CN103642774B - Mixing neutral cellulase and preparation method thereof and the application in papermaking is pulled an oar - Google Patents

Mixing neutral cellulase and preparation method thereof and the application in papermaking is pulled an oar Download PDF

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CN103642774B
CN103642774B CN201310573648.3A CN201310573648A CN103642774B CN 103642774 B CN103642774 B CN 103642774B CN 201310573648 A CN201310573648 A CN 201310573648A CN 103642774 B CN103642774 B CN 103642774B
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bacillus subtilis
fermentation
aspergillus niger
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CN103642774A (en
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赵迎春
王刚
谢合群
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NINGXIA SUNSON INDUSTRIAL GROUP Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • D—TEXTILES; PAPER
    • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
    • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
    • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
    • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes

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Abstract

The invention discloses a kind of mixing neutral cellulase for papermaking making beating, belong to enzymic preparation field.Described mixing neutral cellulase, parts by weight composition is as follows: cellulase 10 40 parts, acidic xylanase 20 30 parts, pectase 5 10 parts, mannase 38 parts, Chinese herbal medicine powder 28 parts.Mixing neutral cellulase prepared by the present invention, effectively make use of the effective ingredient of Chinese herbal medicine powder, and utilizes specific process to overcome the defect that traditional acidic xylanase uses single culture to ferment；Through the defibrination that mixing neutral cellulase processes, beating degree adds 11.76% relative to untreated slurry, and short fine burst index improves 11.98%, and ring crush index improves 13.99%；Long fine pulling force index improves 40.17%, and tear index improves 58.91%, and fiber surface becomes smooth, and enzyme fiber is the most soft；The tear index of paper, tensile index, burst index is become to be respectively increased 8.3%, 10.6%, 16.5%, pulp brightness improves 2% 5%, and sewage quality is substantially improved, and reduces the pollution level to environment.

Description

Mixing neutral cellulase and preparation method thereof and the application in papermaking is pulled an oar
Technical field:
The invention belongs to enzymic preparation field, particularly to paper pulp papermaking field enzyme.
Background technology:
Pulp and paper industry is one of key pillars of the national economy industry, but is also the resource consumptions such as forest, the energy, chemicals Rich and influential family with environmental pollution.The paper industry in the whole world to cut down the forest of several hundred million cubic metres every year, and the most about half becomes discarded Thing is drained back to again, in the air of surrounding and current, cause grave danger and harm to the ecological environment of human survival.Reduce energy Source and chemical cost, raising paper pulp yield, Sewage Biological Treatment etc. are all the fundamental ways overcoming above-mentioned difficulties.And biology skill Art can be fully developed talents the most in these areas.
The complicated enzyme system that cellulase is made up of multiple hydrolytic enzyme, in nature, a lot of funguses can eccrine fiber element enzyme. Traditionally, cellulase is divided into three classes: C1 enzyme, Cx enzyme and β glucosidase.Cellulose is initially worked by C1 enzyme Enzyme, destroys the crystalline texture of cellulose chain.Cx enzyme is to act on cellulose through C1 enzyme activation, decompose β-Isosorbide-5-Nitrae-glycosidic bond Cellulase.Cellobiose, cellotriose and other low molecule cellodextrins can be decomposed into glucose by β glucosidase.
Cellulase reaction and general enzyme reaction are different, and its topmost difference is that cellulase is multicomponent enzyme system, and the end Thing structure is extremely complex.Due to the water-insoluble of substrate, the adsorption of cellulase instead of the ES of enzyme-to-substrate formation and is combined Thing process.Cellulase first specifically adsorbs on substrate cellulose, then by cellulose under the synergism of several components Resolve into glucose.
Nineteen fifty, Reese etc. proposes C1-Cx hypothesis, and this hypothesis is thought must be with different enzyme synergism, could be by fibre Dimension element is hydrolyzed to glucose thoroughly.Synergism is commonly considered as the non-knot of endoglucanase (C1 enzyme) first attack cellulose Crystalline region, forms the new free-end needed for Cx, is then cut fiber two by CX enzyme from reducing end or the non-reducing end of polysaccharide chain Sugar unit, is finally hydrolyzed into two glucoses by beta glucan glycosides enzyme by cellobiose.But, the synergism of cellulase is suitable Sequence is not absolute, finds in research subsequently, and C1-Cx and beta glucan glycosides enzyme must exist simultaneously could hydrolyze natural fiber Element.If first with C1 enzyme effect crystalline cellulose, then removing C1 enzyme, adding Cx enzyme, such sequential action but can not will be tied Crystalline cellulose hydrolyzes.
The factor affecting yield of cellulase and vigor is a lot, outside degerming kind, and also cultivation temperature, pH, moisture, substrate, training The time of supporting etc..These factors are not isolated, but connect each other.Zhang Zhongliang etc. (1997) use uniform Design Cl12 (1210), With Trichoderma viride (T.ViriclePers.expr) as strain, have studied the five big factors affecting cellulase-producing to yield of enzyme and The effect of vigor, it is believed that substrate crude fiber content is 40%, initial pH7.5, add water 4 times, under the conditions of 26-31 DEG C cultivate 45h Maximum yield of enzyme 26mg/g and CMC enzyme activity 20mg/g h can be obtained.Wang Chenghua etc. (1997) also studied its mutagenesis screening The condition of enzyme production of trichoderma reesei 91-3, result show this strain with the powder of straw of 7:3 and Testa Tritici, another add 4% ammonium sulfate, 0.4% potassium dihydrogen phosphate, 0.1% magnesium sulfate are optimal medium, and 28-32 DEG C is suitable cultivation temperature, and 30 DEG C is optimum temperature, 4% For optimum inoculation amount, 96h arrives fermentation peak.Zhang Linghua etc. (1998) have studied with koning trichoderma W-925 for the bacterium that sets out, The optimal conditions of fermentation of the Wu-932 High-Cellulase-Yielding bacterium obtained after mutation.Result shows, with Testa Tritici and the Caulis et Folium Oryzae of 1:2 Powder is culture medium, the inoculum concentration of 5%, and average length 3-5mm pulverized by Caulis et Folium Oryzae, and initial pH4-5, temperature is at 28-35 DEG C, during fermentation Between 72h be optimal conditions of fermentation.
