CN103541259B - The production method of reed, Di Wei paper-making pulping complex enzyme liquid and application - Google Patents
The production method of reed, Di Wei paper-making pulping complex enzyme liquid and application Download PDFInfo
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Abstract
The invention discloses the production method of a kind of reed, Di Wei paper-making pulping complex enzyme liquid, it is characterized in that by each component enzyme through weighing, composite tank mixed preparing, anticorrosion, inspection, packaging after deposit in 5 DEG C of storehouses, described each component enzyme is alkaline pectase, zytase and mannase or alkaline pectase, zytase, mannase and lignoenzyme; Its paper making raw material that is applied as in reed, Di Wei paper-making pulping is sent into digester after pretreatment and is carried out ferment treatment, soak by raw material complex enzyme liquid, ferment treatment condition is: temperature 40 DEG C ~ 60 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.2 ~ 1.8,20 minutes ~ 60 minutes enzyme time of leaching; Boiling in digester after ferment treatment, the vigor of each component enzymes of the present invention is high, and in complex enzyme liquid, each component enzyme does not all have activity to avicel cellulose, carboxymethyl cellulose; Compare with traditional chemical pulp-making method, alkali charge reduces about 50 ﹪, and in waste water, COD content reduces by 30 more than ﹪, and production cost reduces about 20 ﹪, and improves the product quality of paper.
Description
Technical field
The present invention relates to a kind of paper-making pulping, specifically a kind of reed, Di Wei paper-making pulping, particularly relate to a kind of reed, the production method of Di Wei paper-making pulping complex enzyme liquid and application.
Background technology
Along with the development of society and the raising of living standards of the people, China's paper and paper products be consumed in rapid growth, become the consumption market of second-biggest-in-the-world paper and paper products.Paper making raw material 94 ﹪ is timber in the world, because China's Forest Resources is deficient, and the main dependence on import of wood pulp.China lake region reed, Di Wei aboundresources, one of paper making raw material that its Ye Shi China is important.But seriously, NaOH consumption is 14 ﹪ ~ 20 ﹪ of over dry material quality for traditional reed, Di Wei chemical pulping process contamination, and black liquor intractability is large, and costly, general finished paper per ton about needs 200 yuan ~ 300 yuan.Papermaking enterprise is caused to face larger environmental protection pressure and heavy financial burden.
At present, paper-making pulping mainly contains two large class technology paths.1, chemical pulp processes, interpolation auxiliary agent, adjusting process recycle contamination.2, biological pulping method, applying biological pulping technique is from source decreasing pollution.To the former: mainly by the world usual technology---" alkali collection " system solves pollution problem, but " alkali collection " system is mainly developed for large scale pulp mill, if this technology is used for the black liquor pollution administering non-wood pulp, still have that such as calorific value is low, silicon obstacle, wastewater treatment difficulty are large, white clay secondary pollution problems, and " alkali collection " system investments is large, disposal cost is high.For the latter: bio-pulping technology is decreasing pollution from source, improve the utilization rate of raw material, and the black liquor after biological treatment again can be converted into fertilizer and carries out secondary utilization, is a kind of Novel cement slurry preparing technology being paid close attention to exploitation in recent years by experts and scholars and enterprise energetically.
Existing biological pulping method mainly contains two classes: a class adopts microorganism directly to process raw material; One class adopts enzyme treated feed stock.
Employing microorganism directly processes raw material, and if publication number is CN1683711A, denomination of invention is a kind of papermaking bio-pulping process; Publication number is CN1420229A, and denomination of invention is a kind of pulping Process for papermaking straw pulp; Publication number is CN101225614A, and denomination of invention is a kind of paper technology method substituting chemical pulping process with biofermentation pulp-making method; Publication number is CN1188830A, and denomination of invention is biological delignification---the patent applications such as machinery pulping technology are all adopt microorganism directly to process raw material, and their major defect has: the cycle of (1) carrying out a biological disposal upon is long, generally needs several days time; (2), before microbiological treatment, general reply raw material carries out sterilization treatment, adds production cost, likely reduces material quality simultaneously; (3) biological treatment process is difficult to effective control, and usually occur the material quality lack of homogeneity after process, the problems such as carbohydrate degradation is serious, cause pulping yield to reduce; (4) material quality lack of homogeneity after biological treatment, also directly affects slurrying quality, thus affects final product quality; (5) adopt compared with the paper pulp produced of said method starch with general chemistry, the physical property of paper pulp and bleachability are all poor, can not be used for producing the paper of high whiteness, high strength, can only be used for the low-grade paper of production.
Employing enzyme treated feed stock, if publication number is CN1616758A, denomination of invention is bio-pulping process; Publication number is CN1421570A, and denomination of invention is the patents such as the method for enzymatic straw material pulping, and the major defect of this technique is: the cost of enzyme liquid is high, not owning cost advantage in suitability for industrialized production; Meanwhile, lignoenzyme, zytase, the hemicellulase destruction to corresponding composition is difficult to control, and may too much remove lignin, xylan, hemicellulose, affect paper pulp yield.
Publication number is CN101914510A, denomination of invention be a kind of alkaline pectinase production method and the application in paper-making pulping patent discloses a kind of alkaline pectase and the application in paper-making pulping, by paper making raw material after pickling process, with alkaline pectase immersion bubble, boiling in digester again after ferment treatment.But, its complex process, raw material carries out ferment treatment after needing pickling, caustic dip again, has the following disadvantages: one is that enzyme is single, and the leaching enzyme time is long; Two is need newly added equipment, increases cost; Three is need increase pickling, caustic dip step newly, new pollution, is unfavorable for that suitability for industrialized production uses.
