention.
Embodiment 1
In the embodiment of the present invention, a kind of fungi solid state fermentation is produced the method for dormin, and concrete steps are as follows:
1. bacterial classification preparation
The production bacterial classification that the present invention uses is Botrytis cinerea Botrytis Cinerea, is by natural screening, ultraviolet mutagenesis, ethyl sulfate mutagenesis, the laser radiation of YAG double frequency pulse and dynamically screening acquisition in producing.Bacterial classification forms the plate solid state bacterial by activation, expansion.
1.1 culture medium A
Culture medium A, for the first step activation of bacterial classification, is the substratum of first-generation plate bacterial classification.The substratum major ingredient sees the following form, and other compositions are water.The substratum making method similar with the PDA substratum.
1.2 substratum B
Substratum B, for preparing industrial strain in a large number, for solid-state inoculation, is that s-generation plate solid state bacterial is cultivated the substratum used.Major ingredient sees the following form, and other compositions are water.The substratum making method similar with the PDA substratum.Millet, rice need boiling, cross leaching juice.
1.3 the dynamic screening method of bacterial classification
We can use bacterial classification in a large number in the middle of daily production, wherein some mutant strains may occur.In order to filter out better bacterial classification, we have set up a set of distinctive bacterial screening mechanism in this zymotechnique.This mechanism can dynamically be screened mutant strain in the situation that do not affect to produce and carry out, do not expend unnecessary manpower, and concrete steps are as follows:
(1) bacterial classification collection: in daily production process, every batch of random 30 fermentation trays that dish is pollution-free, growing way is good of selecting; From picking bacterial classification the fermentation tray, be placed in the PDA plate, 28 ℃ of static cultivations 6 days, make bacterial classification to be tested;
(2) fermentation culture: cultivate in the middle of bacterial classification is inoculated in to the fermentation tray that contains substratum; Culture condition and daily production are consistent;
(3) mutant strain detects: the ABA content in fermented product is detected, record the fermentation rate of bacterial classification to be tested; If the fermentation rate of bacterial classification to be tested, without considerable change, is given up it, corresponding tunning is incorporated to daily production, extracts wherein dormin; If positive mutant occurs, it is carried out to stability test;
(4) positive mutant stability test: from choosing 5 strains positive mutant, and measure the strain fermentation rate in its 5 generations of going down to posterity; Fermentation culture conditions and daily production are consistent; If obviously decline does not appear in bacterial classification within 5 generations, look it and pass through stability test;
(5) positive mutant comes into operation: will put in production and application by the positive mutant of stability test, and continue above dynamically screening step.
2. fermention medium preparation
Fermention medium is used for continuing growth until the accumulation of PBI 58 product is most important part in the middle of whole production.Its major ingredient such as following table, other compositions are water.Wherein, the purification residue refers in the tunning leaching process and separates the concentrated non-dormin material obtained, component the unknown.
The making method of fermention medium is as follows:
(1) will show Raw and mix, poach, be placed in steam-cooking cabinet and carry out thermal pretreatment;
(2) preheated substratum is broken up, weighing divides to install to cultivates in tray, and every dish dress is got the 600g substratum;
(3) will cultivate tray and be placed in sterilizing cabinet, carry out autoclaving.
3. produce inoculation
The s-generation plate solid state bacterial prepared is pulverized, evenly spread in the middle of the cultivation tray after sterilizing; Each is cultivated tray and uses 3 plate bacterial classifications; Inoculation weight is 8% of substratum.
4. fermentation culture
Vaccinated cultivation tray is moved into to sweathouse, be placed in the fermentation culture device and cultivate; This culturing process utilizes the temperature control system of fermentation culture device and oxygen system accurately to control; Training mode is as shown in the table:
We are divided into four-stage by this fermentation period, and temperature and the air flow in each stage are strictly controlled.By this method, the environment that is beneficial to the PBI 58 accumulation can be artificially created, the fermentation level of Botrytis cinerea can be very effectively improved.
In addition, after cultivating 96 hours, will cultivate the tray left-hand thread, and can effectively reduce because air flow increases the substratum moisture loss brought, guarantee the water activity of late stage of culture substratum.
5. PBI 58 is purified
Solid state fermentation is produced the mode of the extraction employing general chemistry extraction of dormin, and its concrete operations flow process as shown in Figure 2.
Adopt above-mentioned explained hereafter dormin to have the following advantages:
Liquid fermentation method is produced the known fermentation level of dormin and is up to 2.0g/L.And by the method, the average fermentation level of dormin can reach 2934mg/kg, namely in every kilogram of substratum, contains the 2.934g dormin.This is the highest fermentation level that present known industrial level is issued to.