Applicant GuangDong YiDuoLi Biology Science Co., Ltd submit to a kind of neutral cellulase MEG1 optimizing improvement and Its gene and application patent, its application number: 201110362928.0.This invention relates to genetic engineering field, specifically, and this Bright relate to a kind of neutral cellulase MEG1 optimizing improvement and gene thereof and application.The invention provides a kind of optimization in improvement Property cellulase MEG1, invention additionally provides the gene of neutral cellulase MEG1 encoding above-mentioned optimization improvement, and comprises The recombinant vector of this gene and recombinant bacterial strain and application thereof.The neutral cellulase MEG1 optimizing improvement of the present invention is at neutrallty condition Lower enzyme is lived and is improved a lot, and, by the screening of height copy exogenous gene bacterial strain, obtaining can efficient table in the reactor The production bacterial strain reached, meets the requirement of industrialized production further, therefore, the MEG1 optimizing improvement of the present invention can weaving, Washing, papermaking, feedstuff demonstrate huge application potential.
Mixing neutral cellulase is had high synergistic hydrolytic enzyme by many and forms, containing cellulase, xylanase, The bioactive ingredients such as mannase.Cellulase contains again: (1) circumscribed-type glucanase (C1 enzyme)；(2) endo-type Portugal gathers Carbohydrase (Cx enzyme)；(3) three kinds of components of beta-glucosidase (also referred to as cellobiase, CBH enzyme).
Mixing neutral cellulase is mainly by decomposing cellulose inside, promote fiber during (making) defibrination score Silk and brooming, make making beating become easier to, and saves beating energy consumption.By fiber surface is modified, fiber ends is carried out Differentiation, promotes the swollen of fiber, makes fiber surface and the hydroxyl of end and carboxyl increase simultaneously, thus the knot between reinforcing fiber Make a concerted effort, reach to strengthen paper properties, improve into the effect of paper product matter.
By the enzyme modification to cellulose, fibre morphology plays obvious useful change, either before mill or after mill, and its fiber table All there is the wire-dividing broom purification become apparent from face, and therefore the beating degree of slurry is more easy to improve, and the thin fibrillation in surface simultaneously is favorably improved into paper Physical property and improve retention, during sheet formation, fiber combining power is higher.
The profit that sufficiently absorbed water due to fiber rises, and makes fiber become soft and increases slightly.So that becoming the bulk of paper increased, Flexibility improves.There is the effect making fiber fines reconfigure, the beneficially drainage of l Water Paper page simultaneously, reduce in plain boiled water tiny The content of fiber, makes plain boiled water cycling rate increase, thus reduces ton paper oleo stock consumption and a rinse dosage.
Summary of the invention:
Solved by the invention technical problem is that overcomes the defect that in existing pulp and paper technology, waste discharge amount is big, utilizes one Mixing neutral cellulase is crossed and is decomposed cellulose inside, promotes sub-wire and the brooming of fiber during defibrination, makes making beating become Must be easier to, thus save beating energy consumption, make plain boiled water cycling rate increase, thus reduce ton paper oleo stock consumption and a rinse dosage, Improve the quality of sewage.
In order to achieve the above object, the present invention is by the following technical solutions:
A kind of mixing neutral cellulase, for papermaking making beating enzyme；
Described mixing neutral cellulase, parts by weight composition is as follows:
Cellulase 10-40 part, acidic xylanase 20-30 part, pectase 5-10 part, mannase 3-8 part, Chinese herbal medicine powder Agent 2-8 part.
The preparation method of described Chinese herbal medicine powder is as follows: weigh Radix Angelicae Sinensis 20-30 part；Radix Codonopsis 10-18 part；Radix Bupleuri 10-15 part； Radix Scutellariae 10-15 part；It is less than 2 millimeters that said herbal medicine is crushed to particle diameter respectively, then uniformly mixes in container and adds The water of 3-6 times of weight, controls temperature 80 DEG C 95 DEG C and keeps 2 4h, interpolation 2-3 times of w ethanol of mixed material and propanol Mixture, controls temperature and keeps 3 4h to 55 DEG C 65 DEG C, filter；Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder Agent.
