CN108651694A - A kind of method of stalk fermentation - Google Patents
A kind of method of stalk fermentation Download PDFInfo
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Abstract
The invention discloses a kind of methods of stalk fermentation, belong to biotechnology.The present invention is with Magnesium dichloride hexahydrate, sodium sulfite, magnesium bisulfite substance is formed with sulfurous acid, it pre-processes maize straw, lignin in stalk limits the contact of microorganism and enzyme with polysaccharide, inhibit the digestion of cellulose, it has sulfonation to straw lignin, lignin conversion is set to be dissolved out for ligninsulfonate, acid therein can also remove the hemicellulose in stalk, it is small to stalk cellulose destruction, its chemical constitution is not changed, the efficiency that the later stage carries out stalk fermentation conversion is improved, the dissolution that the content of organic matter can be more.The present invention solves in current biological treatment that microorganism used therefor transformation efficiency is low, and processing time is long, and the obtained content of organic matter is few, and active bacteria fermented stalk is affected by the external environment greatly in application process, the problem of being unfavorable for operation.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of method of stalk fermentation.
Background technology
China produces about 700,000,000 tons of crop material, due to the transformation of country labor force transfer and tillage method, most of stalk per year
By on-site incineration or discard.But due to stalk on-site incineration or discarded caused environmental pollution and problem of resource waste
Through becoming regulatory authorities and scientific research personnel's focus of attention.In stalk more than 60% be cellulose and hemicellulose, be very
Excellent biomass resource, by can be widely applied to feed, organic fertilizer, biogas etc. after bioconversion.But stalk
Natural structure it is extremely complex, biotransformation efficiency is relatively low, and the difficulty for being directly used as the exploitation of above Related product is larger, at
This is higher.Therefore, it is a mostly important link during stalk resource utilizes to be effectively treated to stalk.
The processing method of stalk has physical treatment process, method of chemical treatment and biological treatment at present.Wherein biological treatment
Fermentation process is carried out to stalk using efficient degradation bacteria, the siliceous nodular texture of stalk in appearance falls off, and cell wall occurs big
Range is damaged, and entire stalk tissue becomes loose.Straw degradative bacterium can decompose various polymer in its tissue, by the wood in stalk
Quality, cellulose and hemicellulose are decomposed into monomer or oligomer, improve its utility.Biochemical method cost is relatively low,
There is no secondary pollution, but microorganism used therefor transformation efficiency is relatively low in currently used biological treatment, processing time is longer, obtains
The content of organic matter it is low, and active bacteria fermented stalk is affected by the external environment greatly in application process, is unfavorable for operating, affect
The application and popularization of biological treatment.Therefore, it is required to meet to be badly in need of a kind of method of high-efficiency fermenting stalk.
Invention content
The technical problems to be solved by the invention：It is low for microorganism used therefor transformation efficiency in current biological treatment, place
The reason time is long, and the obtained content of organic matter is few, and active bacteria fermented stalk is affected by the external environment greatly in application process, is unfavorable for
The problem of operation, provides a kind of method of stalk fermentation.
