CN100445374C - Composite microorganism beta-dextranase and beta-glucosaccharase production method - Google Patents
Composite microorganism beta-dextranase and beta-glucosaccharase production method Download PDFInfo
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- CN100445374C CN100445374C CNB200610014479XA CN200610014479A CN100445374C CN 100445374 C CN100445374 C CN 100445374C CN B200610014479X A CNB200610014479X A CN B200610014479XA CN 200610014479 A CN200610014479 A CN 200610014479A CN 100445374 C CN100445374 C CN 100445374C
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- 241000228245 Aspergillus niger Species 0.000 claims abstract description 72
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- 238000000855 fermentation Methods 0.000 claims description 20
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention relates to a production method of composite microorganism beta-dextranase and beta-glucosaccharase. Viridin and Aspergillus niger are mixed and fermented to produce composite microorganism beta-dextranase and beta-glucosaccharase which are made into a solid enzyme preparation and a liquid enzyme preparation, inclined-plane culture, shaking culture of a shake flask and enrichment culture in a liquid seed tank are respectively carried out on viridin strain and Aspergillus niger strain, the viridin and the Aspergillus niger are mixed and thrown in a solid fermenting tank to be fermented after enrichment culture is carried out, the viridin and the Aspergillus niger are lixiviated and filtered, are concentrated through an ultra-filtering membrane and are added with a protecting agent after fermented, and then the liquid enzyme preparation is obtained; a concentrated enzyme solution is added with dextrin and then is dried and granulated, and then the solid enzyme preparation is obtained. The present invention has the advantages of scientific and reasonable technological design, low cost, less energy consumption, wide range of application, no discharge of waste liquid and slag and large market prospect, and fills the blank of the enzyme preparation variety in China, and symbiotic mixed culture can be carried out without antagonism reaction with each other on the viridin and the Aspergillus niger which are obtained through mixing, culturing and screening a plurality of fungus.
Description
Technical field
The present invention relates to a kind ofly, relate in particular to the making method of a kind of complex microorganism beta-glucanase and beta-glucosidase.
Background technology
In recent years, research direction for beta-glucan, mainly concentrate on seed selection and produce the high microorganisms producing bacterium of 1,4 beta-glucanase activity, and with the reflection index of activity of beta-glucanase as microorganisms producing bacterium vigor, the activity index of ignoring beta-glucosidase, cause the activity of beta-glucanase produced higher, but in actual applications, hydrolysis effect is undesirable.Trace it to its cause, just be that beta-glucosidase is very low in the enzyme component, in addition detect less than.Yet the shortage beta-glucosidase can not be degraded into glucose fully with cellobiose and procellose, has seriously influenced the hydrolysis efficiency to beta-glucan, has restricted the application of beta-glucanase.
Summary of the invention
Main purpose of the present invention is to solve the problem in the above-mentioned production beta-glucanase, a kind of utilize viride and the mixed fermentation of aspergillus niger complex microorganism are provided, producing beta-glucanase enzyme system constitutes complete, and have the prozyme of higher beta-glucoside enzyme activity, realize the complex microorganism beta-glucanase of complete hydrolysis beta-glucan and the making method of beta-glucosidase.
The technical solution adopted for the present invention to solve the technical problems is:
With viride (Trichoderma viride) and aspergillus niger (Aspergillus niger) mixed fermentation, make complex microorganism beta-glucanase and beta-glucosidase, complex microorganism beta-glucanase and beta-glucosidase are made solid zymin and liquid enzyme formulation, and the physico-chemical property of solid zymin is:
(1) enzyme activity: activity of beta-glucanase 16000mg glucose/gh, beta-glucoside enzyme activity 3300 μ g glucose/gmin,
(2) granularity: preparation can pass through No. 3 standard sieves,
(3) moisture: water content is lower than 8%,
(4) Oranoleptic indicator: yellow pulvis, the color and luster homogeneous,
(5) sanitary index: the every gram total plate count of solid zymin is less than 10,000;
The physico-chemical property of liquid enzyme formulation is:
(1) product enzyme activity: activity of beta-glucanase 4500mg glucose/gh, beta-glucoside enzyme activity 860 μ g glucose/gmin,
(2) Oranoleptic indicator: dark-brown liquid, the color and luster homogeneous,
(3) sanitary index: every milliliter of total plate count is less than 50,000.