The preparation method of described mixing mixing neutral cellulase is as follows: by cellulase, acidic xylanase, pectase, sweet Dew dextranase and Chinese herbal medicine powder mix according to parts by weight；
Described acidic xylanase is prepared by the following technical solutions:
A kind of acidic xylanase preparation method comprises the steps:
(1) aspergillus niger, Bacillus subtilis strain activation
The slant strains of intact aspergillus niger is inoculated in slant medium, cultivates 36-48h for 28-35 DEG C and carry out actication of culture, So activation 2-4 time；
The slant strains of intact bacillus subtilis is inoculated in slant medium, cultivates 24-36h for 34-35 DEG C and carry out bacterium Plant activation, so activation 2-3 time；
Described aspergillus niger slant medium consists of: Rhizoma Solani tuber osi liquor 200g, glucose 20g, agar 20g, herbal mediciment powder 5-20g, distilled water 1000mL, pH value 5.8,121 DEG C of sterilizing 20min；
Described bacillus subtilis slant medium consists of: Carnis Bovis seu Bubali cream 3g, sodium chloride 5g, peptone 10g, agar 15g, Distilled water 1000mL, pH value 6.8,121 DEG C of sterilizing 20min；
(2) aspergillus niger liquid seeds amplification culture
Described aspergillus niger first class seed pot fermentation liquid cell concentration is 7.0x108-8.0x108Individual/ml；
Bacillus subtilis liquid seeds amplification culture
First class seed pot is cultivated: with 10% inoculum concentration, three grades of seeds are accessed the total measurement (volume) first class seed pot as 150L, fermentation training Foster base loading amount 100L, cultivation temperature 32-36 DEG C, mixing speed 200-400rpm, ventilation (V/V) 1:1-2, tank pressure 0.05Mpa, Incubation time 10-20h；
(3) aspergillus niger, the mixing of bacillus subtilis first class seed pot liquid seeds
The bacillus subtilis seed tank liquid strain that aspergillus niger seed tank liquid seeds step (2) obtained and step (2) obtain Son the most uniformly mixes；Described aspergillus niger seed liquor and bacillus subtilis seed liquor volume ratio are 1:0.5-1；
Described mixing liquid seeds cell concentration is: 7.0x108-8.0x108Individual/ml；
(4) secondary seed tank alternate
First class seed pot liquid seeds mixed uniformly in step (3) is accessed two grades as 300L of total measurement (volume) with 10% inoculum concentration Seed tank, alternate culture medium loading amount 240L, secondary seed tank fermentation tank, cultivation temperature 28-35 DEG C, mixing speed 200-700r/m, Ventilation (V/V) 1:1-3, incubation time 15-24h；
Described alternate culture medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, Trehalose 10-30g, yeast powder 4-8g, Semen Maydis pulp 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2 G, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min；
(5) strain mixed fermentation
By secondary seed tank alternate liquid seeds in step (4) with 6% inoculum concentration access fermentation tank, cultivation temperature 28-30 DEG C, Mixing speed 200-700r/m, ventilation (V/V) 1:1-3, incubation time 10-15h；Then delay with 1-2 DEG C/h rate of temperature fall Slowly 10-15 DEG C it is cooled to, constant temperature culture 15-20h；Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, Secondary seed tank alternate liquid seeds in step (4) is added with 4% inoculum concentration and accesses fermentation tank, constant temperature culture 20-30h；? After be to slowly warm up to 10-15 DEG C with 1-2 DEG C/h heating rate, constant temperature culture 15-20h；Continue to delay with 1-2 DEG C/h heating rate Slowly 30-35 DEG C it is warming up to, constant temperature culture 15-20h；
Dissolved oxygen controls: by adjusting speed of agitator and ventilation, control dissolved oxygen 15-30%；
PH controls: by mending ammonia or phosphoric acid,diluted, controls pH value in sweat and is maintained at 5-7；
Control of additive raw material: when the content of reducing sugar in fermentation liquid is down to 3mg/ml-8mg/ml, starts to add supplemented medium, mends Doses is to maintain fermentation liquid content of reducing sugar as 2mg/ml-5mg/ml；
Put tank standard: 60-80% thalline aging self-dissolving, enzyme activity increasess slowly.
Described fermentation medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, Trehalose 30-40g, yeast powder 4-8g, Semen Maydis pulp 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2 G, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min；
Described supplemented medium weight consists of: maltodextrin 20-30%, Semen Maydis powder 10-20%, Semen Glycines powder 15-25%, not foot Pure water is divided to supply, pH value 5-7,121-123 DEG C of sterilizing 30-40min.
(6) fermentation liquor filters, concentrates, allocates, fine straining, is dried and to obtain solid body acidic xylanase.
Beneficial effect:
Mixing neutral cellulase prepared by the present invention, effectively make use of the effective ingredient of Chinese herbal medicine powder, and utilizes special side Method overcomes traditional acidic xylanase and uses the defect of single culture fermentation, by black for the strain producing high vigor acidic xylanase Aspergillosis, with to produce the species Bacillus subtilis combination of sciences of heat-flash stability acidic xylanase, alternate stable, causes mixed bacteria Enzymatic productivity manifest to greatest extent, also make that the present invention produced acidic xylanase type is complete, vigor is high, action condition is wide in range, Stability is strong, be suitable to industrialized production.Fermentation liquid acidic xylan specific activity of enzyme of the present invention is 5300-6800U/mg, than single bacterium Planting fermentation of Aspergillus niger liquid acidic xylanase enzyme activity and averagely improve 2-3 times, heat stability is strong, and applicable range of reaction temperature is 30-78℃；And the defibrination processed through mixing neutral cellulase, beating degree adds 11.76% relative to untreated slurry, short Fine burst index improves 11.98%, and ring crush index improves 13.99%；Long fine pulling force index improves 40.17%, tear index Improve 58.91%, fiber surface becomes smooth, and enzyme fiber is the most soft；Become the tear index of paper, tensile index, resistance to broken finger Number has been respectively increased 8.3%, and 10.6%, 16.5%, decrease the dish end defibrination time, reduce power consumption 10 20%, pulp brightness improves 2%-5%, plain boiled water blood circulation becomes clean, and sewage quality is substantially improved, and reduces the pollution level to environment.
Detailed description of the invention
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention is this Method well known to skilled person.It addition, embodiment is interpreted as illustrative, and unrestricted the scope of the present invention, The spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, real without departing substantially from the present invention On the premise of matter and scope, the various changes or the change that carry out the material component in these embodiments and consumption fall within this Bright protection domain.
Embodiment 1
The Aspergillus niger strain preserving number that the present invention provides is CICC2475, is purchased from Chinese industrial Microbiological Culture Collection administrative center. Described aspergillus niger slant medium consists of: Rhizoma Solani tuber osi liquor 200g, glucose 20g, agar 20g, distilled water 1000mL, PH value 5.8,121 DEG C of sterilizing 20min；
Bacillus subtilis CICC10158 is purchased from Chinese industrial Microbiological Culture Collection administrative center.Described bacillus subtilis is oblique Face culture medium consists of: Carnis Bovis seu Bubali cream 3g, sodium chloride 5g, peptone 10g, agar 15g, distilled water 1000mL, pH value 6.8, 121 DEG C of sterilizing 20min；
Described aspergillus niger one-level, two grades, three grades of seed culture mediums consist of: Rhizoma Solani tuber osi liquor 200g, glucose 20g, Sargassum Sugar 10-30g, distilled water 1000mL, pH value 5.5,121 DEG C of sterilizing 20min；
Described aspergillus niger first class seed pot culture medium consists of: Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, sea Algae sugar 10-30g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2g, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min；
Described bacillus subtilis one-level, two grades, three grades of seed culture medium weight consist of:
Yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, Carnis Bovis seu Bubali cream 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, trehalose 1-3%, insufficient section pure water supplies, pH value 6.8,121-123 DEG C of sterilizing 30-40min.