Utilize traditional organism method of purification, the rate of recovery of dormin can reach 90% in laboratory, can reach 83% in the middle of pilot scale.In pilot process, the PBI 58 yield of a whole set of technique is 2.4g/kg, and namely the per kilogram substratum can finally obtain PBI 58 (purity > 98.5%) 2.4g.
Embodiment 2
In the embodiment of the present invention, a kind of fungi solid state fermentation is produced the method for dormin, and concrete steps are as follows:
1. bacterial classification preparation
The production bacterial classification that the present invention uses is Botrytis cinerea Botrytis Cinerea, is by natural screening, ultraviolet mutagenesis, ethyl sulfate mutagenesis, the laser radiation of YAG double frequency pulse and dynamically screening acquisition in producing.Bacterial classification forms the plate solid state bacterial by activation, expansion.
1.1 culture medium A
Culture medium A, for the first step activation of bacterial classification, is the substratum of first-generation plate bacterial classification.The substratum major ingredient sees the following form, and other compositions are water.The substratum making method similar with the PDA substratum.
1.2 substratum B
Substratum B, for preparing industrial strain in a large number, for solid-state inoculation, is that s-generation plate solid state bacterial is cultivated the substratum used.Major ingredient sees the following form, and other compositions are water.The substratum making method similar with the PDA substratum.Millet, rice need boiling, cross leaching juice.
1.3 the dynamic screening method of bacterial classification
We can use bacterial classification in a large number in the middle of daily production, wherein some mutant strains may occur.In order to filter out better bacterial classification, we have set up a set of distinctive bacterial screening mechanism in this zymotechnique.This mechanism can dynamically be screened mutant strain in the situation that do not affect to produce and carry out, do not expend unnecessary manpower, and concrete steps are as follows:
(1) bacterial classification collection: in daily production process, every batch of random 10 fermentation trays that dish is pollution-free, growing way is good of selecting; From picking bacterial classification the fermentation tray, be placed in the PDA plate, 26 ℃ of static cultivations 5 days, make bacterial classification to be tested;
(2) fermentation culture: cultivate in the middle of bacterial classification is inoculated in to the fermentation tray that contains substratum; Culture condition and daily production are consistent;
(3) mutant strain detects: the ABA content in fermented product is detected, record the fermentation rate of bacterial classification to be tested; If the fermentation rate of bacterial classification to be tested, without considerable change, is given up it, corresponding tunning is incorporated to daily production, extracts wherein dormin; If positive mutant occurs, it is carried out to stability test;
(4) positive mutant stability test: from choosing 3 strains positive mutant, and measure the strain fermentation rate in its 3 generations of going down to posterity; Fermentation culture conditions and daily production are consistent; If obviously decline does not appear in bacterial classification within 3 generations, look it and pass through stability test;
(5) positive mutant comes into operation: will put in production and application by the positive mutant of stability test, and continue above dynamically screening step.
2. fermention medium preparation
Fermention medium is used for continuing growth until the accumulation of PBI 58 product is most important part in the middle of whole production.Its major ingredient such as following table, other compositions are water.Wherein, the purification residue refers in the tunning leaching process and separates the concentrated non-dormin material obtained, component the unknown.
The making method of fermention medium is as follows:
(1) will show Raw and mix, poach, be placed in steam-cooking cabinet and carry out thermal pretreatment;
(2) preheated substratum is broken up, weighing divides to install to cultivates in tray, and every dish dress is got the 400g substratum;
(3) will cultivate tray and be placed in sterilizing cabinet, carry out autoclaving.
3. produce inoculation
The s-generation plate solid state bacterial prepared is pulverized, evenly spread in the middle of the cultivation tray after sterilizing; Each is cultivated tray and uses 2 plate bacterial classifications; Inoculation weight is 6% of substratum.
4. fermentation culture
Vaccinated cultivation tray is moved into to sweathouse, be placed in the fermentation culture device and cultivate; This culturing process utilizes the temperature control system of fermentation culture device and oxygen system accurately to control; Training mode is as shown in the table:
We are divided into four-stage by this fermentation period, and temperature and the air flow in each stage are strictly controlled.By this method, the environment that is beneficial to the PBI 58 accumulation can be artificially created, the fermentation level of Botrytis cinerea can be very effectively improved.
