CN102061264A - Naturally-combined complex enzyme for feeding and preparation method thereof - Google Patents
Naturally-combined complex enzyme for feeding and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the field of feed, in particular relating to a naturally-combined complex enzyme for feeding. The inventor of the complex enzyme firstly reforms Trichoderma sp., Aspergillus usamii and Aspergillus niger in the prior art; ultraviolet mutation, ultraviolet nitrous acid mutation and ultraviolet nitrosoguanidine composite mutation are adopted, and mutant strains are screened and eliminated step by step; and finally, the excellent strains are screened by a fermentation performance test to obtain high-yield enzyme performance strains, i.e. Trichoderma Cx631, Aspergillus usamii By247 and Aspergillus niger M-163. The complex enzyme provided by the invention is main enzyme type of the complex fermentation product of the Trichoderma sp., Aspergillus usamii and Aspergillus niger and is composed of xylanase, mannose, cellulose and alpha-galactosidase. The naturally-combined complex enzyme is from the same fermentation system, and enzymes do not have antagonism and have stronger mutual synergy effect. The complex enzyme has a wide use range when serving as the feed additive for birds, livestock and aquatic cultured animals.
Description
Technical field
The present invention relates to field of fodder, particularly, the present invention relates to a kind of complex enzyme for feed of combination naturally.
Background technology
Complex enzyme for feed is the novel activated feed additive of a class, and it is applied in and starts from the seventies in 20th century abroad, has domesticly begun to be applied in the livestock and poultry cultivation since the eighties in 20th century.
At present, it is less that fodder industry is used the single enzyme preparation, is the compound enzymic preparation product mostly.Prozyme is the uniform mixed system of plurality of enzymes.The production of complex enzyme for feed is mainly by following 3 approach: (1) single enzyme is composite; (2) multiple bacteria compound fermentation of the single enzyme of product; (3) the single culture fermentation of product plurality of enzymes.Three kinds are produced prozyme mode relative merits and are respectively: the composite prozyme enzyme of single enzyme only limits to composite several enzymes, can not satisfy complicated feed diet composition fully, and may be the mixing of complete different sources enzyme, may there be antagonistic action between each enzyme, synergy will weaken greatly mutually, even may lose synergy fully, system stability and enzyme stability alive all will be greatly affected greatly; Advantage is to prepare various prescriptions as required arbitrarily, and is composite flexible.Produce only outstanding a or two kind of enzyme of the general meeting of single culture fermentation of plurality of enzymes, and other related enzyme activity can be lower; Advantage is the more simple to operation and control than multi-strain fermentation aspect the production fermentation, and the successful technical difficulty of fermenting is little.The enzyme process relative complex is produced in each growth of multiple bacteria compound fermentation; Advantage be the enzyme that produces system more comprehensively, derive from the prozyme that same system produces, enzyme system is more stable, collaborative promoter action is stronger between each enzyme, can be better at feedstuff raw material complicated ingredient performance zymin maximum effect.
Mixed fermentation has the symbiosis of many bacterium, and enzyme system is complementary, and energy-conservation, the simplified process equipment of saving of labor overcomes the excessive characteristics of intermediate product concentration.Be suitable at the requirement of feed diet complicated ingredient and produce the comprehensive prozyme of enzyme system.
Summary of the invention
The purpose of this invention is to provide a kind of complex enzyme for feed of combination naturally with the preparation of multiple bacteria compound fermentation technology.
A further object of the present invention provides the method for producing complex enzyme for feed with the multiple bacteria compound fermentation solid fermentation, promptly on solid medium based on the wheat bran dregs of beans, by certain principle inoculation different microorganisms bacterial classification, produce efficient composite enzyme through solid fermentation, can directly apply in poultry, fowl, the aquatic animal feed, eliminate the antinutritional factor in the feed, improve efficiency of feed utilization.
