CN101182470A - Liquid submerged fermentation method for preparing xylanase - Google Patents
Liquid submerged fermentation method for preparing xylanase Download PDFInfo
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- CN101182470A CN101182470A CNA2007101580663A CN200710158066A CN101182470A CN 101182470 A CN101182470 A CN 101182470A CN A2007101580663 A CNA2007101580663 A CN A2007101580663A CN 200710158066 A CN200710158066 A CN 200710158066A CN 101182470 A CN101182470 A CN 101182470A
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Abstract
The invention relates to a liquid deep fermentation method for the preparation of xylanase. The invention applies an Aspergillus niger fermentation method. The invention is characterized in that proper Aspergillus niger strains are sterilely inoculated into a stainless steel mother culture tank; the fermentation culture medium is added into a production fermentation tank to 70 percent to 75 percent of the tank volume; the mother culture is sterilely inoculated into the fermentation tank; the culture fermentation is processed under the condition of 30 degree, 150rpm to 200rpm, 0.1wm to 1.5wm aeration rate, 70 hours to 80 hours; during the middle and late period of 40 hours to 60 hours fermentation, nutritious materials are supplemented; the fermentation process is stopped at the 70 hours to 80 hours period; according to the bacteria morphology examined under microscope, the fermentation process is stopped when the thalli begin to auto-lyze; the refining solid enzyme and the refining liquid enzyme are obtained through three ways for the treatment of the fermentation broth, which are mainly a salting-out method of crude enzyme product, a spray drying method and a flocculation ultra-filtering method. The enzyme activity of the liquid deep fermentation enzyme is 10 percent to 30 percent higher than enzyme activity of the solid fermentation enzyme. And the cost per ton for the production of the crude enzyme is reduced more than 20 percent.
Description
Technical field
The present invention relates to microorganism field, a kind of liquid submerged fermentation method for preparing zytase.
Background technology
The solid-state shallow tray fermentation method of tradition zytase is semi-enclosed equipment at present, can not stop microbiological contamination in the external environment fully, and safety performance can't ensure.Therefore, the fermenting enzyme vigor of solid-state shallow tray fermentation method is low, the cost height, and poor stability, its production efficiency is seen end up two kinds of preparation methods' production test data contrast table of this paper.
Summary of the invention
The purpose of this invention is to provide a kind of liquid submerged fermentation method for preparing zytase, enzyme activity improves 30%, and production cost reduces by 20% at least.
The liquid submerged fermentation method of preparation zytase, the fermentation of employing black-koji mould, it is characterized in that in female jar of the stainless steel grade of the aseptic access of proper amount of strains aspergillus niger strain, plant and add seed culture medium in female jar to tank volume 70 ~ 75%, produce fermentor tank and add the production fermention medium to tank volume 70 ~ 75%, sterile state inoculation mother goes into to produce in the fermentor tank; Cultivation and fermentation: 30 ℃, 150 ~ 200rpm, the 0.1 ~ 1.5vvm that ventilates, 70 ~ 80h, material supplements the nutrients between fermentation middle and later periods 40 ~ 60h; Stop fermentation: fermentation according to the microscopy thalli morphology, promptly stops fermentation if thalline begins self-dissolving to 70 ~ 80h; Fermented liquid mainly contains three kinds of processing modes: the salting-out process of enzyme crude product and spray-drying process, flocculation-ultrafiltration process make refining solid enzyme and refining liquid enzyme.
The present invention prepares the positively effect of the liquid submerged fermentation method of zytase:
1. liquid submerged fermentation will exceed 10-30% than the enzyme activity of solid state fermentation;
2. liquid state fermentation reduces more than 20% than the thick enzyme of solid state fermentation production cost per ton.
3. after the thick enzyme drying of liquid submerged fermentation method preparation, its thick enzyme activity will exceed more than 10 times than the thick enzyme activity of solid-state fermentation process preparation, and is beneficial to storage, transportation and the processing of product enzyme.
4. calculate by fermentation capacity, liquid submerged fermentation is than solid state fermentation production efficiency height in the unit time, and personnel selection is few.
5, tank fermentation method is a jar airtight operation, and strictness is produced under aseptic condition, and goods make in the environment of single fermentation bacterium, and security of products is greatly improved.
Embodiment
This production technique is the change type of aspergillus niger solid state fermentation, i.e. zytase (aspergillus niger strain) liquid submerged fermentation method
One. culture of strains
1. common Cha Shi medium slant is cultivated Aspergillus niger strain, inserts 4-6 and only produces with in the 1000ml triangular flask interior dress nutrient solution 300-400ml 5% bran water solution;
2.30-32 ℃, 200-300rpm cultivates 10-20h.