Described bacillus subtilis seed tank culture basic weight amount consists of:
Maltodextrin 5-15%, yeast powder 0.4-0.8%, trehalose 1-3%, peptone 0.1-0.5%, Semen Maydis pulp 0.1-0.5%, Dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, insufficient section pure water supplies, pH value 6.8,121-123 DEG C of sterilizing 30-40min。
Described alternate culture medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, Trehalose 10-30g, yeast powder 4-8g, Semen Maydis pulp 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2 G, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min；
Described fermentation medium consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, Trehalose 30-40g, yeast powder 4-8g, Semen Maydis pulp 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2 G, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min；
Described supplemented medium weight consists of: maltodextrin 20-30%, Semen Maydis powder 10-20%, Semen Glycines powder 15-25%, not foot Pure water is divided to supply, pH value 5-7,121-123 DEG C of sterilizing 30-40min.
Embodiment 2
The parts by weight composition of mixing neutral cellulase is as follows: cellulase 30 parts, xylanase enzyme 25 parts, pectase 8 Part, mannase 5 parts, Chinese herbal medicine powder 5 parts.
The preparation method of Chinese herbal medicine powder is as follows: weigh Radix Angelicae Sinensis 24 parts；Radix Codonopsis 14 parts；Radix Bupleuri 13 parts；Radix Scutellariae 14 parts；Point It is less than 2 millimeters that said herbal medicine is not crushed to particle diameter, then uniformly mixes and add the water of 4 times of weight in container, control Temperature processed 90 DEG C keeps 4h, adds 3 times of w ethanol of mixed material and the mixture of propanol, controls temperature and keeps 4h to 60 DEG C, Filter；Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
Described acidic xylanase is prepared by the following technical solutions:
(1) aspergillus niger, Bacillus subtilis strain activation
The slant strains of intact aspergillus niger is inoculated in slant medium, cultivates 40h for 30 DEG C and carry out actication of culture, so Activate 3 times；
The slant strains of intact bacillus subtilis is inoculated in slant medium, cultivates 30h for 35 DEG C and carry out actication of culture, So activation 2 times；
Described aspergillus niger slant medium consists of: Rhizoma Solani tuber osi liquor 200g, glucose 20g, agar 20g, distilled water 1000mL, PH value 5.8,121 DEG C of sterilizing 20min；
Described bacillus subtilis slant medium consists of: Carnis Bovis seu Bubali cream 3g, sodium chloride 5g, peptone 10g, agar 15g, Distilled water 1000mL, pH value 6.8,121 DEG C of sterilizing 20min；
(2) aspergillus niger liquid seeds amplification culture
1. first order seed is cultivated: the aspergillus niger slant strains after step (1) being activated, with spore under aseptic washing, accesses 500 In milliliter shaking flask, liquid seed culture medium loading amount 100 milliliters, 30 DEG C, 80rpm shaking table cultivation 40h；
2. secondary seed is cultivated: accesses in 500 milliliters of secondary seed shaking flasks by first order seed according to the inoculum concentration of 10%, cultivates Condition is identical with first order seed；
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 8% inoculum concentration, liquid culture Base loading amount 1000 milliliters, 30 DEG C, 80rpm shaking table cultivation 40h；
4. first class seed pot is cultivated: with 8% inoculum concentration, three grades of seeds are accessed the total measurement (volume) first class seed pot as 150L, fermentation Culture medium loading amount 100L, control ph is 5, cultivation temperature 28 DEG C, mixing speed 200rpm, ventilation (V/V) 1:0.8, Incubation time 36h, dissolved oxygen 10%；
Described aspergillus niger one-level, two grades, three grades of seed culture mediums consist of: Rhizoma Solani tuber osi (liquor) 200g, glucose 20g, Trehalose 10g, distilled water 1000mL, pH value 5.5,121 DEG C of sterilizing 20min；
Described aspergillus niger first class seed pot culture medium consists of: Semen Maydis powder 50g, Semen Glycines powder 15g, wheat bran 10g, trehalose 10g, Ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, pure water 1000mL, pH value 5,121 DEG C of sterilizing 20min；
Described aspergillus niger first class seed pot fermentation liquid cell concentration is 7.0x108Individual/ml；
(3) bacillus subtilis liquid seeds amplification culture
1. first order seed is cultivated: the bacillus subtilis slant strains after step (1) being activated accesses in 500 milliliters of shaking flasks, Culture medium loading amount 100 milliliters, rotary shaker 180 revs/min, cultivation temperature 35 DEG C, incubation time 10h；
2. secondary seed is cultivated: accesses in 500 milliliters of secondary seed shaking flasks by first order seed according to the inoculum concentration of 10%, cultivates bar Part is identical with first order seed；
3. three grades of seed culture: accessing in 5000 milliliters of three grades of seed flask by secondary seed with 10% inoculum concentration, culture medium fills Measure 1000 milliliters, rotary shaker 100 revs/min, cultivation temperature 35 DEG C, incubation time 10h；
4. first class seed pot is cultivated: with 10% inoculum concentration, three grades of seeds are accessed the total measurement (volume) first class seed pot as 150L, fermentation Culture medium loading amount 100L, cultivation temperature 32 DEG C, mixing speed 200rpm, ventilation (V/V) 1:1, tank pressure 0.05Mpa, Incubation time 10h；
Described bacillus subtilis one-level, two grades, three grades of seed culture medium weight consist of:
Yeast powder 0.3%, glucose 1%, peptone 0.3%, Carnis Bovis seu Bubali cream 0.5%, dipotassium hydrogen phosphate 0.8%, trehalose 1%, Insufficient section pure water is supplied, pH value 6.8,121 DEG C of sterilizing 30min.