In order to solve the above technical problems, the present invention is using technical solution as described below：
A kind of method of stalk fermentation, which is characterized in that this method comprises the following steps：
（1）It takes stalk to pulverize and sieve, collects sieving particle, take Magnesium dichloride hexahydrate in mass ratio 2：1：10 addition sodium sulfites,
Distilled water is stirred, and stands, precipitation is taken to be washed with distilled water, and is freeze-dried, is obtained dried object, take dried object in mass ratio 10：
2：5 are added sulfurous acid, distilled water mixing, obtain mixture, take mixture in mass ratio 6：1 is added sieving particle, in 60 ~ 70 DEG C of guarantors
4 ~ 5h of temperature, filtering, takes filter residue through distilled water flushing, obtains washings；
（2）Take filter residue in mass ratio 1：10：3 are added distilled water, citrate buffer solution mixing, adjust pH to 4.5 ~ 4.8, must mix
Liquid takes cellulase, beta-glucosidase, pectase in mass ratio 3：2：1 mixing, obtains mixed enzyme, takes mixed enzyme by 4 ~ 7%
Mixed liquor is added in parts by weight additive amount, is stirred in 45 ~ 50 DEG C, 150rpm, enzyme deactivation, obtains enzyme deactivation object, spare；
（3）Trichoderma viride, Phanerochaete chrysosporium, recombinant yeast pichia pastoris bacterium is taken to be seeded to activating solution by 2% inoculum concentration respectively
In body culture medium, in 25 ~ 30 DEG C of cultures, trichoderma viride activation bacterium solution is obtained, Phanerochaete chrysosporium activates bacterium solution, recombination is complete red
Saccharomycete activates bacterium solution；
（4）Trichoderma viride is taken to activate bacterium solution in mass ratio 7：100 are added fermentation medium, obtain mixture, take mixture by matter
Measure ratio 15：1 is added step（2）Spare enzyme deactivation object obtains fermentation culture medium, takes Huang in 200r/min, 25 ~ 33 DEG C of fermented and cultureds
The flat lead fungi activation bacterium solution of archespore hair, recombinant yeast pichia pastoris activate bacterium solution in mass ratio 1：2 mixing, obtain mixed bacteria liquid, take Mixed Microbes
Liquid is seeded to by 6 ~ 9% inoculum concentration in fermentation culture medium, the Tween-20 of fermentation medium quality 6 ~ 8% is added, in 25 ~ 30
DEG C fermented and cultured, obtains second order fermentation culture, takes second order fermentation culture in mass ratio 100：3 are added methanol, cultivate 12 ~ 15h,
Obtain culture；
（5）Take chitosan in mass ratio 2：5 are added acetic acid solution, are stirred, add the chlorination of chitosan mass 20 ~ 30%
Calcium solution mixes, and obtains mixed solution, takes sodium alginate in mass ratio 3：50：10 are added culture, water mixing, obtain mixed solution A,
Take mixed solution A in mass ratio 10：3 are added mixed solution, are stirred in 40 ~ 50 DEG C, obtain fermented stalk.
The step（1）In stalk be maize straw, wheat stalk, cotton stalk, any one in rice straw
Or it is several.
The step（3）Middle activated liquid culture medium is according to the mass fraction, to take 1000 parts of distilled water, 20 ~ 25 parts of albumen
Peptone, 8 ~ 12 parts of yeast extracts, the mixing of 15 ~ 20 parts of glucose, 121 DEG C of 20 min of sterilizing to get.
The step（4）Middle fermentation medium is according to the mass fraction, to take 20 ~ 25 portions of potatos, 2 ~ 5 parts of glucose, 0.2
~ 0.5 part of ammonium tartrate, 0.25 ~ 0.5 part of manganese sulfate, 0.1 ~ 0.3 part of calcium chloride, 0.001 ~ 0.003 part of vitamin B, 1000 parts of water
Mixing, 121 DEG C sterilizing 20 min to get.