Beta-glucanase belongs to hydrolase, and its enzyme is that family mainly comprises interior β-1,3 dextranase, (E.C.3.2.1.39).Interior β-1,3,1,4 dextranase (E.C.3.2.1.73), outer 3-1,3 dextranases (E.C.3.2.1.58), outer β-1,4 dextranases (E.C.3.2.1.74), beta-glucanase have comprised that all enzymes that can decompose the glucose polymer of β-glycosidic link chain one-tenth are.They have important effect to the beta-glucan in hydrolysis barley, the rye.Hydrolysate mainly is β glucose, cellobiose and procellose.Beta-glucosidase (β-1,4-glucosidase E.C 3.2.1.21) is called for short BG, mainly acts on β-(1,4) glycosidic link, and it can be degraded into glucose with the cellobiose that inscribe, exoglucanase hydrolysis are got off.Beta-glucosidase is very big to the effect of reaction kinetics, because the primary product of inscribe and excision enzyme hydrolysis is a cellobiose, carrying out along with reaction, the product accumulation of cellobiose will make the vigor of excision enzyme be suppressed, reaction is carried out expeditiously, cellobiose must be transformed into other form, and beta-glucosidase has exactly been born this function.
Making method of the present invention is viride bacterial classification and aspergillus niger strain bevel bacterial classification and preservation respectively;
Viride employing czapek's solution (1L) (Czapek`s agar, CZ), substratum is:
Sucrose 30g, SODIUMNITRATE 2.0g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate heptahydrate 0.5g, Repone K 10.5g, ferrous sulfate 0.01g, agar 15g, pH 7.2-7.4;
Aspergillus niger employing potato substratum (1L) (Potato dextrose agar, PDA), substratum is:
Potato 200g, glucose 20g, dipotassium hydrogen phosphate 3g, magnesium sulfate heptahydrate 1.5g, agar 12g, pH nature;
The slant strains of viride and aspergillus niger is cultivated: viride and aspergillus niger use corresponding substratum respectively, inoculum size 5-10%, and static cultivation is 20-48 hour under 28-30 ℃ of condition, puts into 4 ℃ of refrigerators and preserves.
| Bacterium name (Chinese, Latin) | Numbering | Slant medium |
| Viride Trichoderma viride | As 3.3711 | Czapek's solution (1L) (Czapek`s agar.CZ): sucrose 30g, NaNO 3 2.0g，K 2HPO 41.0g， MgSO 4·7H 2O 0.5g，KCl 0.5g，FeSO 40.01g, KCl0.5g, agar 15g, pH 7.2-7.4; |
| Aspergillus niger Aspergillus niger | As 3.1858 | Potato substratum (1L) (Potato dextrose agar.PDA): potato 200g, glucose 20g, KH 2PO 43g， MgSO 4·7H 2O 1.5g, agar 12g, pH nature; |
The slant strains of viride and aspergillus niger is seeded in 500 milliliters of triangles separately respectively shakes in the bottle, shaking culture on shaking table, the be mixed substratum proportioning material of viride and aspergillus niger shakes in the bottle 100 milliliters every bottle to the substratum proportioning material branch triangle of packing in proportion, place in the high-pressure sterilizing pot, 121 ℃ of design temperatures were sterilized 30 minutes, behind the naturally cooling, substratum proportioning material in triangle shakes bottle is inoculated viride bacterial classification, aspergillus niger strain separately respectively, carries out shake-flask culture;
Shake-flask culture condition: temperature 28-30 ℃, rotating speed 180-250 rev/min, inoculum size 1-3% cultivates 48-60h.
Respectively the slant strains of the slant strains of viride and aspergillus niger is seeded in respectively to shake separately and carries out shaking culture in the bottle, the substratum proportioning material of packing in proportion respectively in shaking bottle, the shaking culture of the slant strains of the suitable viride of substratum proportioning material and the slant strains of aspergillus niger is made seed liquor.
Respectively the seed liquor of viride in the liquid shaking bottle and aspergillus niger being inoculated into 1 ton of liquid seeds jar internal breeding more separately respectively cultivates, the substratum proportioning material of viride and aspergillus niger is mixed, in proportion substratum proportioning material branch is packed in the seeding tank, 120 ℃, 3 normal atmosphere of design temperature, autoclave sterilization 45 minutes, behind the naturally cooling, the substratum proportioning material in the liquid seeds jar is inoculated viride bacterial classification, aspergillus niger strain separately respectively, carries out seed tank culture;
The seed tank culture condition; Temperature 28-30 ℃, ventilation 9-13m
3/ h, tank pressure 0.02-0.09Mpa, inoculum size 3-7%, time 60-72 hour.
