CN104357428A - Liquid submerged fermentation method of xylanase - Google Patents
Liquid submerged fermentation method of xylanase Download PDFInfo
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- CN104357428A CN104357428A CN201410638530.9A CN201410638530A CN104357428A CN 104357428 A CN104357428 A CN 104357428A CN 201410638530 A CN201410638530 A CN 201410638530A CN 104357428 A CN104357428 A CN 104357428A
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- zytase
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- corn cob
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- 238000000855 fermentation Methods 0.000 title claims abstract description 97
- 230000004151 fermentation Effects 0.000 title claims abstract description 96
- 239000007788 liquid Substances 0.000 title claims abstract description 77
- 238000000034 method Methods 0.000 title claims abstract description 60
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 title claims abstract description 11
- 239000002253 acid Substances 0.000 claims abstract description 18
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 239000002994 raw material Substances 0.000 claims abstract description 12
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 7
- 240000008042 Zea mays Species 0.000 claims description 60
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 60
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 60
- 235000005822 corn Nutrition 0.000 claims description 60
- 239000002609 medium Substances 0.000 claims description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 238000009423 ventilation Methods 0.000 claims description 20
- 238000001802 infusion Methods 0.000 claims description 16
- 239000002054 inoculum Substances 0.000 claims description 15
- 239000011573 trace mineral Substances 0.000 claims description 15
- 235000013619 trace mineral Nutrition 0.000 claims description 15
- 239000012528 membrane Substances 0.000 claims description 14
- 230000001954 sterilising effect Effects 0.000 claims description 13
- 239000006052 feed supplement Substances 0.000 claims description 12
- 235000015099 wheat brans Nutrition 0.000 claims description 11
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
- 229910019142 PO4 Inorganic materials 0.000 claims description 10
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 239000001110 calcium chloride Substances 0.000 claims description 10
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 10
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 10
- 239000010452 phosphate Substances 0.000 claims description 10
- 239000011148 porous material Substances 0.000 claims description 10
- 239000011591 potassium Substances 0.000 claims description 10
- 229910052700 potassium Inorganic materials 0.000 claims description 10
- 238000011218 seed culture Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 239000000654 additive Substances 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 241000228212 Aspergillus Species 0.000 claims description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 5
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 5
- 239000011609 ammonium molybdate Substances 0.000 claims description 5
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 5
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 5
- 229940010552 ammonium molybdate Drugs 0.000 claims description 5
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 239000011790 ferrous sulphate Substances 0.000 claims description 5
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 5
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 5
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 5
- 235000010234 sodium benzoate Nutrition 0.000 claims description 5
- 239000004299 sodium benzoate Substances 0.000 claims description 5
- 239000001117 sulphuric acid Substances 0.000 claims description 5
- 235000011149 sulphuric acid Nutrition 0.000 claims description 5
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- 229960001763 zinc sulfate Drugs 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 35
- 102000004190 Enzymes Human genes 0.000 abstract description 35
- 230000000694 effects Effects 0.000 abstract description 20
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 239000000463 material Substances 0.000 abstract description 8
- 238000005265 energy consumption Methods 0.000 abstract description 3
- 229940088598 enzyme Drugs 0.000 abstract 3
- 238000004880 explosion Methods 0.000 abstract 2
- 229940079919 digestives enzyme preparation Drugs 0.000 abstract 1
- 238000000605 extraction Methods 0.000 abstract 1
- 238000001914 filtration Methods 0.000 abstract 1
- 239000000411 inducer Substances 0.000 abstract 1
- 239000003755 preservative agent Substances 0.000 abstract 1
- 230000002335 preservative effect Effects 0.000 abstract 1
- 239000000758 substrate Substances 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 229920002678 cellulose Polymers 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 238000010563 solid-state fermentation Methods 0.000 description 4
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 3
- 229960003487 xylose Drugs 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000010298 pulverizing process Methods 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 229920001221 xylan Polymers 0.000 description 2
- 150000004823 xylans Chemical class 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000609240 Ambelania acida Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 240000007582 Corylus avellana Species 0.000 description 1
- 108010001817 Endo-1,4-beta Xylanases Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 239000010905 bagasse Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- -1 mass percent Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000010815 organic waste Substances 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000004076 pulp bleaching Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Classifications
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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)
- C12N9/2477—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
- C12N9/248—Xylanases
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a liquid submerged fermentation method of xylanase and belongs to the technical field of production of bio-enzyme preparations. According to the method, corncobs are taken as main raw material and an aqueous leachate of diluted acid steam explosion material of corncobs is used as an inductor source and an aspergillus niger liquid submerged fermentation method is adopted. The method comprises the following steps: a) preparation and propagation of a culture; b) fed-batch fermentation and cultivation; c) extraction of xylanase and d) treatment of residues of fermentation liquid. Particularly, in the step of b) fed-batch fermentation and cultivation, a culture medium which takes the aqueous leachate of diluted acid steam explosion material of corncobs as an inducer is continuously fed and meanwhile, fermentation mash is intermittently discharged within a fermentation period. A liquid xylanase product is obtained through plate-frame filtration, ultrafiltration and concentration and addition of a preservative. The enzyme activity of xylanase obtained is improved by 60-70% and the yield is improved by 40-50%. Under the condition of not increasing raw material consumption and energy consumption, the enzyme activity and the output of the products are improved to a great extent, so that the productivity is increased, the utilization ratio of equipment is improved and the fermentation cost of unit enzyme activity is lowered.