Summary of the invention
The object of this invention is to provide a kind of reed, the production method of Di Wei paper-making pulping complex enzyme liquid and application, the enzyme activity of described complex enzyme liquid is high, the unit cost of production is low, fermentation period is short, and industrialization degree is high, and technical process is simple and easy to control, and each component enzyme does not all have activity to avicel cellulose, carboxymethyl cellulose in complex enzyme liquid, be applied to paper-making pulping, paper cost and environmental pollution can be reduced, help the product quality improving paper simultaneously.
The present invention adopts following technical scheme to realize its goal of the invention, the production method of a kind of reed, Di Wei paper-making pulping complex enzyme liquid, by each component enzyme through weighing, composite tank mixed preparing, anticorrosion, inspection, packaging after deposit in 5 DEG C of storehouses, described each component enzyme is alkaline pectase, zytase and mannase; Described alkaline pectase be CCTCCNO:M2010004 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described zytase be CGMCCNO:5466 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described mannase be CGMCCNO:6226 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; The vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml, and the vigor of zytase is 1200u/ml ~ 1500u/ml, and the vigor of mannase is 2000u/ml ~ 3000u/ml.
Because biology enzyme has stronger selectivity, the material component in paper making raw material is comparatively complicated.The present invention adds lignoenzyme and directly acts on lignin in raw material, makes it decompose or comes off, and multienzyme coupling synergy raw material contributes to improving the catalytic effect of biology enzyme and the accessibility of industrial chemicals and utilization rate.
Each component enzyme of the present invention is alkaline pectase, zytase, mannase and lignoenzyme; Described lignoenzyme be CGMCCNO:6225 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; The vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml, and the vigor of zytase is 1200u/ml ~ 1500u/ml, and the vigor of mannase is 2000u/ml ~ 3000u/ml, and the vigor of lignoenzyme is 80u/ml ~ 100u/ml.
The application of above-mentioned complex enzyme liquid in reed, Di Wei paper-making pulping, paper making raw material is sent into digester after pretreatment and is carried out ferment treatment, soak by raw material complex enzyme liquid, ferment treatment condition is: pH value 7.0 ~ 9.5, temperature 40 DEG C ~ 60 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.2 ~ 1.8, and the mass ratio of over dry raw material and water is 1:8 ~ 12,20 minutes ~ 60 minutes enzyme time of leaching; Boiling in digester after ferment treatment, conditions of cooking is: NaOH consumption is 7 ﹪ ~ 11 ﹪ of over dry material quality, temperature 120 DEG C ~ 150 DEG C, 70 minutes ~ 120 minutes time.
Ferment treatment condition of the present invention is preferably: pH value 8.0 ~ 9.0, temperature 50 C ~ 55 DEG C, and the mass ratio of over dry raw material and complex enzyme liquid is 100:1.5, and the mass ratio of over dry raw material and water is 1:8 ~ 10,40 minutes ~ 50 minutes enzyme time of leaching.
Owing to adopting technique scheme, the present invention achieves goal of the invention preferably, and the vigor of each component enzymes is high, and unit cost is low, and fermentation period is short, and industrialization degree is high, and in complex enzyme liquid, each component enzyme does not all have activity to avicel cellulose, carboxymethyl cellulose; Be applied to paper-making pulping, without the need to increasing equipment, technique is simple, easy to use, compares with traditional chemical pulp-making method, and alkali charge reduces about 50 ﹪, and in waste water, COD content reduces by 30 more than ﹪, and production cost reduces about 20 ﹪, and improves the product quality of paper.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1:
A production method for reed, Di Wei paper-making pulping complex enzyme liquid, by each component enzyme through weighing, composite tank mixed preparing, anticorrosion, inspection, packaging after deposit in 5 DEG C of storehouses, described each component enzyme is alkaline pectase, zytase and mannase; Described alkaline pectase be CCTCCNO:M2010004 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described zytase be CGMCCNO:5466 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described mannase be CGMCCNO:6226 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; The vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml, and the vigor of zytase is 1200u/ml ~ 1500u/ml, and the vigor of mannase is 2000u/ml ~ 3000u/ml.
CCTCCNO:M2010004 bacterial classification of the present invention is by naturally gathering bacterium sample in Ningxia, China soil, two strain starting strains are obtained through shaking flask screening from nearly hundred strain bacterial strains, and under cellular level, adopt nitrosoguanidine and ultraviolet radiation mutagenesis to obtain nearly ten thousand strain bacterial strains, the bacterial strain that a strain laboratory is numbered MAPLE61 is obtained through shaking flask screening, i.e. heat-resisting Bacillus subtillis (Heat ~ resistantBacillussubtilis), its biological characteristics is: thalline is shaft-like, thalline two ends are more smooth, individual cells (0.7 ~ 0.75) * (2.5 ~ 2.9) micron, without pod membrane, peritrichous, bacterium colony oyster white, Gram-positive, aerobic bacteria, Liquid Culture thalline in growth period majority in chain row example and with three disjunctors or tetrad in the majority, form brood cell, gemma form ellipse is to shaft-like, and (0.6 ~ 0.8) * (1.0 ~ 1.4) micron, is positioned at thalline central authorities or slightly inclined.This bacterial classification submits China typical culture collection center preservation (Wuhan City, Hubei Province Wuhan University) on January 11st, 2010, and preserving number is: CCTCCNO:M2010004, and have submitted preservation survival proof on January 15th, 2010.