Respectively viride seed liquor and aspergillus niger seed liquor are inoculated into respectively separately and carry out multiplication culture in the liquid seeds jar, the substratum proportioning material of in the liquid seeds jar, packing in proportion respectively, the multiplication culture of suitable viride seed liquor of substratum proportioning material and aspergillus niger seed liquor.
Viride behind the multiplication culture and aspergillus niger are mixed the input solid-state fermentation tank, ferment, the solid state fermentation raw material is mixed, solid state fermentation raw material in proportion is mixed, pack into the solid state fermentation raw material in the rotary spherical digester and mix, 120 ℃, 3 normal atmosphere of design temperature, through autoclave sterilization about 60 minutes, make solid state fermentation raw material naturally cooling, again with viride in the seeding tank and aspergillus niger in 2.5～3: 1 ratio is evenly sprayed and is inoculated in the solid state fermentation raw material, and postvaccinal solid state fermentation raw material aseptic delivery in solid-state fermenter, carry out solid state fermentation;
Solid state fermentation conditions: temperature 28-30 ℃, water content: 60%, atmospheric moisture control 80%-90%, ventilation 9-13m
3/ h, tank pressure 0.02-0.09Mpa, inoculum size 10-15%, fermentation time 84-96 hour.
In solid-state fermentation tank, be mixed in proportion the viride and the aspergillus niger that drop into behind the multiplication culture and carry out solid state fermentation, adopt two kinds of microorganism mixed fermentations, these two kinds of microorganisms can symbiosis not have the antagonism reaction, and the enzyme that is produced system is complementary, for obtaining to contain the prozyme that a large amount of beta-glucan materials are degraded into glucose fully with natural, the high viride of activity of beta-glucanase will be produced, the aspergillus niger energetic with producing beta-glucosidase mixes, produce efficient composite enzyme, realize that natural beta-glucan is converted into glucose.The solid state fermentation raw material of packing in proportion in solid-state fermentation tank evenly sprays viride and aspergillus niger in proportion and to be inoculated in the solid state fermentation raw material, carries out solid state fermentation.
After the fermentation, take out the solid fermentation material in solid-state fermentation tank, drop in the lixiviate groove, add flooding, the addition of water is 9 times of solid fermentation material, about 3 hours of lixiviate.
With three～four days song of solid state fermentation taking-up, in the impouring lixiviate groove, add a certain amount of water, the solid fermentation material: water=1: 9 (by weight/volume), carry out about 3 hours lixiviate.
After lixiviate finished, discharging lixiviate groove clear liquid at the middle and upper levels squeezed remaining solid lixiviate material more earlier; Filter supernatant liquid and pressed liquor acquisition filtered liquid with the plate and frame filter, filtered liquid is filtered with the wire wound filter again, the final filtrate that obtains is placed in the storage tank.
After the lixiviate, supernatant liquid is emitted earlier, again remaining solid lixiviate material is squeezed, then collected lixiviate supernatant liquid and pressed liquor are filtered through the plate and frame filter.Filtered liquid through Plate Filtration filters through the wire wound filter once again, and gained filtrate is placed in the storage tank.
Take out filtrate from storage tank, use the ultra-filtration membrane concentrated filtrate, the pressure of ultrafiltration is less than 0.1Mpa, 3 hours/6 tons filtrates of concentration time, and every batch of filtrate is condensed to 300～400 liters concentrated enzyme liquid.
Filtrate in the storage tank is carried out ultra-filtration membrane concentrate, the pressure of ultrafiltration should be less than 0.1Mpa, concentration time 3 hours/batches, and every batch of concentrated enzyme liquid is concentrated about 20 times.
Mix 95% industrial alcohol and glycerine in concentrating enzyme liquid, make 95% industrial alcohol and glycerine, the final concentration in concentrating enzyme liquid reaches 10% and 20% respectively, stirs the back and obtains liquid enzyme formulation, the packing warehouse-in.
Add protective material in concentrated enzyme liquid, protective material is 95% industrial alcohol and glycerine, makes the two final concentration in concentrating enzyme liquid reach 10% and 20% respectively, and in homogenate abundant stirring and evenly mixing, the product of this moment is a liquid enzyme formulation.
In concentrated enzyme liquid, add dextrin as carrier; make the final concentration of dextrin in concentrating enzyme liquid reach 10%; concentrated enzyme liquid after fully stirring is sent into spraying rotary fluid drying-granulating equipment; concentrate enzyme liquid and spray into the drying-granulating device through spraying gun; temperature 45-55 ℃; convection drying becomes solid particulate to obtain the solid zymin, the packing warehouse-in.