Compared with other methods, advantageous effects are the present invention：
（1）The present invention forms magnesium bisulfite substance with Magnesium dichloride hexahydrate, sodium sulfite, with sulfurous acid, by it to corn
Stalk is pre-processed, and the lignin in stalk limits the contact of microorganism and enzyme with polysaccharide, it is suppressed that the digestion of cellulose,
It has sulfonation to straw lignin, and lignin conversion is made to be dissolved out for ligninsulfonate, and acid therein can also remove stalk
In hemicellulose, it is small to stalk cellulose destruction, do not change its chemical constitution, the later stage carries out the effect of stalk fermentation conversion
Rate is improved, the dissolution that the content of organic matter can be more；
（2）Cellulase, beta-glucosidase, pectase is added in the present invention, and cellulase is the main structural polysaccharide of plant
It is translated into soluble sugar, cellobiose and other low molecule cellodextrins can be decomposed into glucose by beta-glucosidase,
Using pectase as digestive ferment, depolymerized pectin matter cracks carbon-chain structure, breaks stalk cell wall by destroying lignocellulosic
Shielding action, be conducive to discharge intracellular nutriment, shorten the time of later stage mixed fungus fermentation stalk, it is preferably interior
Source enzymic digestion improves the digestibility of nutriment in stalk, and improve stalk utilizes conversion ratio, and straw after enzymolysis processing
Stalk enters in animal body, can promote the breeding of Ruminal Fibre degradation bacteria, increases micro organism quantity, to generate more enzymes
Improve the degradation of fiber and the synthesis of microprotein in stalk；
（3）Trichoderma viride is first carried out fermented and cultured by the present invention to the stalk of pre-processing, then through Phanerochaete chrysosporium and
The secondary mixed fermentation of recombinant yeast pichia pastoris bacterium plays the role of symbiosis cooperation between when mixed bacteria liquid culture different bacterial strain, can be with
Ester bond between fibre composition is destroyed, the release phenolic acids such as ferulic acid, promotes hemicellulose, lignin and cellulose
Separation provides condition for the further enzymolysis of cellulose, macromolecular substances is degraded into the small molecule nutrition for being easy to animal digestion
Substance, and Phanerochaete chrysosporium and Trichoderma viride are main lignin-degrading bacteria and cellulose-degrading bacteria respectively, are being fermented
In the process by the organic matters such as cellulose and lignin decomposition and inversion at glucide, carried out through chitosan, sodium alginate after fermentation
Embedding ensure that the activity and stability of active bacteria ingredient in stalk after fermenting, is sustained to it, improve the profit of fermented stalk
Use efficiency.
Specific implementation mode
Activated liquid culture medium：According to the mass fraction, 1000 parts of distilled water, 20 ~ 25 parts of peptones, 8 ~ 12 parts of yeast leachings are taken
Powder, the mixing of 15 ~ 20 parts of glucose, 121 DEG C of 20 min of sterilizing to get.
Fermentation medium：According to the mass fraction, 20 ~ 25 portions of potatos, 2 ~ 5 parts of glucose, 0.2 ~ 0.5 part of tartaric acid are taken
Ammonium, 0.25 ~ 0.5 part of manganese sulfate, 0.1 ~ 0.3 part of calcium chloride, 0.001 ~ 0.003 part of vitamin B, 1000 parts of water mixing, 121 DEG C go out
20 min of bacterium to get.
A kind of method of stalk fermentation, includes the following steps：
（1）It takes stalk crushing to sieve with 100 mesh sieve, collects sieving particle, take Magnesium dichloride hexahydrate in mass ratio 2：1：10 are added sulfurous
Sour sodium, distilled water are stirred 30 ~ 40min, stand 5 ~ 6h, precipitation is taken to be washed with distilled water, and are freeze-dried, obtain dried object, take
Dried object in mass ratio 10：2：5 are added sulfurous acid, distilled water mixing, obtain mixture, take mixture in mass ratio 6：1 was added
Particle is sieved, 4 ~ 5h is kept the temperature in 60 ~ 70 DEG C, filtering takes filter residue through distilled water flushing, obtains washings；
（2）Take filter residue in mass ratio 1：10：3 are added distilled water, citrate buffer solution mixing, adjust pH to 4.5 ~ 4.8, must mix
Liquid takes cellulase, beta-glucosidase, pectase in mass ratio 3：2：1 mixing, obtains mixed enzyme, takes mixed enzyme by 4 ~ 7%
Mixed liquor is added in parts by weight additive amount, is stirred 36 ~ 48h in 45 ~ 50 DEG C, 150rpm, enzyme deactivation obtains enzyme deactivation object, spare；
（3）Trichoderma viride, Phanerochaete chrysosporium, recombinant yeast pichia pastoris bacterium is taken to be seeded to activating solution by 2% inoculum concentration respectively