The slant medium formula of this bacterial classification is: in 1000mL distilled water, adds beef extract 5g ~ 10g, yeast extract 5g ~ 10g, peptone 10g ~ 15g, glucose 5g ~ 10g, sodium chloride 5g ~ 10g, sodium carbonate 0.3 ~ 0.5g, agar powder 20g, adjust pH 6.9 ~ 7.1,0.1MPa sterilizing 20 minutes ~ 30 minutes, make test tube slant, inoculate this bacterial classification, 33 DEG C ~ 35 DEG C, cultivate 18 hours ~ 24 hours.
After this slant strains is qualified by shake flask fermentation inspection bacterial classification enzymatic productivity, can spreads cultivation as seed and use or enzymatic production use.
The condition of culture of shake flask fermentation is: in mass, culture medium prescription (﹪) wheat bran 6 ~ 8, corn flour 0.5 ~ 1, corn steep liquor (nitrogen content 40 ﹪) 1 ~ 2, sodium chloride 0.5 ~ 0.8, sodium carbonate 0.2 ~ 0.3, cellulosic powder 1 ~ 2, water 85.0 ~ 91.0, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0 ~ 8.5,0.1MPa sterilizing 25 minutes ~ 35 minutes, condition of culture: 33 DEG C ~ 35 DEG C, rotating speed 260r/ minute ~ 280r/ minute, cultivate 18 hours ~ 22 hours.
Enzyme liquid is prepared as by eggplant bottle inclined-plane to seeding tank to fermentation tank, second order fermentation, preparation standard enzyme liquid.In mass, its culture medium prescription is (﹪) wheat bran 6 ~ 8, corn flour 0.5 ~ 1, corn steep liquor (nitrogen content 40 ﹪) 1 ~ 2, sodium chloride 0.5 ~ 0.8, sodium carbonate 0.2 ~ 0.3, cellulosic powder 1 ~ 2, water 85.0 ~ 91.0, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0 ~ 8.5,0.1MPa sterilizing 25 minutes ~ 35 minutes, tinning cumulative volume is no more than 70%; The condition of culture of seeding tank: temperature (DEG C): 36 DEG C ~ 38 DEG C, speed of agitator (r/ minute): 220, during throughput (v/v) 0 ~ 5 time, 1:0.40 ~ 0.46,5 time after 1:0.50 ~ 0.56; The condition of culture of fermentation tank: temperature (DEG C): 0 hour ~ 4 hours, 36 ~ 38,4 hours later 33 ~ 35, speed of agitator (r/min): 0 hour ~ 4 hours, 180,4 hours ~ 6 hours, 200,6 hours later 220, throughput (v/v): 0 hour ~ 4 hours, 1:0.25 ~ 0.27,4 hours ~ 6 hours, 1:0.33 ~ 0.36,1:0.5 ~ 0.55 after 6 hours.
After the preparation of enzyme liquid, preparation standard alkaline pectase liquid, fermentation liquid is degerming through plate-frame filtering, diatomite, and obtain under three months conditions are deposited in 15 DEG C of sealings through preservative treatment, enzyme activity conservation rate is the standard alkaline type pectase liquid of 95 more than ﹪.
The enzyme activity determination of alkaline pectase of the present invention is that spectrophotometric colo method records with DNS colour developing.Its condition determination is: with the pectin of 1 ﹪ for substrate was pH9.6,60 DEG C of water-baths 10 minutes, add the colour developing of DNS boiling water bath, 550nm colorimetric.Enzyme activity unit is defined as: under condition determination, and the enzyme amount that hydrolysis of pectin per hour produces needed for 1mg reducing sugar (in galacturonic acid) is defined as an enzyme activity unit.
Above content the patent No. be 201010238630.4, denomination of invention is describe in detail in the patent application of a kind of alkaline pectinase production method and the application in paper-making pulping, its preservation survival proves also to submit to Patent Office of the People's Republic of China with application.
CGMCCNO:5466 bacterial classification of the present invention is in Ningxia, China collection, in strain improvement screening process, have employed the method for test tube Liquid Culture radical reaction DNS colour developing initial characterization screening and shaking flask product enzyme quantitative screening, filter out the superior strain that a strain laboratory is numbered KERR89, according to major physiological biochemical character and 16SrRNA gene order, the experimental datas such as gyrB gene order are comprehensively analyzed and are accredited as husky good fortune bacillus (Bacillussafensis), its biological characteristics is: G+ bacteria, produce gemma, thalline is rod-short, unicellular diameter is less than 1um, breed in binary fission mode, on culture medium of the present invention, bacterium colony is rounded, white, smooth surface, homogeneous, and neat in edge, slightly gloss are sticky.This bacterial classification submits China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on November 17th, 2011, preserving number: CGMCCNO:5466, and proves in have submitted preservation survival on the same day.
The slant medium formula of this bacterial classification is: peptone 0.5 ﹪ ~ 1 ﹪, beef extract 0.3 ﹪ ~ 0.5 ﹪, yeast extract 0.3 ﹪ ~ 0.5 ﹪, glucose 0.3 ﹪ ~ 0.5 ﹪, sodium chloride 0.3 ﹪ ~ 0.5 ﹪, agar 1.8 ﹪ ~ 2.2 ﹪, water 94.8 ﹪ ~ 96.5 ﹪, wherein said each component sum is 100 ﹪, adjust pH 6.9 ~ 7.1,0.1MPa sterilizing 20 minutes ~ 30 minutes, make test tube slant, inoculate this bacterial classification, 32 DEG C ~ 35 DEG C, cultivate 24 hours ~ 26 hours.