It is mould that the present inventor at first transforms wood of the prior art, Aspergillus usamii, and aspergillus niger, with the preservation strain Cx-28 of company, By-31, M-17 is as starting strain, through ultraviolet mutagenesis, the mutagenesis of ultraviolet nitrous acid, ultraviolet ray nitrosoguanidine complex mutation, then the mutant strain step-sizing is eliminated, at last strain excellent being obtained high yield enzyme performance bacterial strain through leavening property test screening is respectively wooden its deposit number of mould Cx631 and is: CGMCC NO.4288, its deposit number of Aspergillus usamii By247 is: CGMCC NO.4290, its deposit number of aspergillus niger M-163 is: CGMCC NO.4289.
According to prozyme of the present invention be that above-mentioned wood is mould, the main enzyme of the complex ferment product of Aspergillus usamii and aspergillus niger consists of zytase, mannase, cellulase, alpha-galactosidase.
The spore of wood of the present invention is mould, Aspergillus usamii, aspergillus niger is pressed different ratios inoculation culture simultaneously, and no obvious antagonistic action between each bacterium can the arbitrary proportion mixed growth, the speed difference of just growing (aspergillus niger＞Aspergillus usamii＞wood is mould).Therefore, can control the growth of each thalline of mixed fermentation by inoculative proportion.For the complexity of simplifying three inoculating proportions changes, behind the suitable proportion of the mixed fermentation of the definite earlier combination of bacterial classification in twos of the present invention, remake comprehensive.As shown in Figure 1, wood inoculative proportion mould and that Aspergillus usamii is suitable is 2.5-2.0: 1; According to shown in Figure 2, the suitable inoculative proportion of Aspergillus usamii and aspergillus niger is 1: 1-0.6; According to shown in Figure 3, the mould inoculative proportion 2.5-1.5 suitable of wood: 1 with aspergillus niger.Comprehensive determine that wood is mould, the inoculative proportion of Aspergillus usamii and aspergillus niger mixed fungus fermentation is 2.0～2.5: 1: 0.6～1.0.
According to prozyme of the present invention, wherein, described wood is mould, the complex ferment culture medium prescription of Aspergillus usamii and aspergillus niger is: wheat bran 70%-80%, dregs of beans 10%-15%, straw powder 5-10%, ammonium sulfate 2-3%, Sodium phosphate dibasic 1-1.5%, sal epsom 0.2%, material-water ratio 1: 1.2-1.5.
According to the complex enzyme for feed of combination naturally of the present invention, wherein, zymotechnique is: adopt bent room multi-layer solid fermentation process, spore suspension is sprayed in the solid fermentation substratum, stir, under the condition of control relative humidity 80%-95% under 30-32 ℃, cultivate 84-96h, then in 45-50 ℃ of following cryodrying.
According to prozyme of the present invention, have synergistic function between each enzyme, promptly be applied to external enzymolysis feedstuff raw material under the same conditions, the prozyme of more single enzyme combination has better hydrolysis result (is index with clean reducing sugar).
The method of the above-mentioned combination naturally of preparation according to the present invention complex enzyme for feed may further comprise the steps:
1) spore liquid of mould, Aspergillus usamii of preparation wood and aspergillus niger, the preparation method of the spore suspension of described three kinds of mould bacterial classifications is: wood is mould, Aspergillus usamii and three kinds of preservation kinds of aspergillus niger insert respectively in the wort agar slant medium, cultivate 84-96h down at 30-32 ℃, sterilized water washes spore, makes spore suspension.5% spore suspension inoculation with the seed culture medium quality stirs, and cultivates 72-86h down in 30-32 ℃, washes spore with sterilized water then, makes spore suspension;
2) preparation solid fermentation substratum, culture medium prescription is: wheat bran 70%-80%, dregs of beans 10%-15%, straw powder 5-10%, ammonium sulfate 2-3%, Sodium phosphate dibasic 1-1.5%, sal epsom 0.2%, material-water ratio 1: 1.2-1.5,121 ℃ of sterilization 30min;
3) solid fermentation: adopt bent room multi-layer solid fermentation process, different spore suspensions are sprayed in the solid fermentation substratum by a certain percentage, stir, under the condition of control relative humidity 80%-95% under 30-32 ℃, cultivate 84-96h, in 45-50 ℃ of following cryodrying, pulverize and obtain complex enzyme for feed of the present invention then.