In addition, after cultivating 96 hours, will cultivate the tray left-hand thread, and can effectively reduce because air flow increases the substratum moisture loss brought, guarantee the water activity of late stage of culture substratum.
5. PBI 58 is purified: technique is with embodiment 1.
Embodiment 3
In the embodiment of the present invention, a kind of fungi solid state fermentation is produced the method for dormin, and concrete steps are as follows:
1. bacterial classification preparation
The production bacterial classification that the present invention uses is Botrytis cinerea Botrytis Cinerea, is by natural screening, ultraviolet mutagenesis, ethyl sulfate mutagenesis, the laser radiation of YAG double frequency pulse and dynamically screening acquisition in producing.Bacterial classification forms the plate solid state bacterial by activation, expansion.
1.1 culture medium A
Culture medium A, for the first step activation of bacterial classification, is the substratum of first-generation plate bacterial classification.The substratum major ingredient sees the following form, and other compositions are water.The substratum making method similar with the PDA substratum.
| Composition | Content (wt%) | Composition | Content (wt%) |
| Potato | 20 | Agar | 3 |
| Glucose | 1 | Lactose | 0.5 |
| Sucrose | 2 | Ca(NO3)2·4H2O | 0.05 |
| Peptone | 2 | Na2HPO4·12H2O | 0.5 |
1.2 substratum B
Substratum B, for preparing industrial strain in a large number, for solid-state inoculation, is that s-generation plate solid state bacterial is cultivated the substratum used.Major ingredient sees the following form, and other compositions are water.The substratum making method similar with the PDA substratum.Millet, rice need boiling, cross leaching juice.
| Composition | Content (wt%) | Composition | Content (wt%) |
| Millet | 10 | Rice | 10 |
| Glucose | 1 | Sucrose | 2 |
| Peptone | 2 | Ca(NO 3) 2·4H 2O | 0.05 |
| Na 2HPO 4·12H 2O | 0.5 | Agar | 3 |
1.3 the dynamic screening method of bacterial classification
We can use bacterial classification in a large number in the middle of daily production, wherein some mutant strains may occur.In order to filter out better bacterial classification, we have set up a set of distinctive bacterial screening mechanism in this zymotechnique.This mechanism can dynamically be screened mutant strain in the situation that do not affect to produce and carry out, do not expend unnecessary manpower, and concrete steps are as follows:
(1) bacterial classification collection: in daily production process, every batch of random 50 fermentation trays that dish is pollution-free, growing way is good of selecting; From picking bacterial classification the fermentation tray, be placed in the PDA plate, 30 ℃ of static cultivations 7 days, make bacterial classification to be tested;
(2) fermentation culture: cultivate in the middle of bacterial classification is inoculated in to the fermentation tray that contains substratum; Culture condition and daily production are consistent;
(3) mutant strain detects: the ABA content in fermented product is detected, record the fermentation rate of bacterial classification to be tested; If the fermentation rate of bacterial classification to be tested, without considerable change, is given up it, corresponding tunning is incorporated to daily production, extracts wherein dormin; If positive mutant occurs, it is carried out to stability test;
(4) positive mutant stability test: from choosing 8 strains positive mutant, and measure the strain fermentation rate in its 8 generations of going down to posterity; Fermentation culture conditions and daily production are consistent; If obviously decline does not appear in bacterial classification within 8 generations, look it and pass through stability test;
(5) positive mutant comes into operation: will put in production and application by the positive mutant of stability test, and continue above dynamically screening step.
2. fermention medium preparation
Fermention medium is used for continuing growth until the accumulation of PBI 58 product is most important part in the middle of whole production.Its major ingredient such as following table, other compositions are water.Wherein, the purification residue refers in the tunning leaching process and separates the concentrated non-dormin material obtained, component the unknown.
| Composition | Content (wt%) | Composition | Content (wt%) |
| Millet | 25 | Rice | 25 |
| Glucose | 2 | Sucrose | 4 |
| Peptone | 4 | CaCO 3 | 1.5 |
| Na 2HPO 4·12H 2O | 0.5 | Ca(HO) 2 | 1.5 |
| MgSO 4 | 0.05 | The purification residue | 1 |
The making method of fermention medium is as follows:
(1) will show Raw and mix, poach, be placed in steam-cooking cabinet and carry out thermal pretreatment;
(2) preheated substratum is broken up, weighing divides to install to cultivates in tray, and every dish dress is got the 800g substratum;
(3) will cultivate tray and be placed in sterilizing cabinet, carry out autoclaving.