It in body culture medium, is cultivated 2 ~ 5 days in 25 ~ 30 DEG C, obtains trichoderma viride activation bacterium solution, Phanerochaete chrysosporium activates bacterium solution, again
Group Pichia yeast activates bacterium solution；
（4）Trichoderma viride is taken to activate bacterium solution in mass ratio 7：100 are added fermentation medium, obtain mixture, take mixture by matter
Measure ratio 15：1 is added step（2）Spare enzyme deactivation object obtained fermentation culture medium in 200r/min, 25 ~ 33 DEG C of fermented and cultureds 2 ~ 3 days,
Take Phanerochaete chrysosporium activation bacterium solution, recombinant yeast pichia pastoris activation bacterium solution in mass ratio 1：2 mixing, obtain mixed bacteria liquid, take mixed
Combined bacteria liquid is seeded to by 6 ~ 9% inoculum concentration in fermentation culture medium, the Tween-20 of fermentation medium quality 6 ~ 8% is added, in 25
~ 30 DEG C of fermented and cultureds 2 ~ 4 days, obtain second order fermentation culture, take second order fermentation culture in mass ratio 100：3 are added methanol, training
12 ~ 15h is supported, culture is obtained；
（5）Take chitosan in mass ratio 2：5 are added the acetic acid solution that mass fraction is 1%, are stirred 30 ~ 40min, add
The calcium chloride solution of chitosan mass 20 ~ 30% mixes, and obtains mixed solution, takes sodium alginate in mass ratio 3：50：10 are added culture
Object, water mixing, obtain mixed solution A, take mixed solution A in mass ratio 10：3 are added mixed solutions, it is stirred 2 in 40 ~ 50 DEG C ~
3h obtains fermented stalk.
Embodiment 1
Activated liquid culture medium：According to the mass fraction, 1000 parts of distilled water, 20 parts of peptones, 8 parts of yeast extracts, 15 parts of Portugals are taken
Grape sugar mix, 121 DEG C sterilizing 20 min to get.
Fermentation medium：According to the mass fraction, 20 portions of potatos, 2 parts of glucose, 0.2 part of ammonium tartrate, 0.25 part of sulphur are taken
Sour manganese, 0.1 part of calcium chloride, 0.001 part of vitamin B, the mixing of 1000 parts of water, 121 DEG C of 20 min of sterilizing to get.
A kind of method of stalk fermentation, includes the following steps：
（1）It takes stalk crushing to sieve with 100 mesh sieve, collects sieving particle, take Magnesium dichloride hexahydrate in mass ratio 2：1：10 are added sulfurous
Sour sodium, distilled water are stirred 30min, stand 5h, precipitation is taken to be washed with distilled water, and are freeze-dried, obtain dried object, take drying
Object in mass ratio 10：2：5 are added sulfurous acid, distilled water mixing, obtain mixture, take mixture in mass ratio 6：1 is added sieving
Grain, 4h is kept the temperature in 60 DEG C, and filtering takes filter residue through distilled water flushing, obtains washings；
（2）Take filter residue in mass ratio 1：10：3 are added distilled water, citrate buffer solution mixing, adjust pH to 4.5, obtain mixed liquor,
Take cellulase, beta-glucosidase, pectase in mass ratio 3：2：1 mixing, obtain mixed enzyme, take mixed enzyme by 4% parts by weight
Mixed liquor is added in additive amount, is stirred 36h in 45 DEG C, 150rpm, enzyme deactivation obtains enzyme deactivation object, spare；
（3）Trichoderma viride, Phanerochaete chrysosporium, recombinant yeast pichia pastoris bacterium is taken to be seeded to activating solution by 2% inoculum concentration respectively
It in body culture medium, is cultivated 2 days in 25 DEG C, obtains trichoderma viride activation bacterium solution, Phanerochaete chrysosporium activates bacterium solution, recombination is complete red
Saccharomycete activates bacterium solution；
（4）Trichoderma viride is taken to activate bacterium solution in mass ratio 7：100 are added fermentation medium, obtain mixture, take mixture by matter
Measure ratio 15：1 is added step（2）Spare enzyme deactivation object obtained fermentation culture medium, takes Huang in 200r/min, 25 DEG C of fermented and cultureds 2 days
The flat lead fungi activation bacterium solution of archespore hair, recombinant yeast pichia pastoris activate bacterium solution in mass ratio 1：2 mixing, obtain mixed bacteria liquid, take Mixed Microbes
Liquid is seeded to by 6% inoculum concentration in fermentation culture medium, adds the Tween-20 of fermentation medium quality 6%, in 25 DEG C of fermentation trainings
It supports 2 days, obtains second order fermentation culture, take second order fermentation culture in mass ratio 100：3 are added methanol, cultivate 12h, must cultivate
Object；
（5）Take chitosan in mass ratio 2：5 are added the acetic acid solution that mass fraction is 1%, are stirred 30min, and it is poly- to add shell
The calcium chloride solution of saccharic amount 20% mixes, and obtains mixed solution, takes sodium alginate in mass ratio 3：50：10 are added culture, water mixes
It closes, obtains mixed solution A, take mixed solution A in mass ratio 10：3 are added mixed solution, are stirred 2h in 40 DEG C, obtain fermentation straw
Stalk.