The main enzyme that described prozyme product comprises is: zytase, mannase, cellulase, alpha-galactosidase etc.
The useful effect that the present invention has is:
1. from a large amount of bacterial classifications, screen the bacterial classification that obtains having the different enzymes of high yield;
2. by the multiple bacteria compound fermentation technology, each bacterial classification can be worked in coordination with the product plurality of enzymes in the solid fermentation substratum, and constitutive enzyme is the compound enzymic preparation of combination naturally that comprehensive enzyme is lived high;
3. this makes up the prozyme same fermentation system of originating naturally, does not have antagonistic action between each enzyme, and synergistic action effect is stronger mutually, and as the fodder additives of the cultivated animals of fowl, poultry, aquatic products, use range is wide;
4. be the main medium composition with vegetable raw material wheat bran, dregs of beans etc., adopt the disposable solid fermentation of many bacterial classifications, production process does not produce waste water and dregs, no secondary environmental pollution, and equipment is simple, and production cost is low.
Description of drawings
Fig. 1 wood mould with the Aspergillus usamii inoculative proportion to producing the influence of enzyme.
Fig. 2 Aspergillus usamii and aspergillus niger inoculative proportion are to producing the influence of enzyme.
Fig. 3 wood mould with the aspergillus niger inoculative proportion to producing the influence of enzyme.
Wood mould (trichoderma sp.Cx631) is stored in China Committee for Culture Collection of Microorganisms common micro-organisms center (preservation centre address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 01st, 2010, postcode: 100101), its deposit number is: CGMCC No.4288.
Aspergillus usamii (Aspergillus usamii By247), be stored in China Committee for Culture Collection of Microorganisms common micro-organisms center (preservation centre address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 01st, 2010, postcode: 100101), its deposit number is: CGMCC NO.4290
Aspergillus niger (Aspergillus niger M-163), be stored in China Committee for Culture Collection of Microorganisms common micro-organisms center (preservation centre address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on November 01st, 2010, postcode: 100101), its deposit number is: CGMCC NO.4289
Embodiment
1, starting strain
Wood is mould: the preservation strain Cx-28 of company (Hunan is opened Zhangjiajie National Forest Park soil sampling sub-sieve and obtained), main cellulase-producing etc.;
Aspergillus usamii: the preservation strain By-31 of company (Yongchuan, Chongqing Rui Te konjak products factory surrounding soil sub-sieve obtains), main product mannase, alpha-galactosidase etc.;
Aspergillus niger: the preservation strain M-17 of company (the normal emerging flour mill wheat bran accumulation surrounding soil in Shanyang County, Shaanxi is adopted the integration sieve and obtained), main product zytase.
The complex mutation breeding of 2 starting strains
Ultraviolet mutagenesis: ultraviolet lamp is opened preheating 20min, adjust spore suspension 10
6～10
7Individual/ml, get 5ml and place aseptic culture dish.Above-mentioned culture dish being placed on earlier on the magnetic stirring apparatus again, opening ware lid, is to shine 10min, dilution coating PDA flat board under the ultraviolet lamp of 20w at distance 30cm, power.
60Co gamma-rays radioinduction: select the enzyme bacterial strain that significantly improves alive behind ultraviolet mutagenesis, coating is sealed the ware mouth, irradiation dose 30 kilorads after cultivating.
Ultraviolet nitrous acid mutagenesis: draw the bacteria suspension 1ml that handles through uviolizing, add pH4.5 acetate buffer solution 2ml and 0.1mol/L sodium nitrite solution 1ml.Uv irradiating dosage: 120S, nitrous enzyme processing time: 30S, 60S, 90S, 120S.Dilution coating PDA flat board.