Description
Technical field
The invention belongs to biological enzyme formulation production technical field, relate to a kind of liquid submerged fermentation method of zytase particularly.
Background technology
Zytase refers to the general name that xylan degrading can be become one group of enzyme of oligose and wood sugar, mainly comprises circumscribed β-Isosorbide-5-Nitrae-zytase, inscribe β-Isosorbide-5-Nitrae-zytase and beta-xylanase.Zytase has a wide range of applications in fields such as feed, food, papermaking, weaving, medicine and the energy.Zytase is used as feed enzyme preparation, can efficient solution except the anti-oxidant action of xylan, promote livestock and poultry digesting and assimilating roughage.Zytase, as novel association with pulp bleaching auxiliary agent, reduces bleaching chlorine, solves the problem of environmental pollution in Pulp industry.In the production practice of cellulose fuel ethanol, add the polymeric form between the peelable xylogen of zytase and Mierocrystalline cellulose, be easy to the abundant contact of the enzyme-to-substrate in enzymolysis process, be beneficial to the generation of enzyme digestion reaction, can decompose hemicellulose becomes the available monose of yeast simultaneously, improve the yield of liquor, reduce the cost of cellulose ethanol.Along with the quickening of Industrialization of Cellulosic Ethanol process, the development research of zytase receives much concern more.
The production bacterial classification of zytase is primarily of bacterium, fungi and mould.Wherein, mould has the gene of many expressed xylanase, produces enzyme level higher than yeast and bacterium, is most widely used in the fermentation of zytase.Zytase is produced mainly through above fermentable and is obtained, and according to the difference of zymotechnique, can be divided into solid state fermentation and liquid state fermentation two kinds.Solid state fermentation is traditional fermentation mode, and bacterial classification is mould mostly, have control simple, cost is lower, pollution-free, productive rate comparatively advantages of higher, but also there is following shortcoming in solid state fermentation: wayward simultaneously, enzyme system is complicated, and the enzyme content such as Mierocrystalline cellulose are high, are difficult to smart enzyme etc.Liquid submerged fermentation is the conventional fermentation mode of liquid state fermentation, has become the main mode of fermentation industry, and the method is convenient to detect in real time in process of production, control, and is easy to expand the scale of production.Meanwhile, because Liquid transfer is convenient, be convenient to mechanized operation, product is also easy to Hydrolysis kinetics.
CN 101182470 A discloses a kind of liquid submerged fermentation method preparing zytase, adopt fermentation of Aspergillus niger method, it is characterized in that, by accessing under appropriate Aspergillus niger strain aseptic condition in the female tank of stainless steel, producing fermentor tank and adding fermention medium to tank volume 70 ~ 75%; Cultivation and fermentation: 30 DEG C, 150 ~ 200r/min, ventilate 0.1 ~ 1.5VVM, 70 ~ 80H, and supplement the nutrients material between fermentation middle and later periods 40 ~ 60H; Stop fermentation: ferment to 70 ~ 80H, according to microscopy thalli morphology, if thalline starts self-dissolving namely stop fermentation; Fermented liquid mainly contains three kinds of processing modes: the salting-out process of enzyme crude product and spray-drying process, the obtained refining solid enzyme of flocculation-ultrafiltration process and refining liquid enzyme.Liquid submerged fermentation will exceed 10 ~ 30% than the enzyme activity of solid state fermentation, and thick enzyme production cost per ton reduces by more than 20%.The enzyme activity of this invention gained zytase improves limited, may be that promoter action that inductor in enzyme stage fermentation tank synthesizes zytase is not obvious to be caused owing to producing.
High-efficient Production zytase a lot of because have, key factor selects suitable inducing substrate and optimal medium composition.During the fermentation, several factors influence the generation of zytase in addition, as the accessibility of substrate, the rate of release of xylo-oligosaccharide and quantity and chemical property, the wood sugar amount etc. that also can produce to some extent.If enzyme-to-substrate combines closely, then can be lost when treat enzyme liquid because being combined on insoluble substrate by some zytase.Therefore the tightness degree that enzyme-to-substrate combines also can have influence on the output of zytase.In addition, the metabolic enzyme (as proteolytic enzyme, FscMⅠ etc.) that microbial cells itself produces all can have influence on the output of zytase.