Described bacillus subtilis seed tank culture basic weight amount consists of:
Maltodextrin 5%, yeast powder 0.4%, trehalose 1%, peptone 0.1%, Semen Maydis pulp 0.1%, dipotassium hydrogen phosphate 0.8%, Magnesium sulfate 0.05%, insufficient section pure water supplies, pH value 6.8,121 DEG C of sterilizing 30min.
Described bacillus subtilis seed tank fermentation liquid cell concentration is 7.0x108Individual/ml；
(4) aspergillus niger, the mixing of bacillus subtilis first class seed pot liquid seeds
The bacillus subtilis seed tank liquid strain that aspergillus niger seed tank liquid seeds step (2) obtained and step (3) obtain Son the most uniformly mixes；
Described aspergillus niger seed liquor and bacillus subtilis seed liquor volume ratio are 1:0.5；
Described mixing liquid seeds cell concentration is: 7.0x108Individual/ml；
(5) secondary seed tank alternate
First class seed pot liquid seeds mixed uniformly in step (4) is accessed two grades as 300L of total measurement (volume) with 10% inoculum concentration Seed tank, alternate culture medium loading amount 240L, secondary seed tank fermentation tank, cultivation temperature 28-35 DEG C, mixing speed 200-700r/m, Ventilation (V/V) 1:1, incubation time 15h；
Described alternate culture medium consists of: maltodextrin 50g, Semen Maydis powder 50g, Semen Glycines powder 15g, wheat bran 10g, trehalose 10g, Yeast powder 4g, Semen Maydis pulp 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, pure water 1000mL, pH value 5,121 DEG C of sterilizing 20min；
(6) strain mixed fermentation
Secondary seed tank alternate liquid seeds in step (5) is accessed fermentation tank, cultivation temperature 28 DEG C, stirring with 6% inoculum concentration Speed 200r/m, ventilation (V/V) 1:1, incubation time 10h；Then with 1 DEG C/h rate of temperature fall slow cooling to 10 DEG C, Constant temperature culture 15h；Continue with 1 DEG C/h rate of temperature fall slow cooling to 2 DEG C, now, by mutual for secondary seed tank in step (5) Raw liquid seeds adds with 4% inoculum concentration and accesses fermentation tank, constant temperature culture 20h；Finally it is to slowly warm up to 1 DEG C/h heating rate 10 DEG C, constant temperature culture 15h；Continue to be to slowly warm up to 30 DEG C with 1 DEG C/h heating rate, constant temperature culture 15h；
Dissolved oxygen controls: by adjusting speed of agitator and ventilation, control dissolved oxygen 15%；
PH controls: by mending ammonia or phosphoric acid,diluted, controls pH value in sweat and is maintained at 5；
Control of additive raw material: when the content of reducing sugar in fermentation liquid is down to 3mg/ml-8mg/ml, starts to add supplemented medium, mends Doses is to maintain fermentation liquid content of reducing sugar as 2mg/ml-5mg/ml；
Put tank standard: 60% thalline aging self-dissolving, enzyme activity increasess slowly.
Described fermentation medium consists of: maltodextrin 50g, Semen Maydis powder 50g, Semen Glycines powder 15g, wheat bran 10g, trehalose 30g, Yeast powder 4g, Semen Maydis pulp 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, pure water 1000mL, pH value 5,121 DEG C of sterilizing 20min；
Described supplemented medium weight consists of: maltodextrin 20%, Semen Maydis powder 10%, Semen Glycines powder 15%, herbal mediciment powder 5%, Insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min.
(7) fermentation liquor filters, concentrates, allocates, fine straining, is dried and to obtain solid body acidic xylanase.
Described allocation process is without any preservative.
The fermentation liquid acidic xylanase enzyme activity obtained through above-mentioned preparation method is 5300U/mg.
Embodiment 3
The usage of described mixing neutral cellulase and consumption:
Select proper product, the adding method of enzyme the most difference according to paper pulp raw material and technique, the different of equipment, can lead Two categories below to be divided into:
(1) place is added in paper plant oneself slurrying, the direct production line for slurry: last one wash engine grout outlet enters to starch with kowtowing forebay Position possible between Kou；Pulp washing disposed slurry obtains suitable temperature and pH value, kowtows forebay and enzyme can be made to obtain enough stops Time.
Adding method: according to sending slurry mode and flow, is configured to be prone to the solution of exact operations enzyme, then uses dosing pump Synchronize to add in slurry.
(2) production line using waste paper and external pulpboard adds place: generally pulper temperature and pH value all can be suitable for raw Thing enzyme uses, and beneficially enzyme is sufficiently stirred and the response time.
Adding method: according to the difference of pulping mode in two kinds of situation: one is continuous pulping: according to pulping yield, biological Enzyme is configured to be prone to the solution of exact operations, then synchronizes to add in pulper with dosing pump；The second is interval pulping: according to Pulping amount weighs enough enzyme every time, direct plunges in pulper.
Addition: 50-200g(ml)/t oven dry stock；
Described mixing neutral cellulase is untouched to be contained under cool place, dry environment, and the shelf-life is 12 months, at 5 DEG C--15 DEG C of low temperature, Under dry environment, the shelf-life is 24 months.