Ultraviolet ray nitrosoguanidine complex mutation: draw the bacteria suspension 1ml that handles through uviolizing, add nitroso guanidine solution 1ml and continue to handle.Uv irradiating dosage: 120S, nitrosoguanidine treatment time: 30S, 60S, 90S, 120S.Dilution coating PDA flat board.
Technological line: starting strain →-preparation of inclined-plane → synchronized culture → unicellular or spore suspension →-mutagenic treatment (mutagenic treatment pilot study) → plate isolation → primary dcreening operation → multiple sieve → (leavening property mensuration) is tested in sieve → expansion again again.
Press the mutagenesis screening scheme, the mutant strain step-sizing is eliminated, at last strain excellent is screened through the leavening property test, obtain high yield enzyme performance bacterial strain and be respectively wooden mould Cx631, cellulase-producing is lived and is 3068U/g, produces enzyme than former bacterial strain Cx-28 and has improved 125.7%; Aspergillus usamii By247 produces the mannase 10180U/g of being alive, produces alpha-galactosidase 492U/g, produces enzyme than former bacterial strain By-31 respectively and has improved 207.6% and 184.5%; Aspergillus niger M-163, the product xylanase activity is 2326U/g, produces enzyme than former bacterial strain M-17 and has improved 84.2%.
In following examples:
Bacterial classification screens preservation for the contriver, and the present invention is aspergillus niger, Aspergillus usamii, wooden mould through screening used bacterial classification; Strain preparation is divided first class inoculum and second class inoculum preparation.
First class inoculum: substratum is the Fructus Hordei Germinatus inclined-plane, with preservation bacterial classification aspergillus niger, Aspergillus usamii, the mould streak inoculation respectively of wood, cultivates 84-96h down for 30-32 ℃, sterilized water washes, be transferred to respectively and be equipped with in the aseptic triangular flask of granulated glass sphere, concussion is broken up, and it is stand-by to make spore suspension.
Second class inoculum: with wheat bran 80%-90%, dregs of beans 10%-20%, ammonium sulfate 2-3%, SODIUM PHOSPHATE, MONOBASIC 1-1.5%, the bottled siccative 20g of every 300mL triangle, material-water ratio 1: 1.0-1.2, add the water mixing, 121 ℃ of sterilization 30min, cooling, inoculate a kind of first order seed suspension 1.0mL respectively, stir, under 30-32 ℃, cultivate about 72-86h, then, wash spore with sterilized water, it is standby to make spore suspension.
Two, fermentative production
1, fermentation mode: adopt bent room multi-layer solid fermentation process, material thickness 3-5cm, temperature control, wet, the maintenance ventilation of control.
2, solid fermentation substratum preparation: the substratum siccative consists of: wheat bran 70%-80%, dregs of beans 10%-16%, straw powder 10-14%, ammonium sulfate 2-3%, SODIUM PHOSPHATE, MONOBASIC 1-1.5%, sal epsom 0.2%, material-water ratio 1: 1.2-1.5 adds the water mixing, 121 ℃ of sterilization 30min;
3, fermentation culture: insert wooden mould kind of suspension, the U.S. aspergillus seed suspension of assistant, the aspergillus niger seed suspension of secondary seed in the fermention medium according to a certain percentage, ferment after stirring, 30-32 ℃ of fermenting process control leavening temperature, relative humidity 80%-95%, fermented incubation time 84-96h.
4, drying:,, pulverize and obtain complex enzyme for feed of the present invention at 45-50 ℃ of following low temperature air flow drying with the fermentation ends solid substrate.