China has the agriculture and forestry organic waste material that abundant corn cob, bagasse, Wheat bran, stalk etc. are rich in hemicellulose class natural resources, is not subject to pay abundant attention and exploitation for a long time.And the cost that prior art produces zytase is still higher, and most of synthesis material price is somewhat expensive, how to utilize cheap cellulose materials High-efficient Production zytase to be the emphasis that investigator and the producer pay close attention to.
Summary of the invention
Technical problem to be solved by this invention is, for the deficiencies in the prior art, a kind of zytase liquid submerged fermentation method is provided, when raw material consumption and energy consumption do not increase, increase substantially product enzyme activity and the rate of output, increase production capacity, improve plant factor, reduce unit enzyme activity fermentation costs.
For solving the problems of the technologies described above, the technical solution adopted in the present invention is: a kind of liquid submerged fermentation method of zytase, take corn cob as main raw material, adopts aspergillus niger liquid submerged fermentation method, specifically comprises the following steps:
A) preparation of bacterial classification and enlarged culturing: by Aspergillus niger strain
aspergillus nigercGMCC No.5833 is forwarded on PDA slant medium, 30 DEG C, cultivates 4 days, obtains slant pore; Then wash described slant pore with sterilized water, obtain spore washings; Aseptically described spore washings is accessed in sterilized triangular flask seed culture medium (121 ~ 124 DEG C, sterilizing 30min), in 180 ~ 220r/min, under 28 ~ 32 DEG C of conditions, cultivate 18 ~ 24h, obtain activated spawn; By described activated spawn with 0.3 ~ 0.5%(v/v) inoculum size access first class seed pot in, in 28 ~ 32 DEG C, ventilation ratio 1:0.6 ~ 0.8(v/v) condition under, cultivate 18 ~ 20h, obtain primary seed solution; By described primary seed solution with 8 ~ 10%(v/v) inoculum size access secondary seed tank, in 28 ~ 32 DEG C, ventilation ratio 1:0.6 ~ 0.8(v/v) condition under, cultivate 8 ~ 12h, obtain secondary seed solution;
B) fed-batch fermentation is cultivated: in fermentor tank, add fermentation basic medium, coefficient is 50 ~ 60%(v/v), adjust ph is 4.5 ~ 5.0, in 121 ~ 124 DEG C, sterilizing 30min; Treat that substratum temperature is down to 30 DEG C, aseptically, the inoculum size with 10 ~ 15% of fermention medium volume accesses described secondary seed solution, 0 ~ 10h after inoculation, ventilation ratio is 1:0.6 ~ 1.5(v/v), mixing speed is 150 ~ 200r/min, and culture temperature is 30 ~ 32 DEG C; After inoculation 10h, ventilation ratio 1:0.5 ~ 1.0(v/v), mixing speed 100 ~ 200r/min, culture temperature 28 ~ 30 DEG C; When concentration of reduced sugar is at 0.1%(w/w) below time, start to add supplemented medium and carry out feed supplement, feed rate is feed supplement amount per hour is 1.2 ~ 1.6%(w/w of fermention medium), control the concentration of reducing sugar at 0.2%(w/w) below; Carry out discharge when fermentating liquid volume reaches 80% of fermenter volume, stop discharge when reaching 70% of fermenter volume to fermentating liquid volume, repeat discharge operation in a fermentation period, fermentation period is 5 ~ 7 days;
C) purification of zytase: fermentation ends, filters fermentation liquor plate-and-frame filter press, and gained filtered liquid organic membrane carries out ultrafiltration, is concentrated into required multiple; Then add sanitas, obtain liquid xylanase product;
D) process of fermented liquid residue: the fermented liquid residue through plate-and-frame filter press gained is carried out drying and processing, making it water content is 10 ~ 15%(w/w), use as fodder additives.
The composition of the seed culture medium of triangular flask described in step a), first class seed pot and secondary seed tank is, by percentage to the quality, glucose 1.5%, wheat bran 4%, corn steep liquor 1%, yeast powder 0.5%, surplus is water;
The basic medium that ferments described in step b) consists of, with mass percent, corn cob 5.0 ~ 7.0%, wheat bran 1.0 ~ 2.0%, corn steep liquor 1.5 ~ 3.0%, ammonium sulfate 0.4 ~ 0.6%, potassium primary phosphate 0.2 ~ 0.4%, magnesium sulfate 0.1 ~ 0.3%, calcium chloride 0.05 ~ 0.15%, trace element 0.05 ~ 0.2%, surplus is water.
Further, the granularity of described corn cob is less than 0.5mm.