In concentrated enzyme liquid, add dextrin as carrier, make its final concentration in concentrating enzyme liquid reach 10%, and in homogenate abundant stirring and evenly mixing.The concentrated enzyme liquid that adds the dextrin carrier is made solid particulate through spraying rotary fluid drying-granulating equipment, the equipment preheating, high temperature drying air (140 ℃) is at system's internal recycle, realize intrasystem sterilization 15 minutes, sterilization reaches job requirement and starts source material delivery system, raw material sprays into drying-granulating device (temperature 45-55 ℃) through spraying gun, and convection drying becomes particle, and the product of acquisition is the solid zymin.
Viride and aspergillus niger need shake bottle shaking culture and the internal breeding of liquid seeds jar respectively and cultivate in the manufacture craft process, and viride and aspergillus niger shake bottle shaking culture and the internal breeding of liquid seeds jar and cultivate employed substratum proportioning material and be every liter and contain:
Straw powder 10-15 part, wheat bran 10-15 part, glucose 5-8 part, yeast powder 1-3 part, dipotassium hydrogen phosphate 3.6-4.6 part, magnesium sulfate heptahydrate 1.5-2.5 part, pH 5.0.
Viride behind the multiplication culture and aspergillus niger mix the input solid-state fermentation tank and ferment, and the solid state fermentation raw material that uses in the solid-state fermentation tank is every kilogram and contains:
Straw powder 270-300 part, wheat bran 80-100 part, brewer's grains 40-80 part, corn steep liquor 2.9-4 part, ammonium sulfate 1.5-3 part, dipotassium hydrogen phosphate 3.6-4.6 part, magnesium sulfate heptahydrate 1.5-2.5 part, water 600-800 part, pH 5.0.
The present invention is the making method of complex microorganism beta-glucanase and beta-glucosidase.Technological design science, rationally, by to many fungal strains mixed culture screening obtain viride and this two strains bacterial classification of aspergillus niger can the symbiosis mixed culture, do not have the antagonism reaction mutually.Substratum proportioning material and solid state fermentation raw material prescription compatibility science, reasonable, cost of manufacture is low, energy consumption is low, pollution-free, energy-conserving and environment-protective are by screening, purifying, adopt viride and two kinds of microorganism mixed fermentations of aspergillus niger, these two kinds of microorganisms must symbiosis not have the antagonism reaction, and the enzyme that is produced system is complementary, and the culture condition advanced person of enzyme is produced in growth.Utilize viride and aspergillus niger mixed culture to produce this prozyme, wherein viride has the advantages that to produce higher 1,4 beta-glucanase activity, aspergillus niger has the advantages that to produce higher activity of beta-glucosidase, mixing can be removed the feedback inhibition of cellobiose to viride secretion beta-glucanase, makes viride produce beta-glucanase efficient and improves.Obtain by condition test several kinds of carbon source and nitrogenous source and inorganic salt, suitable viride and aspergillus niger mixed culture, and the component that helps beta-glucanase and beta-glucosidase excretory substratum proportioning material and solid state fermentation raw material, especially the solid state fermentation raw materials cost is low, less energy consumption, no waste liquid and waste sludge discharge are a kind of modes of production of Sustainable development energy-conserving and environment-protective.
The present invention is directed to solid state fermentation beta-glucanase enzyme system forms unreasonable, shortcoming that can not the complete hydrolysis beta-glucan, utilize viride and the mixed fermentation of aspergillus niger complex microorganism, producing beta-glucanase enzyme system constitutes complete, and have the prozyme of higher beta-glucoside enzyme activity, thereby realize the purpose of complete hydrolysis beta-glucan.Traditional solid-state fermentation process is because working condition is extensive, can not realize sterile culture, so can only produce crude zyme preparation, the present invention is by the innovation of operational path and equipment, realize production food grade and the pure zymin of SILVER REAGENT, improve the quality of product greatly, increased value-added content of product, made it to have more the market competitiveness.
Activity of beta-glucanase that the present invention produces and beta-glucosidase prozyme stable p H scope be at 3-8, and can act on cellulosic material of different nature, so the scope of application is wide in range, can be used for industrial production such as feed, weaving, papermaking.Beta-glucanase and beta-glucosidase prozyme are the important component parts of cotton fabric biological enzyme, mix with amylase, can remove Mierocrystalline cellulose Symbiont on the cotton fibre and the starch size on the cotton fabric warp thread with it under certain condition, improve the wettability of cotton fabric, be beneficial to following process.This prozyme can also be applied to the back arrangement processing of cotton textiles, and cotton fibre is carried out surface modification, with outward appearance, the feel of improving fabric with take performance, improves the class and the added value of product.Beta-glucanase and beta-glucosidase prozyme also can be used for deinking, in conjunction with processing, than chemical deinking better effects if, can also improve the intensity and the drainability thereof of slurry with this prozyme and hemicellulase.The prozyme of beta-glucanase and beta-glucosidase has been filled up the blank of China's zymin kind, and has been had bigger market outlook.