After this slant strains is qualified by shake flask fermentation inspection bacterial classification enzymatic productivity, can spreads cultivation as seed and use or enzymatic production use.
The condition of culture of shake flask fermentation is: by weight, culture medium prescription is wheat bran 5 ﹪ ~ 8 ﹪, cellulosic powder 1.5 ﹪ ~ 2 ﹪, glucose 0.3 ﹪ ~ 0.5 ﹪, ammonium sulfate 0.3 ﹪ ~ 0.5 ﹪, corn steep liquor 1 ﹪ ~ 2 ﹪, dipotassium hydrogen phosphate 0.1 ﹪ ~ 0.2 ﹪, sodium chloride 0.3 ﹪ ~ 0.5 ﹪, water 86.3 ﹪ ~ 91.5 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0 ~ 8.5,0.1MPa sterilizing 20 minutes ~ 35 minutes, condition of culture: 33 DEG C ~ 35 DEG C, shaking speed 260r/min ~ 280r/min, cultivates 48 hours ~ 52 hours.
Enzyme liquid is prepared as by eggplant bottle inclined-plane to seeding tank to fermentation tank, second order fermentation, preparation standard enzyme liquid.
Seed culture based formulas is: by weight, wheat bran 5 ﹪ ~ 8 ﹪, corn flour 1.5 ﹪ ~ 2 ﹪, ammonium sulfate 0.3 ﹪ ~ 0.5 ﹪, corn steep liquor 1 ﹪ ~ 2 ﹪, dipotassium hydrogen phosphate 0.1 ﹪ ~ 0.2 ﹪, sodium chloride 0.3 ﹪ ~ 0.5 ﹪, water 86.8 ﹪ ~ 91.8 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 6.5 ~ 7.0,0.1MPa sterilizing 30 minutes ~ 35 minutes, seeding tank charging cumulative volume is no more than 70%; Condition of culture: temperature 33 DEG C ~ 38 DEG C, speed of agitator 180r/min ~ 220r/min, throughput was in v/v: 0 hour ~ 5 hours, 1:0.40 ~ 0.46, after 5 hours, 1:0.50 ~ 0.56, tank pressure 0.06MPa ~ 0.07MPa, cultivation cycle is 7 hours ~ 9 hours.
The culture medium prescription producing enzyme fermentation tank is: by weight, wheat bran 5 ﹪ ~ 8 ﹪, cellulosic powder 1.5 ﹪ ~ 2 ﹪, glucose 0.3 ~ 0.5 ﹪, ammonium sulfate 0.3 ﹪ ~ 0.5 ﹪, corn steep liquor 1 ﹪ ~ 2 ﹪, dipotassium hydrogen phosphate 0.1 ﹪ ~ 0.2 ﹪, sodium chloride 0.3 ﹪ ~ 0.5 ﹪, water 86.3 ﹪ ~ 91.5 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0 ~ 8.5,0.1MPa sterilizing 30 minutes ~ 35 minutes, charging cumulative volume is no more than 70 ﹪; Condition of culture: temperature 33 DEG C ~ 38 DEG C, speed of agitator 180r/min ~ 220r/min, throughput in v/v: 0 hour ~ 2 hours, 1:0.40 ~ 0.42,2 hours ~ 4 hours, 1:0.50 ~ 0.52,4 hours ~ 6 hours, 1:0.55 ~ 0.68,1:0.60 ~ 0.62 after 6 hours, tank pressure 0.06MPa ~ 0.07MPa, fermentation period is 48 hours ~ 52 hours.
After the preparation of enzyme liquid, preparation standard zytase liquid.By degerming through plate-frame filtering, diatomite for the fermentation liquid producing enzyme fermentation tank, obtain under three months conditions are deposited in 15 DEG C of sealings through preservative treatment, enzyme activity conservation rate is the standard xylanases liquid of 95 more than ﹪.
The vigour-testing method of zytase of the present invention is, is that 2 ﹪ xylans (sigma comes from birch product) solution prepared by solvent with glycine-sodium hydrate buffer solution that pH value is 9.0,0.1mol/L.Draw 0.9ml substrate in 15ml scale test tube, requiring that temperature water bath balances 3min, add the suitable diluted enzyme solution of 0.1ml, reaction 10min, add 3ml3,5 ~ edlefsen's reagent, develop the color in boiling water bath 15min, 15ml is settled to distilled water, spectrophotometer 550nm wavelength light-metering absorption value after cooling.Enzyme activity is defined as, and under condition determination, hydrolysis substrate per hour produces the required enzyme amount of 1mg reducing sugar (in wood sugar) and is defined as an enzyme activity unit (u).
CGMCCNO:6226 bacterial classification of the present invention is in Ningxia, China collection, in strain improvement screening process, filter out the superior strain that a strain laboratory is numbered PARK9639, according to major physiological biochemical character and 16SrRNA gene order, the experimental datas such as gyrB gene order are comprehensively analyzed and are accredited as bacillus subtilis (Bacillussubtilis), this bacterial classification submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on 06 15th, 2012, preserving number: CGMCCNO:6226, and prove in have submitted preservation survival on the same day.