Aspergillus usamii, the wood of refrigerator preservation is mould, aspergillus niger is inoculated respectively on the fresh wort inclined-plane, place constant incubator, cultivate 76h-82h for 30 ℃, treat that the inclined-plane covers with mycelia and covers with spore after, add an amount of sterilized water and wash spore respectively, make spore suspension (one-level kind).The 20g solid fermentation substratum of in the 300mL triangular flask, packing into, comprising wheat bran 17g, dregs of beans 3g, ammonium sulfate 0.5g, SODIUM PHOSPHATE, MONOBASIC 0.2g, water 25mL, 121 ℃ of sterilization 30min, after the cooling, mould, the aspergillus niger of inoculation wood, Aspergillus usamii spore suspension 1.0mL stir respectively, in 30 ℃ of microorganism constant incubators, cultivate, treat behind the 86h that the fermentation culture primary surface covers with spore, wash spore, make Aspergillus usamii, mould, the aspergillus niger spore suspension (secondary kind) of wood respectively with sterilized water.
Preparation 50kg siccative fermention medium (wheat bran 35Kg, dregs of beans 8Kg, straw powder 7Kg, ammonium sulfate 1Kg, Sodium phosphate dibasic 0.5Kg%, sal epsom 0.1Kg), add tap water 65Kg, mixing machine stirs, 121 ℃ of sterilization 30min, the cooling, in 2.5: 1: 1 ratios with the mould seed suspension of wood, Aspergillus usamii seed suspension, aspergillus niger seed suspension 3.2L altogether evenly sprays in the fermention medium, stirs, dress multilayer koji tray, each koji tray thickness 3-4cm, controlled temperature 30-32 ℃, relative humidity is 90%, turn over behind the 20h once, whole process keeps temperature and relative humidity, cultivates 64h again, fermentation ends.The material that will ferment is lower than 50 ℃ of air stream dryings to water content 8-10%, sends into pulverizer and pulverizes, and after the assay was approved, dress 1Kg tin paper bag is packaged into finished product.
After measured, zytase 2413U/g in the products obtained therefrom, mannase 12347U/g, cellulase 3326U/g, polygalacturonase 3612U/g, alpha-galactosidase 647U/g.
Aspergillus usamii, the wood of refrigerator preservation is mould, aspergillus niger is inoculated respectively on the fresh wort inclined-plane, place constant incubator, cultivate 86h for 30 ℃, treat that the inclined-plane covers with mycelia and covers with spore after, add an amount of sterilized water and wash spore respectively, make one-level kind spore suspension.The 20g solid fermentation substratum of in the 300mL triangular flask, packing into, comprising wheat bran 17g, dregs of beans 3g, ammonium sulfate 0.5g, SODIUM PHOSPHATE, MONOBASIC 0.2g, water 25mL, 121 ℃ of sterilization 30min after the cooling, inoculate respectively that one-level kind wood is mould, aspergillus niger, Aspergillus usamii spore suspension 1.0mL, stir, in 30 ℃ of microorganism constant incubators, cultivate, inoculate behind the 86h, treat that the fermentation culture primary surface covers with spore, wash spore with sterilized water, make secondary kind Aspergillus usamii, mould, the aspergillus niger spore suspension of wood respectively.
Preparation 50kg siccative fermention medium (wheat bran 38Kg, dregs of beans 6Kg, straw powder 6Kg, ammonium sulfate 1Kg, SODIUM PHOSPHATE, MONOBASIC 0.5Kg, sal epsom 0.1Kg), add tap water 70Kg, mixing machine stirs, 121 ℃ of sterilization 30min, the cooling, in 2: 1: 0.6 ratios with the mould seed suspension of wood, Aspergillus usamii seed suspension, aspergillus niger seed suspension 3.6L altogether evenly sprays in the fermention medium, stirs, dress multilayer koji tray, each koji tray thickness 3-4cm, controlled temperature 30-32 ℃, relative humidity is 90%, turn over behind the 20h once, whole process keeps temperature and relative humidity, cultivates 68h again, fermentation ends.The material that will ferment is lower than 50 ℃ of air stream dryings to water content 8-10%, sends into pulverizer and pulverizes, and after the assay was approved, dress 1Kg tin paper bag is packaged into finished product.