Claims (4)

1. a mixing neutral cellulase, parts by weight composition is as follows:
Cellulase 10-40 part, acidic xylanase 20-30 part, pectase 5-10 part, mannase 3-8 part, Chinese herbal medicine powder 2-8 part；
Described acidic xylanase is prepared by the following technical solutions:
(1) aspergillus niger, Bacillus subtilis strain activation
The slant strains of intact aspergillus niger is inoculated in slant medium, cultivates 36-48h for 28-35 DEG C and carry out actication of culture, so activation 2-4 time；
The slant strains of intact bacillus subtilis is inoculated in slant medium, cultivates 24-36h for 34-35 DEG C and carry out actication of culture, so activation 2-3 time；
(2) aspergillus niger liquid seeds amplification culture
Described aspergillus niger carries out first class seed pot fermentation, the final concentration of 7.0x10 of first class seed pot fermentation liquid thalline after one, two, three seed culture8-8.0x108Individual/ml；
Bacillus subtilis liquid seeds amplification culture
First class seed pot is cultivated: with 10% inoculum concentration, three grades of seeds of bacillus subtilis are accessed the total measurement (volume) first class seed pot as 150L, fermentation medium loading amount 100L, cultivation temperature 32-36 DEG C, mixing speed 200-400rpm, ventilation volume ratio is 1:1-2, tank pressure 0.05Mpa, incubation time 10-20h；
(3) aspergillus niger, the mixing of bacillus subtilis first class seed pot liquid seeds
The bacillus subtilis seed tank liquid seeds that aspergillus niger seed tank liquid seeds step (2) obtained obtains with step (2) the most uniformly mixes；Described aspergillus niger seed liquor and bacillus subtilis seed liquor volume ratio are 1:0.5-1；
Described mixing liquid seeds cell concentration is: 7.0x108-8.0x108Individual/ml；
(4) secondary seed tank alternate
First class seed pot liquid seeds mixed uniformly in step (3) is accessed the total measurement (volume) secondary seed tank as 300L with 10% inoculum concentration, alternate culture medium loading amount 240L, secondary seed tank fermentation tank, cultivation temperature 28-35 DEG C, mixing speed 200-700r/m, ventilation (V/V) 1:1-3, incubation time 15-24h；
(5) strain mixed fermentation
With 6% inoculum concentration, secondary seed tank alternate liquid seeds in step (4) is accessed fermentation tank, and cultivation temperature 28-30 DEG C, mixing speed 200-700r/m, ventilation volume ratio is 1:1-3, incubation time 10-15h；Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h；Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, secondary seed tank alternate liquid seeds in step (4) is added with 4% inoculum concentration and accesses fermentation tank, constant temperature culture 20-30h；Finally it is to slowly warm up to 10-15 DEG C with 1-2 DEG C/h heating rate, constant temperature culture 15-20h；Continue to be to slowly warm up to 30-35 DEG C with 1-2 DEG C/h heating rate, constant temperature culture 15-20h；
(6) fermentation liquor filters, concentrates, allocates, fine straining, is dried and to obtain solid body acidic xylanase；
The preparation method of described Chinese herbal medicine powder is as follows: weigh Radix Angelicae Sinensis 20-30 part；Radix Codonopsis 10-18 part；Radix Bupleuri 10-15 part；Radix Scutellariae 10-15 part；It is less than 2 millimeters that said herbal medicine is crushed to particle diameter respectively, then in container, uniformly mix and add the water of 3-6 times of weight, control temperature 80 DEG C 95 DEG C and keep 2 4h, add 2-3 times of w ethanol of mixed material and the mixture of propanol, control temperature and keep 3 4h to 55 DEG C 65 DEG C, filter；Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder.
A kind of mixing neutral cellulase the most according to claim 1, it is characterised in that described aspergillus niger one-level, two grades, three grades of seed culture mediums consist of: Rhizoma Solani tuber osi liquor 200g, glucose 20g, trehalose 10-30g, distilled water 1000mL, pH value 5.5,121 DEG C of sterilizing 20min；
The culture medium that described aspergillus niger first class seed pot uses consists of: Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, trehalose 10-30g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2g, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min；
The culture medium weight that three grades of seeds of described bacillus subtilis use consists of:
Yeast powder 0.3-0.5%, glucose 1-1.5%, peptone 0.3-0.5%, Carnis Bovis seu Bubali cream 0.5-0.8%, dipotassium hydrogen phosphate 0.8-1.5%, trehalose 1-3%, insufficient section pure water supplies, pH value 6.8,121-123 DEG C of sterilizing 30-40min；
The culture medium weight that described bacillus subtilis seed tank uses consists of:
Maltodextrin 5-15%, yeast powder 0.4-0.8%, trehalose 1-3%, peptone 0.1-0.5%, Semen Maydis pulp 0.1-0.5%, dipotassium hydrogen phosphate 0.8-1.5%, magnesium sulfate 0.05-0.1%, insufficient section pure water is supplied, pH value 6.8,121-123 DEG C of sterilizing 30-40min；
Described alternate is cultivated the culture medium used and is consisted of: maltodextrin 50-150g, Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, trehalose 10-30g, yeast powder 4-8g, Semen Maydis pulp 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2g, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min；
The culture medium that described mixed fermentation uses consists of: maltodextrin 50-150g, Semen Maydis powder 50-60g, Semen Glycines powder 15-25g, wheat bran 10-15g, trehalose 30-40g, yeast powder 4-8g, Semen Maydis pulp 1-5g, ammonium sulfate 1-3g, dipotassium hydrogen phosphate 1-2g, potassium dihydrogen phosphate 1-2g, pure water 1000mL, pH value 5-7,121 DEG C of sterilizing 20min.