Two. seeding is cultivated (planting female the cultivation)
1. in 1 ton of stainless steel seeding tank, add seed culture medium and cultivate base unit weight and account for 70 ~ 75% tank volume, 121 ℃ of steam sterilizings, 15min, be cooled to 30-32 ℃ standby; The weight percentages of seed culture medium: the aqueous solution contains Semen Maydis powder 1 ~ 8%, corn steep liquor 1 ~ 6%, soybean cake powder 1 ~ 8%, Sodium phosphate dibasic 0.1 ~ 1%, calcium chloride 1 ~ 5%, sulfate of ammoniac 1 ~ 5%.
2. get cultured bacterial classification sterile state and insert seeding tank;
3.30-32 ℃, 200-300rpm cultivates 14-24h.
Three. fermentation
1. fermentor tank add to be produced fermention medium, and tank volume is such as 20 tons, to the 70-75% of tank volume, 121 ℃ of steam sterilizings, 15min, be cooled to 30 ℃ standby; Produce the fermention medium weight percentages: the aqueous solution contains Semen Maydis powder 1 ~ 8%, soybean cake powder 1 ~ 8%, Sodium phosphate dibasic 0.1 ~ 2%, calcium chloride 1 ~ 10%, sulfate of ammoniac 1 ~ 10%.
2. inoculation: sterile state inoculation mother goes in the fermentor tank;
3. cultivation and fermentation: 30 ℃, 150 ~ 200rpm, the 0.1 ~ 1.5vvm that ventilates cultivates 70 ~ 80h;
4. feed supplement: live in order to obtain higher enzyme in process of production, the middle and later periods of need fermenting supplements the nutrients.
A. logical ammonia feed supplement: industrial ammonia feeds in the fermentor tank, 0.5-1h at interval, and the ammonia amount of feeding fluctuates in the 4.5-5.0 scope and is as the criterion to keep fermented liquid pH.
B. nutrition feed supplement: nutritive medium weight percentages: contain Semen Maydis powder 1 ~ 8% in the aqueous solution, soybean cake powder 1 ~ 8%, Sodium phosphate dibasic 0.1 ~ 2%, calcium chloride 1 ~ 10%, sulfate of ammoniac 1 ~ 10%.
C. between fermentation middle and later periods 40-60h according to production requirement, to fermention medium extra-nutrition material (the same substratum of composition and ratio), the thing amount that supplements the nutrients usually is 5L/min, so that good growth of thalline and product enzyme.
5. stop fermentation (putting jar): normally ferment to 70-80h, begin self-dissolving according to the microscopy thalli morphology up to the discovery bacterium and then stop fermentation.
Four. fermentation liquor treatment
1. salting-out process: the fermented liquid of saltouing, enzyme is precipitated out from solution, filter waste liquid through pressure filter, the filter flask oven dry is solid enzyme raw product.
2. spray-drying process: add enzyme stabilizers, filter macrobead after, use the spray tower spraying drying, dried enzyme powder is the powdered raw product.
3. flocculation-ultrafiltration process:
A. flocculation: with flocculation agent particulate matter is flocculated, remove degranulation, receive clarified broth through pressure filter;
B. ultrafiltration: clarified broth is crossed ultrafiltration apparatus and is carried out ultrafiltration, removes the water in the solution and is dissolved in the low minute daughter bacteria metabolite and the pigment of water, obtains transparent, colourless (light color) enzyme concentrated solution;
C. finished product: it is dry that the enzyme concentrated solution loads soma, refining solid enzyme; The enzyme concentrated solution adds stablizer, refining concentrated liquid enzyme.
Five. the examination and test of products
The enzyme raw product meets China forage health standard GB13078-2001; Refining solid enzyme meets China food hygienic standard GB2760; Refining concentrated liquid enzyme meets China food hygienic standard GB2760.
Six. the correlated effect explanation of the inventive method and prior art
Two kinds of preparation methods' production test data contrast table:
| Liquid submerged fermentation | Solid-state shallow tray fermentation | |||||
| Production batch | Fermenting enzyme vigor (U/g) | Spraying drying enzyme activity (U/g) | Thick enzyme production cost per ton (unit) | Fermenting enzyme vigor (U/g) | The air stream drying enzyme activity | Thick enzyme production cost per ton unit |
| ?061208 | ?5485 | ?45600 | ?37700 | ?4197 | ?3510 | ?4120 |
| ?061209 | ?6120 | ?51300 | ?37700 | ?5078 | ?4430 | ?4120 |
| ?061210 | ?5786 | ?46800 | ?37700 | ?4824 | ?4150 | ?4120 |
| ?061211 | ?5927 | ?49700 | ?37700 | ?5136 | ?4560 | ?4120 |
| ?061212 | ?5578 | ?46400 | ?37700 | ?4115 | ?3430 | ?4120 |
Illustrate: above data are the part producing statistic data of continuous 5 production batchs, and every batch bacterial classification is identical.