Described in step b), supplemented medium consists of, mass percent, glucose 3.0 ~ 5.0%, corn steep liquor 2.0 ~ 3.0%, ammonium sulfate 0.6 ~ 0.8%, potassium primary phosphate 0.4 ~ 0.8%, magnesium sulfate 0.2 ~ 0.4%, calcium chloride 0.1 ~ 0.3%, trace element 0.05 ~ 0.1%, all the other disclose infusion for corn cob diluted acid vapour.
Further, consisting of of described trace element, mass percent, ammonium molybdate 0.04%, zinc sulfate 0.2%, ferrous sulfate 0.3%, manganous sulfate 0.1%, cobalt chloride 0.15%, copper sulfate 0.1%, surplus is water.
Further, described corn cob diluted acid vapour discloses that infusion is obtained by following preparation method: corn cob granularity being less than 10mm, in 0.5 ~ 1.0%(w/w) sulphuric acid soln in soak, keep 10 ~ 15min under 6 ~ 10kg pressure, carry out the quick-fried process of vapour, then add moisture, the corn cob solid content after the quick-fried process of vapour is made to remain on 6 ~ 10%(w/w), soak 1 ~ 2h, utilize plate-and-frame filter press to filter, obtain corn cob diluted acid vapour and disclose infusion.
Further, organic membrane described in step c) is membrane molecule amount is 10000 daltonian organic membrane.
Further, the component proportion of sanitas described in step c) is: sodium-chlor 7g ~ 10g, Sodium Benzoate 0.1g ~ 0.3g.
Due to the liquid submerged fermentation method of a kind of zytase of the present invention, comprise preparation and the enlarged culturing of a) bacterial classification, b) fed-batch fermentation is cultivated, c) purification of zytase and the process of d) fermented liquid residue, the method is discharged without waste, environment friendly and pollution-free, has following beneficial effect:
(1) the method when raw material consumption and energy consumption do not increase, can increase production capacity, improves plant factor, reduces unit enzyme activity fermentation costs, and the enzyme activity of products obtained therefrom improves 60 ~ 70%, and the rate of output improves 40 ~ 50%;
(2) the present invention is that main raw material produces zytase by liquid submerged fermentation with corn cob, for the comprehensive exploitation of corn cob and utilization provide novel method and new way;
(3) the corn cob diluted acid vapour that the present invention adopts in feed supplement process is disclosed in infusion and is contained a certain amount of xylo-oligosaccharide, is conducive to induction Aspergillus niger strain and produces enzyme in the fermenting process of zytase; With in the past direct with compared with the corn cob feed supplement after pulverizing, reduce the pulverizing cost of corn cob, avoid the risk of corn cob granule blocking pipe simultaneously with liquid mode feed supplement;
(4) Continuous Flow that the present invention takes adds, interval discharge pattern, efficient solution can remove the scarcity of fermenting process Middle nutrition material and the feedback inhibition of product, improve the fermentation level of zytase;
(5) the raw materials used corn cob of the present invention is renewable resources, and wide material sources, cheap, with it for fermenting raw materials produces zytase, can not only turn waste into wealth, but also reduce the production cost of zytase, the residue obtained fodder additives that also can do uses.
Embodiment
Below in conjunction with embodiment, the present invention is done to the explanation of a step.
Embodiment one
Below with 10m
3fermentor tank is that example is described in detail the present invention.
The liquid submerged fermentation method of zytase of the present invention take corn cob as main raw material, adopts aspergillus niger liquid submerged fermentation method, can carry out according to the following steps:
A) preparation of bacterial classification and enlarged culturing: by Aspergillus niger strain