The slant medium formula of this bacterial classification is: in 1000mL distilled water, add beef extract 3g ~ 8g, yeast extract 3g ~ 8g, peptone 8g ~ 12g, glucose 3g ~ 5g, sodium chloride 3g ~ 5g, adjust pH 6.9 ~ 7.1,0.1MPa sterilizing 28 minutes ~ 32 minutes, make test tube slant, inoculate this bacterial classification, 34 DEG C ~ 36 DEG C, cultivate 20 hours ~ 24 hours.
After this slant strains is qualified by shake flask fermentation inspection bacterial classification enzymatic productivity, can spreads cultivation as seed and use or enzymatic production use.
The condition of culture of shake flask fermentation is: in mass, culture medium prescription is: konjaku flour 3 ﹪ ~ 4 ﹪, wheat bran 2 ﹪ ~ 4 ﹪, corn steep liquor 0.5 ﹪ ~ 1 ﹪ of nitrogen content 40%, yeast extract 0.5 ﹪ ~ 1 ﹪, diammonium hydrogen phosphate 0.2 ﹪ ~ 0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪ ~ 0.05 ﹪, peptone 0.1 ﹪ ~ 0.2 ﹪, magnesium sulfate 0.02 ﹪ ~ 0.05 ﹪, sodium carbonate 0.20 ﹪ ~ 0.25 ﹪, water 89.15 ﹪ ~ 93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is pH8.0 ~ pH8.5, 0.1MPa sterilizing 30 minutes ~ 35 minutes, condition of culture: 32 DEG C ~ 37 DEG C, shaking speed 260r/min ~ 280r/min, cultivate 34 hours ~ 42 hours.
Enzyme liquid is prepared as by eggplant bottle inclined-plane to seeding tank to fermentation tank, second order fermentation, preparation standard enzyme liquid.
Seed culture based formulas is: in mass, corn steep liquor 1 ﹪ ~ 2 ﹪ of wheat bran 5 ﹪ ~ 6 ﹪, corn flour 1 ﹪ ~ 2 ﹪, nitrogen content 40 ﹪, sodium chloride 0.4 ﹪ ~ 0.6 ﹪, sodium carbonate 1 ﹪ ~ 2 ﹪, ammonium sulfate 0.3 ﹪ ~ 0.5 ﹪, water 86.9 ﹪ ~ 91.3 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 6.5 ~ 7.0,0.1MPa sterilizing 30 minutes ~ 35 minutes; Seeding tank charging cumulative volume is no more than 70 ﹪, condition of culture is: temperature 33 DEG C ~ 38 DEG C, speed of agitator 180r/min ~ 220r/min, throughput in v/v: 0 hour ~ 4 hours, 1:0.25 ~ 0.27,4 hours ~ 6 hours, 1:0.33 ~ 0.36, after 6 hours, 1:0.50 ~ 0.55, tank pressure 0.06MPa ~ 0.07MPa, cultivation cycle is 7 hours ~ 9 hours.
The culture medium prescription producing enzyme fermentation tank is: in mass, corn steep liquor 0.5 ﹪ ~ 1 ﹪ of konjaku flour 3 ﹪ ~ 4 ﹪, wheat bran 2 ﹪ ~ 4 ﹪, nitrogen content 40%, yeast extract 0.5 ﹪ ~ 1 ﹪, diammonium hydrogen phosphate 0.2 ﹪ ~ 0.3 ﹪, dipotassium hydrogen phosphate 0.02 ﹪ ~ 0.05 ﹪, peptone 0.1 ﹪ ~ 0.2 ﹪, magnesium sulfate 0.02 ﹪ ~ 0.05 ﹪, sodium carbonate 0.20 ﹪ ~ 0.25 ﹪, water 89.15 ﹪ ~ 93.46 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0 ~ 8.5,0.1MPa sterilizing 30 minutes ~ 35 minutes, charging cumulative volume is no more than 70 ﹪; Condition of culture: temperature 33 DEG C ~ 38 DEG C, speed of agitator 180r/min ~ 220r/min, throughput was in v/v: 0 hour ~ 4 hours, 1:0.30 ~ 0.32,4 hours ~ 6 hours, 1:0.40 ~ 0.42,1:0.50 ~ 0.55 after 6 hours, tank pressure 0.06MPa ~ 0.07MPa, fermentation period is 34 hours ~ 42 hours.
After the preparation of enzyme liquid, preparation standard mannase liquid.By degerming through plate-frame filtering, diatomite for the fermentation liquid producing enzyme fermentation tank, obtain under three months conditions are deposited in 15 DEG C of sealings through preservative treatment, enzyme activity conservation rate is the standard mannase liquid of 95 more than ﹪.
The enzyme activity determination method of mannase is, utilizes the disodium hydrogen phosphate dodecahydrate of 0.05mol/LpH6.0 and the locust bean gum solution of monohydrate potassium buffer 0.5%.In this substrate solution of 0.9ml, add the enzyme solutions that 0.1ml suitably dilutes, 50 DEG C of accurate clock reaction 10min, in 550nm light-metering absorption value.Its enzyme activity is defined as, and under condition determination, hydrolysis locust bean gum per hour produces the required enzyme amount of 1mg reducing sugar (in mannose) and is defined as an enzyme activity unit (u).
By the alkaline pectase liquid prepared, zytase liquid and mannase liquid through weighing, composite tank mixed preparing, anticorrosion, inspection, packaging after deposit in 5 DEG C of storehouses for subsequent use, does is the vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml(the present embodiment 1000u/ml?), does is the vigor of zytase is 1200u/ml ~ 1500u/ml(the present embodiment 1300u/ml?), does is the vigor of mannase is 2000u/ml ~ 3000u/ml(the present embodiment 2200u/ml?).