After measured, zytase 2374U/g in the products obtained therefrom, mannase 12261U/g, cellulase 3408U/g, polygalacturonase 3542U/g, alpha-galactosidase 632U/g.
Embodiment 4
Aspergillus usamii, the wood of refrigerator preservation is mould, aspergillus niger is inoculated respectively on the fresh wort inclined-plane, place constant incubator, cultivate 86h for 30 ℃, treat that the inclined-plane covers with mycelia and covers with spore after, add an amount of sterilized water and wash spore respectively, make one-level kind spore suspension.The 20g solid fermentation substratum of in the 300mL triangular flask, packing into, comprising wheat bran 17g, dregs of beans 3g, ammonium sulfate 0.5g, SODIUM PHOSPHATE, MONOBASIC 0.2g, water 25mL, 121 ℃ of sterilization 30min, after the cooling, mould, the aspergillus niger of inoculation wood, Aspergillus usamii spore suspension 1.5mL respectively, stir, in 30 ℃ of microorganism constant incubators, cultivate, inoculate behind the 86h, treat that the fermentation culture primary surface covers with spore, wash spore with sterilized water, make secondary kind Aspergillus usamii, mould, the aspergillus niger spore suspension of wood respectively.
Preparation 50kg siccative fermention medium (wheat bran 40Kg, dregs of beans 5Kg, straw powder 5Kg, ammonium sulfate 1.5Kg, SODIUM PHOSPHATE, MONOBASIC 0.5Kg%, sal epsom 0.1Kg), add tap water 75Kg, mixing machine stirs, 121 ℃ of sterilization 30min, the cooling, in 2.2: 1: 0.8 ratios with the mould seed suspension of wood, Aspergillus usamii seed suspension, aspergillus niger seed suspension 4.0L altogether evenly sprays in the fermention medium, stirs, dress multilayer koji tray, each koji tray thickness 3-4cm, controlled temperature 30-32 ℃, relative humidity is 90%, turn over behind the 20h once, whole process keeps temperature and relative humidity, cultivates 72h again, fermentation ends.The material that will ferment is lower than 50 ℃ of air stream dryings to water content 8-10%, sends into pulverizer and pulverizes, and after the assay was approved, dress 1Kg tin paper bag is packaged into finished product.
After measured, zytase 2568U/g in the products obtained therefrom, mannase 12176U/g, cellulase 3117U/g, polygalacturonase 3497U/g, alpha-galactosidase 681U/g.
Embodiment 5 compoiste fermented enzymes and single enzyme combination enzyme are to the external enzymolysis of feedstuff raw material relatively
According to prozyme of the present invention, have synergistic function between each enzyme, promptly be applied to external enzymolysis feedstuff raw material under the same conditions, the prozyme of more single enzyme combination has better hydrolysis result (is index with clean reducing sugar).The results are shown in subordinate list 1.
Vitro enzyme is handled feed and raw material method thereof: claim feed or feedstuff raw material 10g in the 250ml iodine flask, press material-water ratio and add pH5.0 acetate buffer 50mL at 1: 10, enzyme concentration is 0.02% of a feedstuff raw material, behind 39 ℃ of isothermal vibration enzymolysis 5h, add the 5ml trichoroacetic acid(TCA) and handle 5min, enzymolysis solution is collected clear liquid in the centrifugal 10min of 4000r/min, and the DNS method is measured reducing sugar.
The compoiste fermented enzyme of table 1 and single enzyme combination enzyme are to the external enzymolysis comparative result of feedstuff raw material
Annotate: feed resource is full of in Zhuhai contains feed corporation,Ltd, and feed 1 is the aquatic products material, produces article No.: 312, and the date manufactured 20091222; Feed 2 is poultry (pig) material, produces article No.: 503, and the date manufactured: 20100115; Feed 3 is that fowl (chicken) material is produced article No.: 882, and the date manufactured 20100326.