aspergillus nigercGMCC No.5833 is forwarded on PDA slant medium, cultivates 4 days, obtain slant pore under 30 DEG C of conditions; Then wash described slant pore with sterilized water, obtain spore washings; Aseptically accessed by described spore washings in sterilized triangular flask seed culture medium (at 121 ~ 124 DEG C, sterilizing 30 min), shaking speed is 180r/min, cultivates 24h, obtain activated spawn under 28 DEG C of conditions; By described activated spawn with 0.3%(v/v) inoculum size access first class seed pot in, in 28 DEG C, ventilation ratio 1:0.8(v/v) condition under, cultivate 20h, obtain primary seed solution; By described primary seed solution with 8%(v/v) inoculum size access secondary seed tank, in 28 DEG C, ventilation ratio 1:0.6(v/v) condition under, cultivate 10h, obtain secondary seed solution; In this step, the composition of described triangular flask seed culture medium, first class seed pot and first class seed pot is, with mass percent, and glucose 1.5%, wheat bran 4%, corn steep liquor 1%, yeast powder 0.5%, surplus is water;
B) fed-batch fermentation is cultivated: at 10m
3in fermentor tank, load 5m
3fermentation basic medium (by percentage to the quality, granularity is less than the corn cob 6.0% of 0.5mm, wheat bran 1.0%, corn steep liquor 2.0%, ammonium sulfate 0.4%, potassium primary phosphate 0.3%, magnesium sulfate 0.1%, calcium chloride 0.12%, trace element 0.05%, surplus is water), the pH value of described fermentation basic medium is adjusted to 5.0, sterilizing 30min at 121 ~ 124 DEG C; Treat that substratum temperature is down to 30 DEG C, aseptically, access described secondary seed solution with the inoculum size of fermention medium volume 10%, 6h after inoculation, ventilation ratio is 1:0.6(v/v), mixing speed is 200r/min, and culture temperature is 32 DEG C; 20h after inoculation, ventilation ratio is 1:0.5(v/v), mixing speed is 150r/min, and culture temperature is 29 DEG C; During fermentation 35h, concentration of reduced sugar is 0.05% (w/w), adds supplemented medium and carries out feed supplement, and feed rate is 60L/h, and the concentration of reducing sugar controls at 0.15%(w/w); When fermentating liquid volume reaches 8m
3time, start to carry out discharge, reach 7m to fermentating liquid volume
3time, stop discharge; Repeat discharge operation in a fermentation period, fermentation period is 6 days;
Wherein, the mass percent of described supplemented medium consists of, glucose 3.0%, corn steep liquor 2.5%, ammonium sulfate 0.6%, potassium primary phosphate 0.5%, magnesium sulfate 0.3%, calcium chloride 0.1%, trace element 0.08%, all the other disclose infusion for corn cob diluted acid vapour, and adjust ph is 4.7, sterilizing 30min at 121 ~ 124 DEG C.The mass percent of described trace element consists of, ammonium molybdate 0.04%, zinc sulfate 0.2%, ferrous sulfate 0.3%, manganous sulfate 0.1%, cobalt chloride 0.15%, copper sulfate 0.1%.Described corn cob diluted acid vapour discloses that infusion is obtained by following preparation method: corn cob granularity being less than 10mm, in 0.7%(w/w) soak in sulphuric acid soln, 12min is kept under 8kg pressure, carry out the quick-fried process of vapour, then add a certain amount of moisture, make the corn cob solid content after process remain on 8%(w/w), soak 1.5h, utilize plate-and-frame filter press to filter, obtain corn cob diluted acid vapour and disclose infusion;
C) purification of zytase: fermentation ends, filters fermentation liquor plate-and-frame filter press, and gained filtered liquid membrane molecule amount is that 10000 daltonian organic membrane carry out ultrafiltration, is concentrated into required multiple; Then add sanitas, its component proportion is sodium-chlor 7g, and Sodium Benzoate 0.1g obtains liquid xylanase product;
D) process of fermented liquid residue: the fermented liquid residue through plate-and-frame filter press gained is dried, makes it water content at 10%(w/w), use as fodder additives.
The enzyme activity unit unified definition (U) of zytase: at pH=4.8, under 50 DEG C of conditions, the per minute degraded substrate enzyme amount generated needed for 1 μm of oL wood sugar is 1 unit of activity.
After measured, the enzyme activity of the present embodiment gained zytase is 2568.35U/mL, single tank single batch fermentation liquid total amount 12.4 tons; Compared with existing zytase liquid submerged fermentation technique, the enzyme activity of zytase improves 60%, and the rate of output improves 40%, and unit enzyme activity fermentation costs reduces by 8%.
Embodiment two
Below with 20m
3fermentor tank is that example is described in detail the present invention.
