CN101659949B - Preparation method of liquid cellulase - Google Patents
Preparation method of liquid cellulase Download PDFInfo
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- CN101659949B CN101659949B CN2009100939343A CN200910093934A CN101659949B CN 101659949 B CN101659949 B CN 101659949B CN 2009100939343 A CN2009100939343 A CN 2009100939343A CN 200910093934 A CN200910093934 A CN 200910093934A CN 101659949 B CN101659949 B CN 101659949B
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Abstract
The invention relates to a preparation method of liquid cellulase. The invention adopts a semicontinuous fermentation mode to utilize microorganism fermentation so as to produce cellulase in culture medium. The preparation method comprises the following steps of: (I) inoculating the microorganism into a fermentation medium, insulating and culturing; and (II) discharging 10 percent to 80 percent of culture liquid obtained by the step (I), and simultaneously supplying new fermentation medium with same volume, and culturing; and circling the step (II) for more than 10 to 15 times, and discharging the tank. The method is applicable to industrial production, can improve utilization ratio of equipment, and simultaneously reduce the cost of preparing seeds; in addition, the cellulase and microcrystalline cellulose degradation products in the fermentation liquid also provide good external environment for the induction of cellulase of the following semicontinuous fermentation, therefore, the cellulase can be prepared with high yield.
Description
Technical field
The present invention relates to the biological fermentation field, specifically, relate to a kind of method of utilizing semi-continuous type to produce liquid cellulase.
Background technology
Mierocrystalline cellulose is recyclability natural resource the abundantest on the earth as the main polyose product of photosynthesis of plant.According to estimates, the energy of being contained in the Mierocrystalline cellulose on the earth is about as much as 6,400 hundred million tons the contained energy of oil, and cellulosic recyclability is that the mineral energy such as oil are incomparable.Make full use of the cellulose treatment Mierocrystalline cellulose, might fundamentally solve the human energy and the food problem that is faced, its profound influence is immeasurable.
Yet about 80% Mierocrystalline cellulose is not exploited at present, has very tempting prospect.In recent years, the applied research of cellulase both at home and abroad is very active, has screened a collection of superior strain.Mierocrystalline cellulose is as a kind of renewable resources with great potential; Utilize the cellulase of microorganisms producing to be translated into the human energy, food and the industrial chemicals of being badly in need of, solve environmental pollution, be short of food and energy dilemma is of great immediate significance for human society.
Cellulase has widely at numerous areas such as weaving, food fermentation, feed, papermaking, daily-use chemical industry, Chinese herbal medicine extracting to be used; Particularly aspect stalk production alcohol; Have huge potential value, its prospect is very wide, and Study on Cellulase has become a strategic problem.
The U.S. is outstanding company of section to cooperate with American National renewable energy resources laboratory; Also obtaining important breakthrough aspect the production cost that reduces cellulase; Make the cost of production of cellulose enzyme reduce to 10 cents～20 cents/gallon, reached the target of 30 times of the reductions of expection.Mean that the commercialization popularization that realizes the bio-based alcohol fuel is no longer remote, this achievement in research will be verified on testing apparatus soon.
Cellulase has tens yuan the market sales revenue every year in China; Has application widely in industry such as weaving, alcohol, feeds; But the domestic market share more than 90% is captured by external product at present; Therefore carry out the cellulase fermentations Study on Technology, improve the liquid fermenting enzyme activity, reducing production costs has very important practical sense.
The production of cellulase is the same with other zymin, mainly contains two kinds of solid fermentation and liquid submerged fermentations, has done many valuable work both at home and abroad in this respect.Solid fermentation method is to be main raw material with agricultural crop straws such as corns, its less investment, and technology is simple, and product price is cheap, and present domestic most cellulase production producer all adopts should technology production of cellulose enzyme.But solid fermentation method exists certain defective, is that the solid fermentation method of the raw material cellulase level of automation of producing is low with the stalk, and labour intensity is big, unstable product quality.
Therefore, along with the development of liquid fermenting enzymatic process and the raising of bacterial classification performance, adopting solution fermentation producd fibers enzyme element is inexorable trend.Batch fermentation is mainly adopted in the fermentation of liquid cellulase at present, and fermentation period is longer, reaches more than 7 days; Plant factor is lower, and power consumption is bigger, and the preparation cost of seed is higher; Consumption to raw material Microcrystalline Cellulose and lactose is bigger, so cost is high.