The application of above-mentioned complex enzyme liquid in reed, Di Wei paper-making pulping, paper making raw material is sent into digester after pretreatment and is carried out ferment treatment, soak by raw material complex enzyme liquid, ferment treatment condition is: pH value 7.0 ~ 9.5, temperature 40 DEG C ~ 60 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.2 ~ 1.8, and the mass ratio of over dry raw material and water is 1:8 ~ 12,20 minutes ~ 60 minutes enzyme time of leaching; Boiling in digester after ferment treatment, conditions of cooking is: NaOH consumption is 7 ﹪ ~ 11 ﹪ of over dry material quality, temperature 120 DEG C ~ 150 DEG C, 70 minutes ~ 120 minutes time.
Ferment treatment condition of the present invention is preferably: pH value 8.0 ~ 9.0, temperature 50 C ~ 55 DEG C, and the mass ratio of over dry raw material and complex enzyme liquid is 100:1.5, and the mass ratio of over dry raw material and water is 1:8 ~ 10,40 minutes ~ 50 minutes enzyme time of leaching.
The present embodiment pH value 8.5, temperature 55 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.5, and the mass ratio of over dry raw material and water is 1:8, the 50 minutes enzyme time of leaching.Enter the boiling stage after enzymolysis completes, conditions of cooking during boiling is: NaOH consumption is 10 ﹪ of over dry material quality, temperature 150 DEG C, 110 minutes time.In digestion process, enzyme is by High Temperature High Pressure deactivation, does not produce any harmful effect to subsequent technique.
Complex enzyme liquid is applied in reed, Di Wei paper-making pulping by the present invention, pectin, the pectic substance of high consumption chemical industry material in the effective hydrolysis material of pectase in complex enzyme liquid, the materials such as heteroglycan such as the xylan in xylanase hydrolysis raw material, directly can reduce chemical industry material consumption; Meanwhile, pectin, pectic substance in the mesoglea of phase adhesion between cell with cell in each component enzyme enzymolysis of plants tissue, the heteroglycan such as xylan, mannosan makes cell and cell estranged or separate, and lignin fragment comes off, and forms pore; Enzymolysis causes cell wall structure to loosen, and occurs crack, for the direct rapid permeability of chemical industry material directly acts on lignin create conditions to plant cavity, thus improves chemical industry material utilization rate, reaches reduction chemical industry material consumption object.
And zytase not cellulase-producing, do not damage fiber, do not affect pulp strength.
Pectic substance in alkaline pectase effect raw material, xylan in zytase effect raw material, mannosan in mannase effect raw material, decreases the consumption of the heteroglycan such as chemical industry material effect pectin, xylan and mannosan such as alkali, reaches the object reducing chemical industry material; Additionally by the hydrolysis to heteroglycan such as pectic substance, xylan and mannosans, make the pectic substance in spore interbed, xylan, the adhesion material such as mannosan reduces, and make cell and cell estranged or separate, pore increases increase, be beneficial to the infiltration of the chemical industry material such as alkali, increase their accessibility, improve its functioning efficiency, thus reach the object reducing chemical industry material consumption.
The present invention is through showing at the paper mill Industrialized Production Practice being raw material with reed, Di Wei, compare with traditional chemical pulp-making method, alkali charge reduces about 50 ﹪, digestion time also declines more than 30 minutes, in waste water, COD content reduces by 30 more than ﹪, production cost reduces about 20 ﹪, and improves the product quality of paper.
Embodiment 2:
Each component enzyme of the present invention is alkaline pectase, zytase, mannase and lignoenzyme; Described lignoenzyme be CGMCCNO:6225 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; The vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml, and the vigor of zytase is 1200u/ml ~ 1500u/ml, and the vigor of mannase is 2000u/ml ~ 3000u/ml, and the vigor of lignoenzyme is 80u/ml ~ 100u/ml.
CGMCCNO:6225 bacterial classification of the present invention is in Ningxia, China collection, in strain improvement screening process, filter out the superior strain that a strain laboratory is numbered KERR7989, according to major physiological biochemical character and 16SrRNA gene order, the experimental datas such as gyrB gene order are comprehensively analyzed and are accredited as me and carry bacillus (Bacillusaltitudinis), this bacterial classification submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on 06 15th, 2012, preserving number: CGMCCNO:6225, and prove in have submitted preservation survival on the same day.
The slant medium formula of this bacterial classification is: in 1000mL distilled water, add beef extract 5g ~ 10g, yeast extract 5g ~ 10g, peptone 10g ~ 15g, glucose 5g ~ 10g, sodium chloride 3g ~ 8g, adjust pH 6.9 ~ 7.1,0.1MPa sterilizing 20 minutes ~ 30 minutes, make test tube slant, inoculate this bacterial classification, 33 DEG C ~ 35 DEG C, cultivate 18 hours ~ 24 hours.
After this slant strains is qualified by shake flask fermentation inspection bacterial classification enzymatic productivity, can spreads cultivation as seed and use or enzymatic production use.