Claims (8)
1. mould Cx631 of wood, its deposit number is: CGMCC NO.4288.
2. Aspergillus usamii By247, its deposit number is: CGMCC NO.4290.
3. aspergillus niger M-163, its deposit number is: CGMCC NO.4289.
4. a combination complex enzyme for feed naturally is characterized in that, described prozyme is that the described wood of claim 1 is mould, the complex ferment product of the described Aspergillus usamii of claim 2 and the described aspergillus niger of claim 3.
5. the complex enzyme for feed of combination naturally according to claim 4, it is characterized in that, described wood is mould, the complex ferment culture medium prescription of Aspergillus usamii and aspergillus niger is: wheat bran 70%-80%, dregs of beans 10%-15%, straw powder 5-10%, ammonium sulfate 2-3%, Sodium phosphate dibasic 1-1.5%, sal epsom 0.2%, material-water ratio 1: 1.2-1.5.
6. the complex enzyme for feed of combination naturally according to claim 4 is characterized in that the ratio of wherein wooden mould, Aspergillus usamii and aspergillus niger is 2.0～2.5: 1: 0.6～1.0.
7. the complex enzyme for feed of combination naturally according to claim 4, it is characterized in that, zymotechnique is: adopt bent room multi-layer solid fermentation process, spore suspension is sprayed in the solid fermentation substratum, stir, under the condition of control relative humidity 80%-95% under 30-32 ℃, cultivate 84-96h, then in 45-50 ℃ of following cryodrying.
8. a method for preparing the described combination naturally of claim 4 complex enzyme for feed is characterized in that, said method comprising the steps of:
1) spore liquid of mould, Aspergillus usamii of preparation wood and aspergillus niger; Wood is mould, Aspergillus usamii and three kinds of preservation bacterial classifications of aspergillus niger are received respectively in the wort agar slant medium, cultivate 84-96h down at 30-32 ℃, and sterilized water washes spore, makes spore suspension.5% spore suspension inoculation with the seed culture medium quality stirs, and cultivates 72-86h down in 30-32 ℃, washes spore with sterilized water then, makes spore suspension;
2) preparation solid fermentation substratum, culture medium prescription is: wheat bran 70%-80%, dregs of beans 10%-15%, straw powder 5-10%, ammonium sulfate 2-3%, Sodium phosphate dibasic 1-1.5%, sal epsom 0.2%, material-water ratio 1: 1.2-1.5;
3) spore suspension is sprayed in the solid fermentation substratum, stirs, and cultivates 84-96h under the condition of control relative humidity 80%-95% under 30-32 ℃, then in 45-50 ℃ of following cryodrying, pulverizing, promptly obtains nature combination complex enzyme for feed.
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| CN104630069A (en) * | 2013-11-12 | 2015-05-20 | 天津工业大学 | Aspergillus niger capable of producing cellulase at high yield |
| CN109055333A (en) * | 2018-07-26 | 2018-12-21 | 天津科技大学 | A kind of application of glycoside hydrolase and its complex enzyme in galactomannan degradation |
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| CN103045564A (en) * | 2012-12-10 | 2013-04-17 | 广东溢多利生物科技股份有限公司 | Method for fermentation production of beta-mannase |
| CN103045564B (en) * | 2012-12-10 | 2015-01-07 | 广东溢多利生物科技股份有限公司 | Method for fermentation production of beta-mannase |
| CN104630069A (en) * | 2013-11-12 | 2015-05-20 | 天津工业大学 | Aspergillus niger capable of producing cellulase at high yield |
| CN104630069B (en) * | 2013-11-12 | 2017-11-28 | 天津工业大学 | The aspergillus niger of one plant height cellulase-producing |
| CN109055333A (en) * | 2018-07-26 | 2018-12-21 | 天津科技大学 | A kind of application of glycoside hydrolase and its complex enzyme in galactomannan degradation |
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| CN102061264B (en) | 2012-09-19 |
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