The present invention is directed to above defective; A kind of method of utilizing the semi-continuous type preparing liquid cellulase is proposed; Can improve plant factor; Reduce the expense of preparation seed simultaneously, and existing cellulase also is that inducing of cellulase provides a good outside atmosphere in the follow-up semicontinuous fermentation with the Microcrystalline Cellulose degradation product in the fermented liquid, reduces the cost.
Summary of the invention
The objective of the invention is the deficiency to the existing fermentation technique of cellulase, provide a kind of easy and simple to handle, cost is low, the method for utilizing the semi-continuous type preparing liquid cellulase that enzyme activity is stable.
The method of preparing liquid cellulase of the present invention is to adopt the semicontinuous fermentation mode in substratum, to utilize microbial fermentation production of cellulose enzyme, comprising: (I) with microbial inoculant heat insulating culture in the fermention medium; The 10%-80% of the nutrient solution that (II) step (I) is obtained emits, and replenishes the fresh fermention medium of equal volume simultaneously and cultivates; Step (II) is carried out in circulation, puts jar.
This method is applicable to the mould that viride, black mold, Trichodermareesei etc. can adopt this method to prepare cellulase, preferred viride;
Contain 1-3% Microcrystalline Cellulose, 2～4% steeping waters, 1～3% urea, 2～5% ammonium sulfate, 2～5% potassium hydrogenphosphates in the said fermention medium.
Said culture temperature is 25-32 ℃;
The described incubation time of step (I) is 3-7 days;
The described incubation time of step (II) is 12-48 hour;
The cycle index of said step (II) 10～15 times.
Method of the present invention in step (I) before, also comprises seed activation and cultivation.
The temperature of said seed culture is 30-32 ℃.
Specifically, said seed activation and cultured method are following:
1) slant culture: under aseptic technique, original seed is inserted in the PDA test tube slant substratum, cultivated 4～8 days for 30-32 ℃;
2) shaking culture: under aseptic condition, slant strains is inserted in the seed culture medium of triangular flask, put on the bottle swingging machine shaking culture 24～48 hours, the temperature of cultivation is 30-32 ℃;
3) enlarged culturing: then the shake-flask seed seeding tank of transferring continue was cultivated 24～48 hours.Culture temperature is 30-32 ℃.
Said seed culture medium contains 3～5% glucose, 2～4% steeping waters, 0.5～1% urea, 2～4% ammonium sulfate, 1～3% potassium hydrogenphosphate, 0.5～1% sal epsom, and surplus is a water.
The method of semicontinuous cultivation preparing liquid cellulase of the present invention is applicable to industrial production, can improve plant factor, reduces the expense of preparation seed simultaneously; In addition, existing cellulase and Microcrystalline Cellulose degradation product can the produced in high yields cellulases also for inducing of cellulase in the follow-up semicontinuous fermentation provides a good outside atmosphere in the fermented liquid.
Description of drawings
Fig. 1 produces the liquid cellulase process route chart for semicontinuous fermentation.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.These embodiment just play the effect of explanation, and the present invention is not limited to these embodiment.Employed method is the normal experiment method like no specified otherwise among the following embodiment.
Embodiment 1
With viride (Chinese industrial microbial strains management preservation center; CICC 40635), liquid amount utilizes Microcrystalline Cellulose semicontinuous fermentation production of cellulose enzyme in being the 250mL triangular flask of 50mL; Circulating fermentation 10 times, leavening temperature are 30 ℃, and rotating speed is 250 rev/mins.
Wherein contain 3% Microcrystalline Cellulose, 3% steeping water, 3% urea, 4% ammonium sulfate, 4% potassium hydrogenphosphate in the fermention medium.
Emit 50% nutrient solution after fermenting first 96 hours, add the fresh culture of equivalent then.Later whenever emitted 50% nutrient solution, add the fresh culture of equivalent again, altogether stop fermentation behind the circulating fermentation 10 times at a distance from 24 hours.Composition is 3% Microcrystalline Cellulose, 2% steeping water, 1% urea, 2% ammonium sulfate, 0.5% potassium hydrogenphosphate in the substratum of adding.