A kind of mixing neutral cellulase the most according to claim 1, it is characterised in that the parts by weight composition of described mixing neutral cellulase is as follows: cellulase 30 parts, xylanase 25 parts, pectase 8 parts, mannase 5 parts, Chinese herbal medicine powder 5 parts；
The preparation method of Chinese herbal medicine powder is as follows: weigh Radix Angelicae Sinensis 24 parts；Radix Codonopsis 14 parts；Radix Bupleuri 13 parts；Radix Scutellariae 14 parts；It is less than 2 millimeters that said herbal medicine is crushed to particle diameter respectively, then uniformly mixes and add the water of 4 times of weight in container, controls temperature 90 DEG C and keeps 4h, adds 3 times of w ethanol of mixed material and the mixture of propanol, controls temperature and keeps 4h to 60 DEG C, filters；Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder；
Described acidic xylanase is prepared by the following technical solutions:
(1) aspergillus niger, Bacillus subtilis strain activation
The slant strains of intact aspergillus niger is inoculated in slant medium, cultivates 40h for 30 DEG C and carry out actication of culture, so activation 3 times；
The slant strains of intact bacillus subtilis is inoculated in slant medium, cultivates 30h for 35 DEG C and carry out actication of culture, so activation 2 times；
Described aspergillus niger slant medium consists of: Rhizoma Solani tuber osi liquor 200g, glucose 20g, agar 20g, distilled water 1000mL, pH value 5.8,121 DEG C of sterilizing 20min；
Described bacillus subtilis slant medium consists of: Carnis Bovis seu Bubali cream 3g, sodium chloride 5g, peptone 10g, agar 15g, distilled water 1000mL, pH value 6.8,121 DEG C of sterilizing 20min；
(2) aspergillus niger liquid seeds amplification culture
1. first order seed is cultivated: the aspergillus niger slant strains after step (1) being activated, with spore under aseptic washing, accesses in 500 milliliters of shaking flasks, liquid seed culture medium loading amount 100 milliliters, 30 DEG C, 80rpm shaking table cultivation 40h；
2. secondary seed is cultivated: being accessed in 500 milliliters of secondary seed shaking flasks according to the inoculum concentration of 10% by first order seed, condition of culture is identical with first order seed；
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 8% inoculum concentration, fluid medium loading amount 1000 milliliters, 30 DEG C, 80rpm shaking table cultivation 40h；
4. first class seed pot is cultivated: with 8% inoculum concentration, three grades of seeds are accessed the total measurement (volume) first class seed pot as 150L, fermentation medium loading amount 100L, and control ph is 5, cultivation temperature 28 DEG C, mixing speed 200rpm, ventilation volume ratio is 1:0.8, incubation time 36h, dissolved oxygen 10%；
Described aspergillus niger one-level, two grades, three grades of seed culture mediums consist of: Rhizoma Solani tuber osi liquor 200g, glucose 20g, trehalose 10g, distilled water 1000mL, pH value 5.5,121 DEG C of sterilizing 20min；
Described aspergillus niger first class seed pot culture medium consists of: Semen Maydis powder 50g, Semen Glycines powder 15g, wheat bran 10g, trehalose 10g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, pure water 1000mL, pH value 5,121 DEG C of sterilizing 20min；
Described aspergillus niger first class seed pot fermentation liquid cell concentration is 7.0x108Individual/ml；
(3) bacillus subtilis liquid seeds amplification culture
1. first order seed is cultivated: the bacillus subtilis slant strains after step (1) being activated accesses in 500 milliliters of shaking flasks, culture medium loading amount 100 milliliters, rotary shaker 180 revs/min, cultivation temperature 35 DEG C, incubation time 10h；
2. secondary seed is cultivated: being accessed in 500 milliliters of secondary seed shaking flasks according to the inoculum concentration of 10% by first order seed, condition of culture is identical with first order seed；
3. three grades of seed culture: secondary seed is accessed in 5000 milliliters of three grades of seed flask with 10% inoculum concentration, culture medium loading amount 1000 milliliters, rotary shaker 100 revs/min, cultivation temperature 35 DEG C, incubation time 10h；
4. first class seed pot is cultivated: with 10% inoculum concentration, three grades of seeds are accessed the total measurement (volume) first class seed pot as 150L, and fermentation medium loading amount 100L, cultivation temperature 32 DEG C, mixing speed 200rpm, ventilation volume ratio is 1:1, tank pressure 0.05Mpa, incubation time 10h；
Described bacillus subtilis one-level, two grades, three grades of seed culture medium weight consist of:
Yeast powder 0.3%, glucose 1%, peptone 0.3%, Carnis Bovis seu Bubali cream 0.5%, dipotassium hydrogen phosphate 0.8%, trehalose 1%, insufficient section pure water supplies, pH value 6.8,121 DEG C of sterilizing 30min；
Described bacillus subtilis seed tank culture basic weight amount consists of:
Maltodextrin 5%, yeast powder 0.4%, trehalose 1%, peptone 0.1%, Semen Maydis pulp 0.1%, dipotassium hydrogen phosphate 0.8%, magnesium sulfate 0.05%, insufficient section pure water supplies, pH value 6.8,121 DEG C of sterilizing 30min；
Described bacillus subtilis seed tank fermentation liquid cell concentration is 7.0x108Individual/ml；
(4) aspergillus niger, the mixing of bacillus subtilis first class seed pot liquid seeds
The bacillus subtilis seed tank liquid seeds that aspergillus niger seed tank liquid seeds step (2) obtained obtains with step (3) the most uniformly mixes；
Described aspergillus niger seed liquor and bacillus subtilis seed liquor volume ratio are 1:0.5；
Described mixing liquid seeds cell concentration is: 7.0x108Individual/ml；
(5) secondary seed tank alternate
First class seed pot liquid seeds mixed uniformly in step (4) is accessed the total measurement (volume) secondary seed tank as 300L with 10% inoculum concentration, alternate culture medium loading amount 240L, secondary seed tank fermentation tank, cultivation temperature 28-35 DEG C, mixing speed 200-700r/m, ventilation volume ratio is 1:1, incubation time 15h；