3. produce inoculation
The s-generation plate solid state bacterial prepared is pulverized, evenly spread in the middle of the cultivation tray after sterilizing; Each is cultivated tray and uses 4 plate bacterial classifications; Inoculation weight is 10% of substratum.
4. fermentation culture
Vaccinated cultivation tray is moved into to sweathouse, be placed in the fermentation culture device and cultivate; This culturing process utilizes the temperature control system of fermentation culture device and oxygen system accurately to control; Training mode is as shown in the table:
| Incubation time (hour) | Medial temperature (℃) | Average ventilation (L/(100 coils * days)) |
| 0-48 | 28.5 | 10 |
| 48-96 | 28 | 20 |
| 96-216 | 24.5 | 50 |
| 216-288 | 26.5 | 20 |
We are divided into four-stage by this fermentation period, and temperature and the air flow in each stage are strictly controlled.By this method, the environment that is beneficial to the PBI 58 accumulation can be artificially created, the fermentation level of Botrytis cinerea can be very effectively improved.
In addition, after cultivating 96 hours, will cultivate the tray left-hand thread, and can effectively reduce because air flow increases the substratum moisture loss brought, guarantee the water activity of late stage of culture substratum.
5. PBI 58 is purified: the concrete operations flow process is with embodiment 1.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and in the situation that do not deviate from spirit of the present invention or essential characteristic, can realize the present invention with other specific form.Therefore, no matter from which point, all should regard embodiment as exemplary, and be nonrestrictive, scope of the present invention is limited by claims rather than above-mentioned explanation, therefore is intended to include in the present invention dropping on the implication that is equal to important document of claim and all changes in scope.Any Reference numeral in claim should be considered as limit related claim.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should make specification sheets as a whole, and the technical scheme in each embodiment also can, through appropriate combination, form other embodiments that it will be appreciated by those skilled in the art that.
Claims (9)
1. the method that the fungi solid state fermentation is produced dormin, is characterized in that, concrete steps are as follows:
(1) bacterial classification preparation
Screen the Botrytis cinerea of positive mutagenesis for producing bacterial classification, then, by activation and enlarged culturing, form the plate solid state bacterial, standby;
(2) fermention medium preparation
Fermention medium according to the raw material of weight percent is: millet 15-25%, rice 15-25%, glucose 0.1-2%, sucrose 0.1-4%, peptone 1-4%, CaCO
30.5-1.5%, Na
2HPO
412H
2O 0.1-0.5%, Ca (HO)
20.5-1.5%, MgSO
40.01-0.05%, purification residue 0.5-1%, other compositions are water; Wherein, the purification residue refers in the tunning leaching process and separates the concentrated non-dormin material obtained;
The making method of fermention medium: all raw materials are mixed, and poach, be placed in steam-cooking cabinet and carry out thermal pretreatment; Preheated substratum is broken up, and weighing divides to install to cultivates in tray, and every dish dress is got the 400-800g substratum; To cultivate tray and be placed in sterilizing cabinet, carry out autoclaving, standby;
(3) produce inoculation
Plate solid state bacterial prepared by step (1) pulverizes, and evenly spreads in the middle of the cultivation tray of step (2) after sterilizing; Each is cultivated tray and uses 2-4 plate bacterial classification; Inoculation weight is the 6-10% of substratum;
(4) fermentation culture
Vaccinated cultivation tray is moved into to sweathouse, be placed in the fermentation culture device and cultivate; Training mode is as follows:
The medial temperature of cultivating 0-48 hour be 26.5-28.5 ℃, average ventilation be the 5-10L/100 dish/day;
The medial temperature of cultivating 48-96 hour be 26-28 ℃, average ventilation be the 15-20L/100 dish/day;
The medial temperature of cultivating 96-216 hour be 22-24.5 ℃, average ventilation be the 40-50L/100 dish/day;
The medial temperature of cultivating 216-288 hour be 25.5-26.5 ℃, average ventilation be the 10-20L/100 dish/day;
(5) PBI 58 is purified: the tunning of step (4) is purified and obtained PBI 58.
2. fungi solid state fermentation according to claim 1 is produced the method for dormin, it is characterized in that, by natural screening, ultraviolet mutagenesis, ethyl sulfate mutagenesis, the laser radiation of YAG double frequency pulse and in producing, dynamically screen the Botrytis cinerea that obtains positive mutagenesis in step (1).