The liquid submerged fermentation method of zytase of the present invention take corn cob as main raw material, adopts aspergillus niger liquid submerged fermentation method, can carry out according to the following steps:
A) preparation of bacterial classification and enlarged culturing: by Aspergillus niger strain
aspergillus nigercGMCC No.5833 is forwarded on PDA slant medium, cultivates 4 days, obtain slant pore under 30 DEG C of conditions; Then wash described slant pore with sterilized water, obtain spore washings; Aseptically accessed by described spore washings in sterilized triangular flask seed culture medium (at 121 ~ 124 DEG C, sterilizing 30 min), shaking speed is 200r/min, cultivates 20h, obtain activated spawn under 30 DEG C of conditions; By described activated spawn with 0.5%(v/v) inoculum size access in described first class seed pot, in 30 DEG C, under the condition of ventilation ratio 1:0.7, cultivate 18h, obtain primary seed solution; By described primary seed solution with 10%(v/v) inoculum size access secondary seed tank, in 30 DEG C, under the condition of ventilation ratio 1:0.7, cultivate 12h, obtain secondary seed solution.Wherein, the composition of described triangular flask seed culture medium, first class seed pot and secondary seed tank is: with mass percent, glucose 1.5%, wheat bran 4%, corn steep liquor 1%, yeast powder 0.5%, and surplus is water;
B) fed-batch fermentation is cultivated: at 20m
3in fermentor tank, load fermentation basic medium 12m
3(with mass percent, grinding particle size is less than the corn cob 7.0% of 0.5mm, wheat bran 1.5%, corn steep liquor 3.0%, ammonium sulfate 0.5%, potassium primary phosphate 0.2%, magnesium sulfate 0.2%, calcium chloride 0.05%, trace element 0.1%, surplus is water), be 4.8 by above-mentioned substratum adjust ph, sterilizing 30min at 121 ~ 124 DEG C; Treat that substratum temperature is down to 30 DEG C, aseptically, the 12%(v/v with fermention medium volume) inoculum size access described secondary seed solution, 9h after inoculation, ventilation ratio is 1:1, and mixing speed is 160r/min, and culture temperature is 30 DEG C; 15h after inoculation, ventilation ratio is 1:0.8, and mixing speed is 110r/min, and culture temperature is 28 DEG C; Fermentation 32h, concentration of reduced sugar is 0.08%, adds supplemented medium and carries out feed supplement, and feed rate is feed supplement amount per hour is fermentation basic medium 1.4%(w/w), reducing sugar controls at 0.1%(w/w); Discharge is carried out when fermentating liquid volume reaches 80% of fermenter volume, discharge is stopped when reaching 70% of fermenter volume to fermentating liquid volume, repeat discharge operation in a fermentation period, maintain fermentating liquid volume between 70% of fermenter volume, fermentation period is 7 days;
Wherein, the mass percent of described supplemented medium consists of, glucose 4.0%, corn steep liquor 2.0%, ammonium sulfate 0.7%, potassium primary phosphate 0.7%, magnesium sulfate 0.4%, calcium chloride 0.2%, trace element 0.06%, all the other disclose infusion for corn cob diluted acid vapour, and adjust ph is 4.5, sterilizing 30min at 121 ~ 124 DEG C.The mass percent of described trace element consists of, ammonium molybdate 0.04%, zinc sulfate 0.2%, ferrous sulfate 0.3%, manganous sulfate 0.1%, cobalt chloride 0.15%, copper sulfate 0.1%.Described corn cob diluted acid vapour discloses that infusion is obtained by following preparation method: corn cob granularity being less than 10mm, in 0.5%(w/w) soak in sulphuric acid soln, 10min is kept under 6kg pressure, carry out the quick-fried process of vapour, then add a certain amount of moisture, make the corn cob solid content after process remain on 6%(w/w), soak 1h, utilize plate-and-frame filter press to filter, obtain corn cob diluted acid vapour and disclose infusion;
C) purification of zytase: fermentation ends, filters fermentation liquor plate-and-frame filter press, and gained filtered liquid membrane molecule amount is that 10000 daltonian organic membrane carry out ultrafiltration, is concentrated into required multiple; Then add sanitas, its component proportion is sodium-chlor 8g, and Sodium Benzoate 0.2g obtains liquid xylanase product;
D) process of fermented liquid residue: the fermented liquid residue through plate-and-frame filter press gained is dried, makes it water content at 13%(w/w), use as fodder additives.
After measured, the enzyme activity of the present embodiment gained zytase is 2968.04U/mL, single tank single batch fermentation liquid total amount 32.5 tons; Compared with existing zytase liquid submerged fermentation technique, the enzyme activity of zytase improves 65%, and the rate of output improves 46%, and unit enzyme activity fermentation costs reduces by 6%.
Embodiment three
Below with 50m
3fermentor tank is that example is described in detail the present invention.