Description of drawings
Below in conjunction with drawings and Examples to the detailed description of the invention.
The manufacture craft schema of Fig. 1 complex microorganism beta-glucanase and beta-glucosidase
Embodiment
Embodiment 1
With viride (Trichoderma viride) and aspergillus niger (Aspergillus niger) mixed fermentation, make complex microorganism beta-glucanase and beta-glucosidase, complex microorganism beta-glucanase and beta-glucosidase are made solid zymin and liquid enzyme formulation, and the physico-chemical property of solid zymin is:
(1) enzyme activity: activity of beta-glucanase 16000mg glucose/gh, beta-glucoside enzyme activity 3300 μ g glucose/gmin,
(2) granularity: preparation can pass through No. 3 standard sieves,
(3) moisture: water content is lower than 8%,
(4) Oranoleptic indicator: yellow pulvis, the color and luster homogeneous,
(5) sanitary index: the every gram total plate count of solid zymin is less than 10,000;
The physico-chemical property of liquid enzyme formulation is:
(1) product enzyme activity: activity of beta-glucanase 4500mg glucose/gh, beta-glucoside enzyme activity 860 μ g glucose/gmin,
(2) Oranoleptic indicator: dark-brown liquid, the color and luster homogeneous,
(3) sanitary index: every milliliter of total plate count is less than 50,000, as shown in Figure 1.
Embodiment 2
Viride bacterial classification and aspergillus niger strain bevel bacterial classification and preservation respectively;
Viride employing czapek's solution (1L) (Czapek`s agar, CZ), substratum is:
Sucrose 30g, SODIUMNITRATE 2.0g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate heptahydrate 0.5g, Repone K 10.5g, ferrous sulfate 0.01g, agar 15g, pH 7.2-7.4;
Aspergillus niger employing potato substratum (1L) (Potato dextrose agar, PDA), substratum is:
Potato 200g, glucose 20g, dipotassium hydrogen phosphate 3g, magnesium sulfate heptahydrate 1.5g, agar 12g, pH nature;
The slant strains of viride and aspergillus niger is cultivated: viride and aspergillus niger use corresponding substratum respectively, inoculum size 5-10%, and static cultivation is 20-48 hour under 28-30 ℃ of condition, puts into 4 ℃ of refrigerators and preserves, as shown in Figure 1.
Embodiment 3
The slant strains of viride and aspergillus niger is seeded in 500 milliliters of triangles separately respectively shakes in the bottle, shaking culture on shaking table, the be mixed substratum proportioning material of viride and aspergillus niger shakes in the bottle 100 milliliters every bottle to the substratum proportioning material branch triangle of packing in proportion, place in the high-pressure sterilizing pot, 121 ℃ of design temperatures were sterilized 30 minutes, behind the naturally cooling, substratum proportioning material in triangle shakes bottle is inoculated viride bacterial classification, aspergillus niger strain separately respectively, carries out shake-flask culture;
Shake-flask culture condition: temperature 28-30 ℃, rotating speed 180-250 rev/min, inoculum size 1-3% cultivates 48-60h, as shown in Figure 1.
Embodiment 4
Respectively the seed liquor of viride in the liquid shaking bottle and aspergillus niger being inoculated into 1 ton of liquid seeds jar internal breeding more separately respectively cultivates, the substratum proportioning material of viride and aspergillus niger is mixed, in proportion substratum proportioning material branch is packed in the seeding tank, 120 ℃, 3 normal atmosphere of design temperature, autoclave sterilization 45 minutes, behind the naturally cooling, the substratum proportioning material in the liquid seeds jar is inoculated viride bacterial classification, aspergillus niger strain separately respectively, carries out seed tank culture;
Seed tank culture condition: temperature 28-30 ℃, ventilation 9-13m
3/ h, tank pressure 0.02-0.09Mpa, inoculum size 3-7%, time 60-72 hour, as shown in Figure 1.