3. fungi solid state fermentation according to claim 2 is produced the method for dormin, it is characterized in that, in described production, dynamically the concrete steps of screening are as follows:
Bacterial classification gathers: in daily production process, every batch of random 10-50 of selection coils the fermentation tray pollution-free, that growing way is good; From picking bacterial classification the fermentation tray, be placed in the PDA plate, 26-30 ℃ of static cultivation 5-7 days, make bacterial classification to be tested;
Fermentation culture: cultivate in the middle of bacterial classification is inoculated in to the fermentation tray that contains substratum; Culture condition and daily production are consistent;
Mutant strain detects: the ABA content in fermented product is detected, record the fermentation rate of bacterial classification to be tested; If the fermentation rate of bacterial classification to be tested, without considerable change, is given up it, corresponding tunning is incorporated to daily production, extracts wherein dormin; If positive mutant occurs, it is carried out to stability test;
Positive mutant stability test: from choosing the 3-8 strain positive mutant, and measure its 3-8 strain fermentation rate in generation that goes down to posterity; Fermentation culture conditions and daily production are consistent; If bacterial classification, in obviously decline of interior appearance of 3-8 generation, is looked it and is passed through stability test;
Positive mutant comes into operation: will put in production and application by the positive mutant of stability test, and continue above dynamically screening step.
4. fungi solid state fermentation according to claim 1 is produced the method for dormin, it is characterized in that, step is carried out activation culture by culture medium A to Botrytis cinerea in (1), described culture medium A according to the raw material of weight percent is: potato 10-20%, agar 1-3%, glucose 0.1-1%, lactose 0.1-0.5%, sucrose 1-2%, Ca (NO3) 24H2O 0.01-0.05%, peptone 1-2%, Na2HPO412H2O 0.1-0.5%, other compositions are water.
5. fungi solid state fermentation according to claim 1 is produced the method for dormin, it is characterized in that, by substratum B, the botrytis sp bacterium activated is carried out to enlarged culturing in step (1), and substratum B is for solid-state inoculation; Described substratum B according to the raw material of weight percent is: millet 5-10%, rice 5-10%, glucose 0.1-1%, sucrose 1-2%, peptone 1-2%, Ca (NO
3)
24H
2O 0.01-0.05%, Na
2HPO
412H
2O 0.1-0.5%, agar 1-3%, other compositions are water; Described millet, rice need boiling, cross leaching juice.
6. fungi solid state fermentation according to claim 1 is produced the method for dormin, it is characterized in that, the fermention medium described in step (2) according to the raw material of weight percent is: millet 20%, rice 20%, glucose 1%, sucrose 2%, peptone 2.5%, CaCO
31%, Na
2HPO
412H
2O 0.3%, Ca (HO)
21%, MgSO
40.03%, the purification residue 0.75%, and other compositions are water; In the making method of fermention medium, every dish dress is got the 600g substratum.
7. fungi solid state fermentation according to claim 1 is produced the method for dormin, it is characterized in that, in step (3), each is cultivated tray and uses 3 plate bacterial classifications; Inoculation weight is 8% of substratum.
8. fungi solid state fermentation according to claim 1 is produced the method for dormin, it is characterized in that, the training mode in step (4) is as follows:
The medial temperature of cultivating 0-48 hour be 27 ℃, average ventilation be the 7.5L/100 dish/day;
The medial temperature of cultivating 48-96 hour be 27 ℃, average ventilation be the 17.5L/100 dish/day;
The medial temperature of cultivating 96-216 hour be 23 ℃, average ventilation be the 45L/100 dish/day;
The medial temperature of cultivating 216-288 hour be 26 ℃, average ventilation be the 15L/100 dish/day.
9. the method for producing dormin according to the described fungi solid state fermentation of claim 1 or 8, is characterized in that, after cultivating 96 hours, will cultivate the tray left-hand thread.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106995826A (en) * | 2017-04-18 | 2017-08-01 | 四川龙蟒福生科技有限责任公司 | A kind of fermentation medium for being used to produce S abscisic acids |
| CN107056485A (en) * | 2017-05-22 | 2017-08-18 | 重庆聚立信生物工程有限公司 | A kind of entomogenous fungi granule and preparation method thereof |
-
2013
- 2013-07-10 CN CN2013102888840A patent/CN103409474A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106995826A (en) * | 2017-04-18 | 2017-08-01 | 四川龙蟒福生科技有限责任公司 | A kind of fermentation medium for being used to produce S abscisic acids |
| CN107056485A (en) * | 2017-05-22 | 2017-08-18 | 重庆聚立信生物工程有限公司 | A kind of entomogenous fungi granule and preparation method thereof |
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