The liquid submerged fermentation method of zytase of the present invention take corn cob as main raw material, adopts aspergillus niger liquid submerged fermentation method, can carry out according to the following steps:
A) preparation of bacterial classification and enlarged culturing: by Aspergillus niger strain
aspergillus nigercGMCC No.5833 is forwarded on PDA slant medium, cultivates 4 days, obtain slant pore under 30 DEG C of conditions; Then wash described slant pore with sterilized water, obtain spore washings; Aseptically accessed by described spore washings in sterilized triangular flask seed culture medium (at 121 ~ 124 DEG C, sterilizing 30 min), shaking speed is 220r/min, cultivates 18h, obtain activated spawn under 32 DEG C of conditions; By described activated spawn with 0.4%(v/v) inoculum size access in described first class seed pot, in 32 DEG C, under the condition of ventilation ratio 1:0.6, cultivate 20h, obtain primary seed solution; By described primary seed solution with 9%(v/v) inoculum size access secondary seed tank, in 32 DEG C, under the condition of ventilation ratio 1:0.8, cultivate 8h, obtain secondary seed solution.Wherein, the composition of described triangular flask seed culture medium, first class seed pot and secondary seed tank is: with mass percent, glucose 1.5%, wheat bran 4%, corn steep liquor 1%, yeast powder 0.5%, and surplus is water;
B) fed-batch fermentation is cultivated: at 60m
3in fermentor tank, load fermentation basic medium 36m
3(with mass percent, grinding particle size is less than 0.5mm corn cob 5.0%, wheat bran 2.0%, corn steep liquor 1.5%, ammonium sulfate 0.6%, potassium primary phosphate 0.4%, magnesium sulfate 0.3%, calcium chloride 0.15%, trace element 0.2%, surplus is water), be 4.5 by above-mentioned substratum adjust ph, sterilizing 30min at 121 ~ 124 DEG C; Treat that substratum temperature is down to 30 DEG C, aseptically, the 15%(v/v with fermention medium volume) inoculum size access described secondary seed solution, 4h after inoculation, ventilation ratio is 1:1.5, and mixing speed is 150r/min, and culture temperature is 32 DEG C; 11h after inoculation, ventilation ratio is 1:1, and mixing speed is 100r/min, and culture temperature is 29 DEG C; Fermentation 30h, concentration of reduced sugar is 0.02%, adds supplemented medium and carries out feed supplement, and feed rate is feed supplement amount per hour is fermentation basic medium 1.6%(w/w), reducing sugar controls at 0.12%(w/w); Discharge is carried out when fermentating liquid volume reaches 80% of fermenter volume, discharge is stopped when reaching 70% of fermenter volume to fermentating liquid volume, repeat discharge operation in a fermentation period, maintain fermentating liquid volume between 80% of fermenter volume, fermentation period is 5 days;
Wherein, the mass percent of described supplemented medium consists of, glucose 5.0%, corn steep liquor 3.0%, ammonium sulfate 0.6%, potassium primary phosphate 0.4%, magnesium sulfate 0.4%, calcium chloride 0.3%, trace element 0.1%, all the other disclose infusion for corn cob diluted acid vapour, and adjust ph is 4.5, sterilizing 30min at 121 ~ 124 DEG C.The mass percent of described trace element consists of, ammonium molybdate 0.04%, zinc sulfate 0.2%, ferrous sulfate 0.3%, manganous sulfate 0.1%, cobalt chloride 0.15%, copper sulfate 0.1%.Described corn cob diluted acid vapour discloses that infusion is obtained by following preparation method: corn cob granularity being less than 10mm, in 1.0%(w/w) soak in sulphuric acid soln, 15min is kept under 10kg pressure, carry out the quick-fried process of vapour, then add a certain amount of moisture, make the corn cob solid content after process remain on 10%(w/w), soak 2h, utilize plate-and-frame filter press to filter, obtain corn cob diluted acid vapour and disclose infusion;
C) purification of zytase: fermentation ends, filters fermentation liquor plate-and-frame filter press, and gained filtered liquid membrane molecule amount is that 10000 daltonian organic membrane carry out ultrafiltration, is concentrated into required multiple; Then add sanitas, its component proportion is sodium-chlor 10g, and Sodium Benzoate 0.3g obtains liquid xylanase product;
D) process of fermented liquid residue: the fermented liquid residue through plate-and-frame filter press gained is dried, makes it water content at 15%(w/w), use as fodder additives.
After measured, the enzyme activity of the present embodiment gained zytase is 3485.65U/mL, single tank single batch fermentation liquid total amount 97.6 tons; Compared with existing zytase liquid submerged fermentation technique, the enzyme activity of zytase improves 70%, and the rate of output improves 50%, and unit enzyme activity fermentation costs reduces by 7.5%.