Embodiment 2
Activated liquid culture medium：According to the mass fraction, 1000 parts of distilled water, 25 parts of peptones, 12 parts of yeast extracts, 20 parts of Portugals are taken
Grape sugar mix, 121 DEG C sterilizing 20 min to get.
Fermentation medium：According to the mass fraction, 25 portions of potatos, 5 parts of glucose, 0.5 part of ammonium tartrate, 0.5 part of sulphur are taken
Sour manganese, 0.3 part of calcium chloride, 0.003 part of vitamin B, the mixing of 1000 parts of water, 121 DEG C of 20 min of sterilizing to get.
A kind of method of stalk fermentation, includes the following steps：
（1）It takes stalk crushing to sieve with 100 mesh sieve, collects sieving particle, take Magnesium dichloride hexahydrate in mass ratio 2：1：10 are added sulfurous
Sour sodium, distilled water are stirred 40min, stand 6h, precipitation is taken to be washed with distilled water, and are freeze-dried, obtain dried object, take drying
Object in mass ratio 10：2：5 are added sulfurous acid, distilled water mixing, obtain mixture, take mixture in mass ratio 6：1 is added sieving
Grain, 5h is kept the temperature in 70 DEG C, and filtering takes filter residue through distilled water flushing, obtains washings；
（2）Take filter residue in mass ratio 1：10：3 are added distilled water, citrate buffer solution mixing, adjust pH to 4.8, obtain mixed liquor,
Take cellulase, beta-glucosidase, pectase in mass ratio 3：2：1 mixing, obtain mixed enzyme, take mixed enzyme by 7% parts by weight
Mixed liquor is added in additive amount, is stirred 48h in 50 DEG C, 150rpm, enzyme deactivation obtains enzyme deactivation object, spare；
（3）Trichoderma viride, Phanerochaete chrysosporium, recombinant yeast pichia pastoris bacterium is taken to be seeded to activating solution by 2% inoculum concentration respectively
It in body culture medium, is cultivated 5 days in 30 DEG C, obtains trichoderma viride activation bacterium solution, Phanerochaete chrysosporium activates bacterium solution, recombination is complete red
Saccharomycete activates bacterium solution；
（4）Trichoderma viride is taken to activate bacterium solution in mass ratio 7：100 are added fermentation medium, obtain mixture, take mixture by matter
Measure ratio 15：1 is added step（2）Spare enzyme deactivation object obtained fermentation culture medium, takes Huang in 200r/min, 33 DEG C of fermented and cultureds 3 days
The flat lead fungi activation bacterium solution of archespore hair, recombinant yeast pichia pastoris activate bacterium solution in mass ratio 1：2 mixing, obtain mixed bacteria liquid, take Mixed Microbes
Liquid is seeded to by 9% inoculum concentration in fermentation culture medium, adds the Tween-20 of fermentation medium quality 8%, in 30 DEG C of fermentation trainings
It supports 4 days, obtains second order fermentation culture, take second order fermentation culture in mass ratio 100：3 are added methanol, cultivate 15h, must cultivate
Object；
（5）Take chitosan in mass ratio 2：5 are added the acetic acid solution that mass fraction is 1%, are stirred 40min, and it is poly- to add shell
The calcium chloride solution of saccharic amount 30% mixes, and obtains mixed solution, takes sodium alginate in mass ratio 3：50：10 are added culture, water mixes
It closes, obtains mixed solution A, take mixed solution A in mass ratio 10：3 are added mixed solution, are stirred 3h in 50 DEG C, obtain fermentation straw
Stalk.