The condition of culture of shake flask fermentation is: in mass, culture medium prescription is: wheat bran 6 ﹪ ~ 8 ﹪, cellulosic powder 1 ﹪ ~ 2 ﹪, corn steep liquor 1 ﹪ ~ 2 ﹪ of nitrogen content 40 ﹪, ammonium sulfate 0.3 ﹪ ~ 0.5 ﹪, dipotassium hydrogen phosphate 0.1 ﹪ ~ 0.2 ﹪, peptone 0.5 ﹪ ~ 1 ﹪, magnesium sulfate 0.03 ﹪ ~ 0.05 ﹪, sodium carbonate 0.2 ﹪ ~ 0.3 ﹪, water 85.95 ﹪ ~ 90.7 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0 ~ 8.5, 0.1MPa sterilizing 25 minutes ~ 35 minutes, condition of culture: 33 DEG C ~ 35 DEG C, shaking speed 260r/ minute ~ 280r/ minute, cultivate 48 hours ~ 52 hours.
Enzyme liquid is prepared as and obtains cellular liquid seed to seeding tank by eggplant bottle inclined-plane, then to producing enzyme fermentation tank, second order fermentation, preparation standard enzyme liquid.
Seed culture based formulas is: in mass, corn steep liquor 1 ﹪ ~ 2 ﹪ of wheat bran 5 ﹪ ~ 6 ﹪, corn flour 1 ﹪ ~ 2 ﹪, nitrogen content 40 ﹪, sodium chloride 0.4 ﹪ ~ 0.6 ﹪, sodium carbonate 1 ﹪ ~ 2 ﹪, ammonium sulfate 0.3 ﹪ ~ 0.5 ﹪, water 86.9 ﹪ ~ 91.3 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 6.5 ~ 7.0,0.1MPa sterilizing 25 minutes ~ 35 minutes; Seeding tank charging cumulative volume is no more than 70 ﹪, condition of culture is: temperature 33 DEG C ~ 38 DEG C, speed of agitator 180r/min ~ 220r/min, throughput in v/v: 0 hour ~ 4 hours, 1:0.25 ~ 0.27,4 hours ~ 6 hours, 1:0.33 ~ 0.36, after 6 hours, 1:0.50 ~ 0.55, tank pressure 0.06MPa ~ 0.08MPa, cultivation cycle is 7 hours ~ 9 hours.
The culture medium prescription producing enzyme fermentation tank is: in mass, corn steep liquor 1 ﹪ ~ 2 ﹪ of wheat bran 6 ﹪ ~ 8 ﹪, cellulosic powder 1 ﹪ ~ 2 ﹪, nitrogen content 40 ﹪, ammonium sulfate 0.3 ﹪ ~ 0.5 ﹪, dipotassium hydrogen phosphate 0.1 ﹪ ~ 0.2 ﹪, peptone 0.5 ﹪ ~ 1 ﹪, magnesium sulfate 0.03 ﹪ ~ 0.05 ﹪, sodium carbonate 0.2 ﹪ ~ 0.3 ﹪, water 85.95 ﹪ ~ 90.87 ﹪, wherein said each component sum is 100 ﹪, before sterilizing, adjust pH is 8.0 ~ 8.5,0.1MPa sterilizing 25 minutes ~ 35 minutes, charging cumulative volume is no more than 70 ﹪; Condition of culture: temperature 33 DEG C ~ 38 DEG C, speed of agitator 180r/min ~ 220r/min, throughput was in v/v: 0 hour ~ 4 hours, 1:0.30 ~ 0.32,4 hours ~ 6 hours, 1:0.40 ~ 0.42,1:0.50 ~ 0.55 after 6 hours, tank pressure 0.06MPa ~ 0.07MPa, fermentation period is 36 hours ~ 42 hours.
After the preparation of enzyme liquid, preparation standard lignoenzyme liquid.By degerming through plate-frame filtering, diatomite for the fermentation liquid producing enzyme fermentation tank, obtain under three months conditions are deposited in 15 DEG C of sealings through preservative treatment, enzyme activity conservation rate is the standard lignoenzyme liquid of 95 more than ﹪.
The assay method of the enzyme activity of lignoenzyme: the syringaldazine solution of configuration 0.2mg/L to load in black agents bottle in Refrigerator store 5 days effectively.During use, the Tris buffer solution of 25mmol/LpH7.5 dilutes 10 times, and draw in this substrate solution of 2.5ml the enzyme solutions adding 0.5ml and suitably dilute, 30 DEG C of accurate clock reaction 5min, in 530nm light-metering absorption value.Enzyme activity is defined as, and is defined as 1 enzyme activity unit (u) when enzyme substrate specificity per hour under condition determination makes light absorption value often increase by 1.0 units.
By the alkaline pectase liquid prepared, zytase liquid, mannase liquid and lignoenzyme liquid are through weighing, composite tank mixed preparing, anticorrosion, inspection, deposit in 5 DEG C of storehouses for subsequent use after packaging, the vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml(the present embodiment is 900u/ml), the vigor of zytase is 1200u/ml ~ 1500u/ml(the present embodiment is 1300u/ml), the vigor of mannase is 2000u/ml ~ 3000u/ml(the present embodiment is 2200u/ml), the vigor of lignoenzyme is 80u/ml ~ 100u/ml(the present embodiment is 90u/ml).
The application of above-mentioned complex enzyme liquid in reed, Di Wei paper-making pulping, paper making raw material is sent into digester after pretreatment and is carried out ferment treatment, soak by raw material complex enzyme liquid, ferment treatment condition is: pH value 7.0 ~ 9.5, temperature 40 DEG C ~ 60 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.2 ~ 1.8, and the mass ratio of over dry raw material and water is 1:8 ~ 12,20 minutes ~ 60 minutes enzyme time of leaching; Boiling in digester after ferment treatment, conditions of cooking is: NaOH consumption is 7 ﹪ ~ 11 ﹪ of over dry material quality, temperature 120 DEG C ~ 150 DEG C, 70 minutes ~ 120 minutes time.