Embodiment 5
Viride behind the multiplication culture and aspergillus niger are mixed the input solid-state fermentation tank, ferment, the solid state fermentation raw material is mixed, solid state fermentation raw material in proportion is mixed, pack into the solid state fermentation raw material in the rotary spherical digester and mix, 120 ℃, 3 normal atmosphere of design temperature, through autoclave sterilization about 60 minutes, make solid state fermentation raw material naturally cooling, again with viride in the seeding tank and aspergillus niger in 2.5～3: 1 ratio is evenly sprayed and is inoculated in the solid state fermentation raw material, and postvaccinal solid state fermentation raw material aseptic delivery in solid-state fermenter, carry out solid state fermentation;
Solid state fermentation conditions: temperature 28-30 ℃, water content: 60%, atmospheric moisture control 80%-90%, ventilation 9-13m
3/ h, tank pressure 0.02-0.09Mpa, inoculum size 10-15%, fermentation time 84-96 hour, as shown in Figure 1.
Embodiment 6
After the fermentation, take out the solid fermentation material in solid-state fermentation tank, drop in the lixiviate groove, add flooding, the addition of water is 9 times of solid fermentation material, about 3 hours of lixiviate.
After lixiviate finished, discharging lixiviate groove clear liquid at the middle and upper levels squeezed remaining solid lixiviate material more earlier; Filter supernatant liquid and pressed liquor acquisition filtered liquid with the plate and frame filter, filtered liquid is filtered with the wire wound filter again, the final filtrate that obtains is placed in the storage tank.
Take out filtrate from storage tank, use the ultra-filtration membrane concentrated filtrate, the pressure of ultrafiltration is less than 0.1Mpa, 3 hours/6 tons filtrates of concentration time, and every batch of filtrate is condensed to 300～400 liters concentrated enzyme liquid.
Mix 95% industrial alcohol and glycerine in concentrating enzyme liquid, make 95% industrial alcohol and glycerine, the final concentration in concentrating enzyme liquid reaches 10% and 20% respectively, and stir the back and obtain liquid enzyme formulation, the packing warehouse-in, as shown in Figure 1.
Embodiment 7
In concentrated enzyme liquid, add dextrin as carrier; make the final concentration of dextrin in concentrating enzyme liquid reach 10%; concentrated enzyme liquid after fully stirring is sent into spraying rotary fluid drying-granulating equipment; concentrate enzyme liquid and spray into the drying-granulating device through spraying gun; temperature 45-55 ℃; convection drying becomes solid particulate to obtain the solid zymin, the packing warehouse-in, as shown in Figure 1.
Embodiment 8 (high dosage)
Viride and aspergillus niger need shake bottle shaking culture and liquid seeds jar internal breeding cultivation respectively in the manufacture craft process, viride and aspergillus niger shake bottle shaking culture and the internal breeding of liquid seeds jar and cultivate employed substratum proportioning material and be every liter and contain (weight ratio, every part is 1g):
Straw powder 15g, wheat bran 15g, glucose 8g, yeast powder 3g, dipotassium hydrogen phosphate 4.6g, magnesium sulfate heptahydrate 2.5g, pH 5.0, as shown in Figure 1.
Embodiment 9 (low dosage)
Viride and aspergillus niger need shake bottle shaking culture and liquid seeds jar internal breeding cultivation respectively in the manufacture craft process, viride and aspergillus niger shake bottle shaking culture and the internal breeding of liquid seeds jar and cultivate employed substratum proportioning material and be every liter and contain (weight ratio, every part is 1g):
Straw powder 10g, wheat bran 10g, glucose 5g, yeast powder 1g, dipotassium hydrogen phosphate 3.6g, magnesium sulfate heptahydrate 1.5g, pH 5.0, as shown in Figure 1.
Embodiment 10 (median dose)
Viride and aspergillus niger need shake bottle shaking culture and liquid seeds jar internal breeding cultivation respectively in the manufacture craft process, viride and aspergillus niger shake bottle shaking culture and the internal breeding of liquid seeds jar and cultivate employed substratum proportioning material and be every liter and contain (weight ratio, every part is 1g):
Straw powder 12.5g, wheat bran 12.5g, glucose 6.5g, yeast powder 2g, dipotassium hydrogen phosphate 4.1g, magnesium sulfate heptahydrate 2g, pH 5.0, as shown in Figure 1.
Embodiment 11 (high dosage)
Viride behind the multiplication culture and aspergillus niger mix the input solid-state fermentation tank and ferment, and the solid state fermentation raw material that uses in the solid-state fermentation tank is every kilogram and contains (weight ratio, every part is 1g):
Straw powder 300g, wheat bran 100g, brewer's grains 80g, corn steep liquor 4g, ammonium sulfate 3g, dipotassium hydrogen phosphate 4.6g, magnesium sulfate heptahydrate 2.5g, water 800g, pH 5.0, as shown in Figure 1.