Claims (5)
1. the liquid submerged fermentation method for preparing zytase, adopt the fermentation of Aspergillus niger method, it is characterized in that and to cultivate in the female jar of the stainless steel grade of the aseptic access of proper amount of strains aspergillus niger strain that produce the fermentor tank adding and produce fermention medium to tank volume 70 ~ 75%, sterile state is inoculated mother and gone into to produce in the fermentor tank; Cultivation and fermentation: 30 ℃, 150-200rpm, ventilation 0.1-1.5vvm, 70-80h, material supplements the nutrients between fermentation middle and later periods 40-60h; Stop fermentation: ferment to 70-80h,, promptly stop fermentation if thalline begins self-dissolving according to the microscopy thalli morphology; Fermented liquid mainly contains three kinds of processing modes: the salting-out process of enzyme crude product and spray-drying process, flocculation-ultrafiltration process make refining solid enzyme and refining liquid enzyme.
2. the liquid submerged fermentation method of preparation zytase according to claim 1 is characterized in that kind of mother's cultivation:
A. in 1 ton of stainless steel seeding tank, add seed culture medium, cultivate base unit weight and account for 70 ~ 75% tank volume, 121 ℃ of steam sterilizings, 15min, be cooled to 30-32 ℃ standby;
B. get cultured bacterial classification aspergillus niger strain sterile state and insert seeding tank;
C.30-32 ℃, 200-300rpm cultivates 14-24h.
3. the liquid submerged fermentation method of preparation zytase according to claim 2, it is characterized in that the weight percentages of kind of mother's substratum: the aqueous solution contains Semen Maydis powder 1 ~ 8%, corn steep liquor 1 ~ 6%, soybean cake powder 5%, Sodium phosphate dibasic 0.1 ~ 1%, calcium chloride 1 ~ 5%, sulfate of ammoniac 1 ~ 5%.
4. the liquid submerged fermentation method of preparation zytase according to claim 1 is characterized in that the material that supplements the nutrients between fermentation middle and later periods 40 ~ 60h.
5. the liquid submerged fermentation method of preparation zytase according to claim 4, the weight percentages of material is characterized in that supplementing the nutrients: contain Semen Maydis powder 1 ~ 8% in the nutrient solution water, soybean cake powder 1 ~ 8%, Sodium phosphate dibasic 0.1 ~ 2%, calcium chloride 1 ~ 10%, sulfate of ammoniac 1 ~ 10%.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101580663A CN101182470A (en) | 2007-11-12 | 2007-11-12 | Liquid submerged fermentation method for preparing xylanase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101580663A CN101182470A (en) | 2007-11-12 | 2007-11-12 | Liquid submerged fermentation method for preparing xylanase |
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| CN101182470A true CN101182470A (en) | 2008-05-21 |
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| CNA2007101580663A Pending CN101182470A (en) | 2007-11-12 | 2007-11-12 | Liquid submerged fermentation method for preparing xylanase |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776166A (en) * | 2011-05-09 | 2012-11-14 | 白银赛诺生物科技有限公司 | Production method for xylanase |
| CN104141233A (en) * | 2013-05-06 | 2014-11-12 | 上海瑞鹰化工有限公司 | A co-bath enzyme agent used for printing and dyeing, a preparing method thereof and applications of the co-bath enzyme agent |
| CN104357428A (en) * | 2014-11-13 | 2015-02-18 | 河南天冠纤维乙醇有限公司 | Liquid submerged fermentation method of xylanase |
| CN105369634A (en) * | 2015-11-18 | 2016-03-02 | 德清县伊得利丝绸有限公司 | Cotton and linen interwoven fabric bleaching dyeing deoxygenization process |
| CN105369633A (en) * | 2015-11-18 | 2016-03-02 | 德清县伊得利丝绸有限公司 | Cotton and linen bleaching and dyeing deoxygenization finishing agent |
-
2007
- 2007-11-12 CN CNA2007101580663A patent/CN101182470A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102776166A (en) * | 2011-05-09 | 2012-11-14 | 白银赛诺生物科技有限公司 | Production method for xylanase |
| CN102776166B (en) * | 2011-05-09 | 2014-07-02 | 白银赛诺生物科技有限公司 | Production method for xylanase |
| CN104141233A (en) * | 2013-05-06 | 2014-11-12 | 上海瑞鹰化工有限公司 | A co-bath enzyme agent used for printing and dyeing, a preparing method thereof and applications of the co-bath enzyme agent |
| CN104141233B (en) * | 2013-05-06 | 2018-01-09 | 广州金瑞鹰生物科技有限公司 | Printing and dyeing are with the same as bath enzyme agent and its preparation method and application |
| CN104357428A (en) * | 2014-11-13 | 2015-02-18 | 河南天冠纤维乙醇有限公司 | Liquid submerged fermentation method of xylanase |
| CN105369634A (en) * | 2015-11-18 | 2016-03-02 | 德清县伊得利丝绸有限公司 | Cotton and linen interwoven fabric bleaching dyeing deoxygenization process |
| CN105369633A (en) * | 2015-11-18 | 2016-03-02 | 德清县伊得利丝绸有限公司 | Cotton and linen bleaching and dyeing deoxygenization finishing agent |
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