Ferment treatment condition of the present invention is preferably: pH value 8.0 ~ 9.0, temperature 50 C ~ 55 DEG C, and the mass ratio of over dry raw material and complex enzyme liquid is 100:1.5, and the mass ratio of over dry raw material and water is 1:8 ~ 10,40 minutes ~ 50 minutes enzyme time of leaching.
The present embodiment pH value 8.5, temperature 55 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.5, and the mass ratio of over dry raw material and water is 1:8, the 50 minutes enzyme time of leaching.Enter the boiling stage after enzymolysis completes, conditions of cooking during boiling is: NaOH consumption is 8 ﹪ of over dry material quality, temperature 145 DEG C, 110 minutes time.In digestion process, enzyme is by High Temperature High Pressure deactivation, does not produce any harmful effect to subsequent technique.
Because biology enzyme has stronger selectivity, the material component in paper making raw material is comparatively complicated.The effect of lignoenzyme of the present invention mainly contains two aspects: one is lignoenzyme lignin degrading or lignin fragments is come off, and decreases the consumption that the chemical industry material such as alkali directly act on lignin; Two is lignoenzyme xylogen degradations or after lignin fragments is come off, and the lignin wrapped up on cellulose is reduced or loose, and cellulose comes out, and is beneficial to the further effect of the chemical industry material such as alkali, and the accessibility that improve chemical industry material reduces the consumption of chemical industry material.
Zytase is cellulase-producing not, does not damage fiber, and mannonase lignoenzyme is alkaline enzyme, is adapted to alkaline environment, Heat stability is good, does not all have activity, do not affect pulp strength to avicel cellulose, carboxymethyl cellulose.
The present invention tests through suitability for industrialized production, compares with traditional chemical pulp-making method, and alkali charge reduces about 50 ﹪, and digestion time also declines more than 30 minutes, and in waste water, COD content reduces by 30 more than ﹪, and production cost reduces about 20 ﹪, and improves the product quality of paper.
Remaining with embodiment 1.
Claims (3)
1. the application of complex enzyme liquid in reed, Di Wei paper-making pulping, it is characterized in that paper making raw material is sent into digester after pretreatment and carried out ferment treatment, soak by raw material complex enzyme liquid, ferment treatment condition is: pH value 7.0 ~ 9.5, temperature 40 DEG C ~ 60 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.2 ~ 1.8, and the mass ratio of over dry raw material and water is 1:8 ~ 12,20 minutes ~ 60 minutes enzyme time of leaching; Boiling in digester after ferment treatment, conditions of cooking is: NaOH consumption is 7 ﹪ ~ 11 ﹪ of over dry material quality, temperature 120 DEG C ~ 150 DEG C, 70 minutes ~ 120 minutes time; To be each component enzyme deposit in 5 DEG C of storehouses described complex enzyme liquid after weighing, composite tank mixed preparing, anticorrosion, inspection, packaging, and described each component enzyme is alkaline pectase, zytase and mannase; Described alkaline pectase be CCTCCNO:M2010004 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described zytase be CGMCCNO:5466 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described mannase be CGMCCNO:6226 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; The vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml, and the vigor of zytase is 1200u/ml ~ 1500u/ml, and the vigor of mannase is 2000u/ml ~ 3000u/ml.
2. the application of complex enzyme liquid in reed, Di Wei paper-making pulping, it is characterized in that paper making raw material is sent into digester after pretreatment and carried out ferment treatment, soak by raw material complex enzyme liquid, ferment treatment condition is: pH value 7.0 ~ 9.5, temperature 40 DEG C ~ 60 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.2 ~ 1.8, and the mass ratio of over dry raw material and water is 1:8 ~ 12,20 minutes ~ 60 minutes enzyme time of leaching; Boiling in digester after ferment treatment, conditions of cooking is: NaOH consumption is 7 ﹪ ~ 11 ﹪ of over dry material quality, temperature 120 DEG C ~ 150 DEG C, 70 minutes ~ 120 minutes time; To be each component enzyme deposit in 5 DEG C of storehouses described complex enzyme liquid after weighing, composite tank mixed preparing, anticorrosion, inspection, packaging, and described each component enzyme is alkaline pectase, zytase, mannase and lignoenzyme; Described alkaline pectase be CCTCCNO:M2010004 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described zytase be CGMCCNO:5466 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described mannase be CGMCCNO:6226 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; Described lignoenzyme be CGMCCNO:6225 bacterial classification through Spawn incubation, shake flask fermentation, seeding tank is prepared after fermentation tank, filtration, degerming, preservative treatment; The vigor of described complex enzyme liquid neutral and alkali pectase is 800u/ml ~ 1200u/ml, and the vigor of zytase is 1200u/ml ~ 1500u/ml, and the vigor of mannase is 2000u/ml ~ 3000u/ml, and the vigor of lignoenzyme is 80u/ml ~ 100u/ml.
3. the application of complex enzyme liquid in reed, Di Wei paper-making pulping according to claim 1 or 2, it is characterized in that ferment treatment condition is preferably: pH value 8.0 ~ 9.0, temperature 50 C ~ 55 DEG C, the mass ratio of over dry raw material and complex enzyme liquid is 100:1.5, the mass ratio of over dry raw material and water is 1:8 ~ 10,40 minutes ~ 50 minutes enzyme time of leaching.
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