Embodiment 3
Activated liquid culture medium：According to the mass fraction, 1000 parts of distilled water, 23 parts of peptones, 10 parts of yeast extracts, 17 parts of Portugals are taken
Grape sugar mix, 121 DEG C sterilizing 20 min to get.
Fermentation medium：According to the mass fraction, 23 portions of potatos, 3 parts of glucose, 0.3 part of ammonium tartrate, 0.4 part of sulphur are taken
Sour manganese, 0.2 part of calcium chloride, 0.002 part of vitamin B, the mixing of 1000 parts of water, 121 DEG C of 20 min of sterilizing to get.
A kind of method of stalk fermentation, includes the following steps：
（1）It takes stalk crushing to sieve with 100 mesh sieve, collects sieving particle, take Magnesium dichloride hexahydrate in mass ratio 2：1：10 are added sulfurous
Sour sodium, distilled water are stirred 35min, stand 5h, precipitation is taken to be washed with distilled water, and are freeze-dried, obtain dried object, take drying
Object in mass ratio 10：2：5 are added sulfurous acid, distilled water mixing, obtain mixture, take mixture in mass ratio 6：1 is added sieving
Grain, 4h is kept the temperature in 65 DEG C, and filtering takes filter residue through distilled water flushing, obtains washings；
（2）Take filter residue in mass ratio 1：10：3 are added distilled water, citrate buffer solution mixing, adjust pH to 4.7, obtain mixed liquor,
Take cellulase, beta-glucosidase, pectase in mass ratio 3：2：1 mixing, obtain mixed enzyme, take mixed enzyme by 5% parts by weight
Mixed liquor is added in additive amount, is stirred 42h in 47 DEG C, 150rpm, enzyme deactivation obtains enzyme deactivation object, spare；
（3）Trichoderma viride, Phanerochaete chrysosporium, recombinant yeast pichia pastoris bacterium is taken to be seeded to activating solution by 2% inoculum concentration respectively
It in body culture medium, is cultivated 3 days in 27 DEG C, obtains trichoderma viride activation bacterium solution, Phanerochaete chrysosporium activates bacterium solution, recombination is complete red
Saccharomycete activates bacterium solution；
（4）Trichoderma viride is taken to activate bacterium solution in mass ratio 7：100 are added fermentation medium, obtain mixture, take mixture by matter
Measure ratio 15：1 is added step（2）Spare enzyme deactivation object obtained fermentation culture medium, takes Huang in 200r/min, 28 DEG C of fermented and cultureds 2 days
The flat lead fungi activation bacterium solution of archespore hair, recombinant yeast pichia pastoris activate bacterium solution in mass ratio 1：2 mixing, obtain mixed bacteria liquid, take Mixed Microbes
Liquid is seeded to by 7% inoculum concentration in fermentation culture medium, adds the Tween-20 of fermentation medium quality 7%, in 27 DEG C of fermentation trainings
It supports 3 days, obtains second order fermentation culture, take second order fermentation culture in mass ratio 100：3 are added methanol, cultivate 14h, must cultivate
Object；
（5）Take chitosan in mass ratio 2：5 are added the acetic acid solution that mass fraction is 1%, are stirred 35min, and it is poly- to add shell
The calcium chloride solution of saccharic amount 25% mixes, and obtains mixed solution, takes sodium alginate in mass ratio 3：50：10 are added culture, water mixes
It closes, obtains mixed solution A, take mixed solution A in mass ratio 10：3 are added mixed solution, are stirred 2h in 45 DEG C, obtain fermentation straw
Stalk.
Comparative example：The stalk fermentation method that company of Zhengzhou City provides.
The stalk fermentation method of embodiment and comparative example is subjected to fermenting property test respectively, observes the straw decomposing time,
Time is shorter, and the efficiency for illustrating to ferment is higher, and specific test result is shown in Table 1.
Table 1：
In summary, stalk fermentation method fermentation efficiency of the invention is high, and obtained organic matter has very well also superior to commercial product
Market prospects.