Claims (9)
1. a liquid submerged fermentation method for zytase take corn cob as main raw material, and adopt aspergillus niger liquid submerged fermentation method, it is characterized in that, it specifically comprises the following steps:
A) preparation of bacterial classification and enlarged culturing: by Aspergillus niger strain
aspergillus nigercGMCC No.5833 is forwarded on PDA slant medium, 30 DEG C, cultivates 4 days, obtains slant pore; Then wash described slant pore with sterilized water, obtain spore washings; Aseptically described spore washings is accessed in 121 ~ 124 DEG C, in the triangular flask seed culture medium of sterilizing 30min, in 180 ~ 220r/min, under 28 ~ 32 DEG C of conditions, cultivate 18 ~ 24h, obtain activated spawn; By described activated spawn with 0.3 ~ 0.5%(v/v) inoculum size access in described first class seed pot, in 28 ~ 32 DEG C, under the condition of ventilation ratio 1:0.6 ~ 0.8, cultivate 18 ~ 20h, obtain primary seed solution; By described primary seed solution with 8 ~ 10%(v/v) inoculum size access secondary seed tank, in 28 ~ 32 DEG C, under the condition of ventilation ratio 1:0.6 ~ 0.8, cultivate 8 ~ 12h, obtain secondary seed solution;
B) fed-batch fermentation is cultivated: in fermentor tank, add fermentation basic medium, coefficient is 50 ~ 60%(v/v), adjust ph is 4.5 ~ 5.0,121 ~ 124 DEG C, sterilizing 30min; Treat that substratum temperature is down to 30 DEG C, aseptically, 10 ~ 15%(v/v with fermention medium volume) inoculum size access described secondary seed solution, 0 ~ 10h after inoculation, ventilation ratio is 1:0.6 ~ 1.5, and mixing speed is 150 ~ 200r/min, and culture temperature is 30 ~ 32 DEG C; After inoculation 10h, ventilation ratio 1:0.5 ~ 1.0, mixing speed 100 ~ 200r/min, culture temperature 28 ~ 30 DEG C; When reducing sugar content is at 0.1%(w/w) below time, start to add supplemented medium and carry out feed supplement, feed rate is feed supplement amount per hour is fermention medium 1.2 ~ 1.6%(w/w), control the content of reducing sugar at 0.2%(w/w) below; Carry out discharge when fermentating liquid volume reaches 80% of fermenter volume, stop discharge when reaching 70% of fermenter volume to fermentating liquid volume, repeat discharge operation in a fermentation period, fermentation period is 5 ~ 7 days;
C) purification of zytase: fermentation ends, filters fermentation liquor plate-and-frame filter press, and gained filtered liquid organic membrane carries out ultrafiltration, is concentrated into required multiple; Add sanitas, obtain liquid xylanase product;
D) process of fermented liquid residue: the fermented liquid residue through plate-and-frame filter press gained is dried, makes it water content at 10 ~ 15%(w/w), use as fodder additives.
2. the liquid submerged fermentation method of zytase as claimed in claim 1, it is characterized in that: the mass percent composition of the seed culture medium of triangular flask described in step a), first class seed pot and secondary seed tank is, 1.5% glucose, 4% wheat bran, 1% corn steep liquor, 0.5% yeast powder, surplus is water.
3. the liquid submerged fermentation method of zytase as claimed in claim 1, it is characterized in that: the mass percent of the basic medium that ferments described in step b) consists of, corn cob 5.0 ~ 7.0%, wheat bran 1.0 ~ 2.0%, corn steep liquor 1.5 ~ 3.0%, ammonium sulfate 0.4 ~ 0.6%, potassium primary phosphate 0.2 ~ 0.4%, magnesium sulfate 0.1 ~ 0.3%, calcium chloride 0.05 ~ 0.15%, trace element 0.05 ~ 0.2%, surplus is water.
4. the liquid submerged fermentation method of zytase as claimed in claim 3, is characterized in that: the granularity of described corn cob is less than 0.5mm.
5. the liquid submerged fermentation method of zytase as claimed in claim 1, it is characterized in that: the mass percent of supplemented medium described in step b) consists of, glucose 3.0 ~ 5.0%, corn steep liquor 2.0 ~ 3.0%, ammonium sulfate 0.6 ~ 0.8%, potassium primary phosphate 0.4 ~ 0.8%, magnesium sulfate 0.2 ~ 0.4%, calcium chloride 0.1 ~ 0.3%, trace element 0.05 ~ 0.1%, all the other disclose infusion for corn cob diluted acid vapour.
6. the liquid submerged fermentation method of zytase as claimed in claim 5, is characterized in that: the mass percent of described trace element consists of, ammonium molybdate 0.04%, zinc sulfate 0.2%, ferrous sulfate 0.3%, manganous sulfate 0.1%, cobalt chloride 0.15%, copper sulfate 0.1%.
7. the liquid submerged fermentation method of zytase as claimed in claim 5, it is characterized in that: described corn cob diluted acid vapour discloses that infusion is obtained by following preparation method: corn cob granularity being less than 10mm, in 0.5 ~ 1.0%(w/w) soak in sulphuric acid soln, 10 ~ 15min is kept under 6 ~ 10kg pressure, carry out the quick-fried process of vapour, then a certain amount of moisture is added, the corn cob solid content after process is made to remain on 6 ~ 10%(w/w), soak 1 ~ 2h, utilize plate-and-frame filter press to filter, obtain corn cob diluted acid vapour and disclose infusion.
8. the liquid submerged fermentation method of zytase as claimed in claim 1, is characterized in that: organic membrane described in step c) is membrane molecule amount is 10000 daltonian organic membrane.
9. the liquid submerged fermentation method of zytase as claimed in claim 1, is characterized in that: the component proportion of sanitas described in step c) is: sodium-chlor 7g ~ 10g, Sodium Benzoate 0.1g ~ 0.3g.
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