Embodiment 12 (low dosage)
Viride behind the multiplication culture and aspergillus niger mix the input solid-state fermentation tank and ferment, and the solid state fermentation raw material that uses in the solid-state fermentation tank is every kilogram and contains (weight ratio, every part is 1g):
Straw powder 270g, wheat bran 80g, brewer's grains 40g, corn steep liquor 2.9g, ammonium sulfate 1.5g, dipotassium hydrogen phosphate 3.6g, magnesium sulfate heptahydrate 1.5g, water 600g, pH 5.0, as shown in Figure 1.
Embodiment 13 (median dose)
Viride behind the multiplication culture and aspergillus niger mix the input solid-state fermentation tank and ferment, and the solid state fermentation raw material that uses in the solid-state fermentation tank is every kilogram and contains (weight ratio, every part is 1g):
Straw powder 285g, wheat bran 90g, brewer's grains 60g, corn steep liquor 3.45g, ammonium sulfate 2.25g, dipotassium hydrogen phosphate 4.1g, magnesium sulfate heptahydrate 4g, water 700g, pH 5.0, as shown in Figure 1.
Claims (1)
1, the making method of a kind of complex microorganism beta-glucanase and beta-glucosidase, it is characterized in that viride (Trichoderma viride) and aspergillus niger (Aspergillus niger) mixed fermentation, make complex microorganism beta-glucanase and beta-glucosidase, complex microorganism beta-glucanase and beta-glucosidase are made solid zymin and liquid enzyme formulation, and the physico-chemical property of solid zymin is:
(1) enzyme activity: activity of beta-glucanase 16000mg glucose/gh, beta-glucoside enzyme activity 3300 μ g glucose/gmin,
(2) granularity: preparation can pass through No. 3 standard sieves,
(3) moisture: water content is lower than 8%,
(4) Oranoleptic indicator: yellow pulvis, the color and luster homogeneous,
(5) sanitary index: the every gram total plate count of solid zymin is less than 10,000;
The physico-chemical property of liquid enzyme formulation is:
(1) product enzyme activity: activity of beta-glucanase 4500mg glucose/gh, beta-glucoside enzyme activity 860 μ g glucose/gmin,
(2) Oranoleptic indicator: dark-brown liquid, the color and luster homogeneous, tart flavour slightly, pH value 4～5,
(3) sanitary index: every milliliter of total plate count is less than 50,000;
This making method is viride bacterial classification and aspergillus niger strain bevel bacterial classification and preservation respectively;
Viride adopts 1 liter of czapek's solution, and this czapek's solution is:
Sucrose 30g, SODIUMNITRATE 2.0g, dipotassium hydrogen phosphate 1.0g, magnesium sulfate heptahydrate 0.5g, Repone K 10.5g, ferrous sulfate 0.01g, agar 15g, pH 7.2-7.4;
Aspergillus niger adopts 1 liter of potato substratum, and this potato substratum is:
Potato 200g, glucose 20g, dipotassium hydrogen phosphate 3g, magnesium sulfate heptahydrate 1.5g, agar 12g, pH nature;
Viride and aspergillus niger use corresponding substratum respectively, inoculum size 5-10%, and static cultivation is 20-48 hour under 28-30 ℃ of condition, puts into 4 ℃ of refrigerators and preserves;
The slant strains of viride and aspergillus niger is seeded in 500 milliliters of triangles separately respectively shakes in the bottle, shaking culture on shaking table, the substratum proportioning material of be mixed viride and aspergillus niger, substratum proportioning material is every liter and contains:
Straw powder 10-15 part, wheat bran 10-15 part, glucose 5-8 part, yeast powder 1-3 part, dipotassium hydrogen phosphate 3.6-4.6 part, magnesium sulfate heptahydrate 1.5-2.5 part, pH 5.0;
In proportion the substratum proportioning material branch triangle of packing into is shaken in the bottle, 100 milliliters every bottle, place in the high-pressure sterilizing pot, 121 ℃ of design temperatures, sterilized 30 minutes, behind the naturally cooling, the substratum proportioning material in triangle shakes bottle is inoculated viride bacterial classification, aspergillus niger strain separately respectively, carries out shake-flask culture;
Shake-flask culture condition: temperature 28-30 ℃, rotating speed 180-250 rev/min, inoculum size 1-3% cultivates 48-60h;