Described alternate culture medium consists of: maltodextrin 50g, Semen Maydis powder 50g, Semen Glycines powder 15g, wheat bran 10g, trehalose 10g, yeast powder 4g, Semen Maydis pulp 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, pure water 1000mL, pH value 5,121 DEG C of sterilizing 20min；
(6) strain mixed fermentation
With 6% inoculum concentration, secondary seed tank alternate liquid seeds in step (5) is accessed fermentation tank, and cultivation temperature 28 DEG C, mixing speed 200r/m, ventilation volume ratio is 1:1, incubation time 10h；Then with 1 DEG C/h rate of temperature fall slow cooling to 10 DEG C, constant temperature culture 15h；Continue with 1 DEG C/h rate of temperature fall slow cooling to 2 DEG C, now, secondary seed tank alternate liquid seeds in step (5) is added with 4% inoculum concentration and accesses fermentation tank, constant temperature culture 20h；Finally it is to slowly warm up to 10 DEG C with 1 DEG C/h heating rate, constant temperature culture 15h；Continue to be to slowly warm up to 30 DEG C with 1 DEG C/h heating rate, constant temperature culture 15h；
Dissolved oxygen controls: by adjusting speed of agitator and ventilation, control dissolved oxygen 15%；
PH controls: by mending ammonia or phosphoric acid,diluted, controls pH value in sweat and is maintained at 5；
Control of additive raw material: when the content of reducing sugar in fermentation liquid is down to 3mg/ml-8mg/ml, starts to add supplemented medium, and feed supplement amount is to maintain fermentation liquid content of reducing sugar as 2mg/ml-5mg/ml；
Put tank standard: 60% thalline aging self-dissolving, enzyme activity increasess slowly；
Described fermentation medium consists of: maltodextrin 50g, Semen Maydis powder 50g, Semen Glycines powder 15g, wheat bran 10g, trehalose 30g, yeast powder 4g, Semen Maydis pulp 1g, ammonium sulfate 1g, dipotassium hydrogen phosphate 1g, potassium dihydrogen phosphate 1g, pure water 1000mL, pH value 5,121 DEG C of sterilizing 20min；
Described supplemented medium weight consists of: maltodextrin 20%, Semen Maydis powder 10%, Semen Glycines powder 15%, herbal mediciment powder 5%, and insufficient section pure water is supplied, pH value 5,121 DEG C of sterilizing 30min；
(7) fermentation liquor filters, concentrates, allocates, fine straining, is dried and to obtain solid body acidic xylanase；
Described allocation process is without any preservative；
The fermentation liquid acidic xylanase enzyme activity obtained through above-mentioned preparation method is 5300U/mg.
4. according to claim 1-3 any one, mix the preparation method of neutral cellulase, comprise the steps: that the preparation method of described mixing neutral cellulase is as follows: cellulase, acidic xylanase, pectase, mannase and Chinese herbal medicine powder are mixed according to parts by weight；
The preparation method of described Chinese herbal medicine powder is as follows: weigh Radix Angelicae Sinensis 20-30 part；Radix Codonopsis 10-18 part；Radix Bupleuri 10-15 part；Radix Scutellariae 10-15 part；It is less than 2 millimeters that said herbal medicine is crushed to particle diameter respectively, then in container, uniformly mix and add the water of 3-6 times of weight, control temperature 80 DEG C 95 DEG C and keep 2 4h, add 2-3 times of w ethanol of mixed material and the mixture of propanol, control temperature and keep 3 4h to 55 DEG C 65 DEG C, filter；Filter vacuum concentrates postlyophilization and obtains Chinese herbal medicine powder；
Described acidic xylanase is prepared by the following technical solutions:
(1) aspergillus niger, Bacillus subtilis strain activation
The slant strains of intact aspergillus niger is inoculated in slant medium, cultivates 36-48h for 28-35 DEG C and carry out actication of culture, so activation 2-4 time；
The slant strains of intact bacillus subtilis is inoculated in slant medium, cultivates 24-36h for 34-35 DEG C and carry out actication of culture, so activation 2-3 time；
(2) aspergillus niger liquid seeds amplification culture
The described aspergillus niger first class seed pot final concentration of 7.0x10 of fermentation liquid thalline8-8.0x108Individual/ml；
Bacillus subtilis liquid seeds amplification culture
First class seed pot is cultivated: with 10% inoculum concentration, three grades of seeds of bacillus subtilis are accessed the total measurement (volume) first class seed pot as 150L, fermentation medium loading amount 100L, cultivation temperature 32-36 DEG C, mixing speed 200-400rpm, ventilation volume ratio is 1:1-2, tank pressure 0.05Mpa, incubation time 10-20h；
(3) aspergillus niger, the mixing of bacillus subtilis first class seed pot liquid seeds
The bacillus subtilis seed tank liquid seeds that aspergillus niger seed tank liquid seeds step (2) obtained obtains with step (2) the most uniformly mixes；Described aspergillus niger seed liquor and bacillus subtilis seed liquor volume ratio are 1:0.5-1；
Described mixing liquid seeds cell concentration is: 7.0x108-8.0x108Individual/ml；
(4) secondary seed tank alternate
First class seed pot liquid seeds mixed uniformly in step (3) is accessed the total measurement (volume) secondary seed tank as 300L with 10% inoculum concentration, alternate culture medium loading amount 240L, secondary seed tank fermentation tank, cultivation temperature 28-35 DEG C, mixing speed 200-700r/m, ventilation volume ratio is 1:1-3, incubation time 15-24h；
(5) strain mixed fermentation
With 6% inoculum concentration, secondary seed tank alternate liquid seeds in step (4) is accessed fermentation tank, and cultivation temperature 28-30 DEG C, mixing speed 200-700r/m, ventilation volume ratio is 1:1-3, incubation time 10-15h；Then with 1-2 DEG C/h rate of temperature fall slow cooling to 10-15 DEG C, constant temperature culture 15-20h；Continue with 1-2 DEG C/h rate of temperature fall slow cooling to 2-5 DEG C, now, secondary seed tank alternate liquid seeds in step (4) is added with 4% inoculum concentration and accesses fermentation tank, constant temperature culture 20-30h；Finally it is to slowly warm up to 10-15 DEG C with 1-2 DEG C/h heating rate, constant temperature culture 15-20h；Continue to be to slowly warm up to 30-35 DEG C with 1-2 DEG C/h heating rate, constant temperature culture 15-20h；
(6) fermentation liquor filters, concentrates, allocates, fine straining, is dried and to obtain solid body acidic xylanase.
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