Claims (4)
1. a kind of method of stalk fermentation, which is characterized in that this method comprises the following steps：
It takes stalk to pulverize and sieve, collects sieving particle, take Magnesium dichloride hexahydrate in mass ratio 2：1：10 are added sodium sulfite, distillation
Water is stirred, and stands, precipitation is taken to be washed with distilled water, and is freeze-dried, is obtained dried object, take dried object in mass ratio 10：2：5
Sulfurous acid, distilled water mixing is added, obtains mixture, takes mixture in mass ratio 6：1 is added sieving particle, and 4 are kept the temperature in 60 ~ 70 DEG C
~ 5h, filtering, takes filter residue through distilled water flushing, obtains washings；
Take filter residue in mass ratio 1：10：3 are added distilled water, citrate buffer solution mixing, adjust pH to 4.5 ~ 4.8, obtain mixed liquor,
Take cellulase, beta-glucosidase, pectase in mass ratio 3：2：1 mixing, obtain mixed enzyme, take mixed enzyme by 4 ~ 7% weight
Mixed liquor is added in part additive amount, is stirred in 45 ~ 50 DEG C, 150rpm, enzyme deactivation, obtains enzyme deactivation object, spare；
Trichoderma viride, Phanerochaete chrysosporium, recombinant yeast pichia pastoris bacterium is taken to be seeded to activated liquid by 2% inoculum concentration respectively
In culture medium, in 25 ~ 30 DEG C of cultures, trichoderma viride activation bacterium solution, Phanerochaete chrysosporium activation bacterium solution, the complete red ferment of recombination are obtained
Female bacterium activates bacterium solution；
Trichoderma viride is taken to activate bacterium solution in mass ratio 7：100 are added fermentation medium, obtain mixture, take mixture in mass ratio
15：1 is added step（2）Spare enzyme deactivation object obtains fermentation culture medium, takes yellow archespore in 200r/min, 25 ~ 33 DEG C of fermented and cultureds
The flat lead fungi activation bacterium solution of hair, recombinant yeast pichia pastoris activate bacterium solution in mass ratio 1：2 mixing, obtain mixed bacteria liquid, take mixed bacteria liquid by 6
~ 9% inoculum concentration is seeded in fermentation culture medium, adds the Tween-20 of fermentation medium quality 6 ~ 8%, in 25 ~ 30 DEG C of fermentations
Culture, obtains second order fermentation culture, takes second order fermentation culture in mass ratio 100：3 are added methanol, cultivate 12 ~ 15h, must cultivate
Object；
Take chitosan in mass ratio 2：5 are added acetic acid solution, are stirred, the calcium chloride for adding chitosan mass 20 ~ 30% is molten
Liquid mixes, and obtains mixed solution, takes sodium alginate in mass ratio 3：50：10 are added culture, water mixing, obtain mixed solution A, take mixed
Close solution A in mass ratio 10：3 are added mixed solution, are stirred in 40 ~ 50 DEG C, obtain fermented stalk.
2. the method for stalk fermentation according to claim 1, which is characterized in that the step（1）In stalk be corn
Any one or a few in stalk, wheat stalk, cotton stalk, rice straw.
3. the method for stalk fermentation according to claim 1, which is characterized in that the step（3）Middle activated liquid culture
Base is according to the mass fraction, to take 1000 parts of distilled water, 20 ~ 25 parts of peptones, 8 ~ 12 parts of yeast extracts, 15 ~ 20 parts of glucose are mixed
Close, 121 DEG C sterilizing 20 min to get.
4. the method for stalk fermentation according to claim 1, which is characterized in that the step（4）Middle fermentation medium is
According to the mass fraction, take 20 ~ 25 portions of potatos, 2 ~ 5 parts of glucose, 0.2 ~ 0.5 part of ammonium tartrate, 0.25 ~ 0.5 part of manganese sulfate,
0.1 ~ 0.3 part of calcium chloride, 0.001 ~ 0.003 part of vitamin B, the mixing of 1000 parts of water, 121 DEG C of 20 min of sterilizing to get.
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