Respectively the seed liquor of viride in the liquid shaking bottle and aspergillus niger being inoculated into 1 ton of liquid seeds jar internal breeding more separately respectively cultivates, the substratum proportioning material of viride and aspergillus niger is mixed, in proportion substratum proportioning material branch is packed in the seeding tank, 120 ℃, 3 normal atmosphere of design temperature, autoclave sterilization 45 minutes, behind the naturally cooling, the substratum proportioning material in the liquid seeds jar is inoculated viride bacterial classification, aspergillus niger strain separately respectively, carries out seed tank culture;
Seed tank culture condition: temperature 28-30 ℃, ventilation 9-13m
3/ h, tank pressure 0.02-0.09Mpa, inoculum size 3-7%, time 60-72 hour;
The solid state fermentation raw material that is mixed, the solid state fermentation raw material is every kilogram and contains:
Straw powder 270-300 part, wheat bran 80-100 part, brewer's grains 40-80 part, corn steep liquor 2.9-4 part, ammonium sulfate 1.5-3 part, dipotassium hydrogen phosphate 3.6-4.6 part, magnesium sulfate heptahydrate 1.5-2.5 part, water 600-800 part, pH 5.0;
Solid state fermentation raw material in proportion is mixed, pack into the solid state fermentation raw material in the rotary spherical digester and mix, 120 ℃, 3 normal atmosphere of design temperature, through autoclave sterilization 60 minutes, make solid state fermentation raw material naturally cooling, again with viride in the seeding tank and aspergillus niger in 2.5～3: 1 ratio is evenly sprayed and is inoculated in the solid state fermentation raw material, and postvaccinal solid state fermentation raw material aseptic delivery in solid-state fermenter, carry out solid state fermentation;
Solid state fermentation conditions: temperature 28-30 ℃, water content: 60%, atmospheric moisture control 80%-90%, ventilation 9-13m
3/ h, tank pressure 0.02-0.09Mpa, inoculum size 10-15%, fermentation time 84-96 hour;
After the fermentation, take out the solid fermentation material in solid-state fermentation tank, drop in the lixiviate groove, add flooding, the addition of water is 9 times of solid fermentation material, lixiviate 3 hours;
After lixiviate finished, discharging lixiviate groove clear liquid at the middle and upper levels squeezed remaining solid lixiviate material more earlier; Filter supernatant liquid and pressed liquor acquisition filtered liquid with the plate and frame filter, filtered liquid is filtered with the wire wound filter again, the final filtrate that obtains is placed in the storage tank;
Take out filtrate from storage tank, use the ultra-filtration membrane concentrated filtrate, the pressure of ultrafiltration is less than 0.1Mpa, 3 hours/6 tons filtrates of concentration time, and every batch of filtrate is condensed to 300～400 liters concentrated enzyme liquid;
In concentrating enzyme liquid, mix 95% industrial alcohol and glycerine, make 95% industrial alcohol and the final concentration of glycerine in concentrating enzyme liquid reach 10% and 20% respectively, stir the back and obtain liquid enzyme formulation, the packing warehouse-in;
In concentrated enzyme liquid, add dextrin as carrier; make the final concentration of dextrin in concentrating enzyme liquid reach 10%; concentrated enzyme liquid after fully stirring is sent into spraying rotary fluid drying-granulating equipment; concentrate enzyme liquid and spray into the drying-granulating device through spraying gun; temperature 45-55 ℃; convection drying becomes solid particulate to obtain the solid zymin, the packing warehouse-in.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1385519A (en) * | 2002-04-19 | 2002-12-18 | 浙江大学 | Li's Trichoderma strains and use thereof |
| WO2004044130A2 (en) * | 2002-11-13 | 2004-05-27 | Kemin Industries, Inc. | Microbial enzymes having multiple improved characteristics |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1385519A (en) * | 2002-04-19 | 2002-12-18 | 浙江大学 | Li's Trichoderma strains and use thereof |
| WO2004044130A2 (en) * | 2002-11-13 | 2004-05-27 | Kemin Industries, Inc. | Microbial enzymes having multiple improved characteristics |
Non-Patent Citations (2)
| Title |
|---|
| Enhanced cellobiohydrolase production from Aspergillus ustus and Trichoderma harzianum. Macris, BJ., et al.Enzyme and Microbial Technology,Vol.8 No.3. 1986 |
| Enhanced cellobiohydrolase production from Aspergillus ustus and Trichoderma harzianum. Macris, BJ., et al.Enzyme and Microbial Technology,Vol.8 No.3. 1986 * |
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