The fermented liquid of emitting at last need be measured cellulase activity and residual sugar.Enzyme work is 60 ius, and residual sugar is 0.05%
Embodiment 2
In 50L stainless steel standard fermentor tank, liquid amount is 30L with viride (Chinese industrial microbial strains management preservation center, CICC 41495).Utilize Microcrystalline Cellulose semicontinuous fermentation production of cellulose enzyme, fermentation circulation 10 times, leavening temperature is 30 ℃, mixing speed is 300 rev/mins, ventilates to being that 0.8vvm, tank pressure are 0.05Mpa.
Wherein contain in the fermention medium and contain 2% Microcrystalline Cellulose, 2% steeping water, 3% urea, 5% ammonium sulfate, 2% potassium hydrogenphosphate in the fermention medium, 0.001% bubble enemy.
Emit 30% nutrient solution after fermenting first 85 hours, add the fresh culture of equivalent then.Later on whenever, emitted 20% at a distance from 24 hours, the 30%......80% nutrient solution, add the fresh culture of equivalent again, circulating fermentation 7 times, every then nutrient solution of 60% of emitting at a distance from 24 hours replenishes the fresh culture of equivalent simultaneously, end is fermented after 7 times.Composition is 3% Microcrystalline Cellulose, 2% steeping water, 1% urea, 2% ammonium sulfate, 2% potassium hydrogenphosphate in the substratum of adding, 0.001% bubble enemy, 0.0015% tween 80.
The fermented liquid of emitting at last all will be measured cellulase activity and residual sugar immediately.Enzyme work is 59 ius, and residual sugar is 0.04%.
Embodiment 3
In 1T stainless steel standard fermentor tank, liquid amount is 750L with viride (Chinese industrial microbial strains management preservation center, CICC 40492).Utilize Microcrystalline Cellulose semicontinuous fermentation production of cellulose enzyme, fermentation circulation 10 times, leavening temperature is 30 ℃, mixing speed is 200 rev/mins, ventilates to being that 0.7vvm, tank pressure are 0.05Mpa.
Wherein contain 2% Microcrystalline Cellulose, 2% steeping water, 12% urea, 2.5% ammonium sulfate, 2.5% potassium hydrogenphosphate in the fermention medium, 0.001% bubble enemy.
Emit 40% nutrient solution after fermenting first 75 hours, add the fresh culture of equivalent then.Later whenever emitted 40% nutrient solution, add the fresh culture of equivalent again, altogether finish fermentation behind the circulating fermentation 12 times at a distance from 24 hours.Composition is 2% Microcrystalline Cellulose, 2% steeping water, 1.5% urea, 2% ammonium sulfate, 2% potassium hydrogenphosphate in the substratum of adding, 0.001% bubble enemy, 0.0015% tween 80.
The fermented liquid of emitting at last all will be measured cellulase activity and residual sugar immediately.Enzyme work is 58 ius, and residual sugar is 0.03%
Embodiment 4
In 10T stainless steel standard fermentor tank, liquid amount is 7.5T with viride (Chinese industrial microbial strains management preservation center, CICC 40636).Utilize Microcrystalline Cellulose semicontinuous fermentation production of cellulose enzyme, fermentation circulation 10 times, leavening temperature is 30 ℃, mixing speed is 120 rev/mins, ventilates to being that 0.5vvm, tank pressure are 0.03Mpa.
Wherein contain 2% Microcrystalline Cellulose, 4% steeping water, 3% urea, 5% ammonium sulfate, 5% potassium hydrogenphosphate in the fermention medium.
Emit 40% nutrient solution after fermenting first 75 hours, add the fresh culture of equivalent then.Later whenever emitted 40% nutrient solution, add the fresh culture of equivalent again, altogether finish fermentation behind the circulating fermentation 10 times at a distance from 24 hours.Composition is 1% Microcrystalline Cellulose, 2% steeping water, 1% urea, 2% ammonium sulfate, 2% potassium hydrogenphosphate in the substratum of adding, 0.001% bubble enemy, 0.0015% tween 80.
The fermented liquid of at every turn emitting all will be measured cellulase activity and residual sugar immediately.Enzyme work is 46 ius, and residual sugar is 0.01%.
Claims (5)
1. a preparation method of liquid cellulase is characterized in that, is to adopt the semicontinuous fermentation mode in substratum, to utilize microbial fermentation production of cellulose enzyme, comprising:
(I) viride is inoculated into heat insulating culture in the fermention medium, contains 1-3% Microcrystalline Cellulose, 2～4% steeping waters, 1～3% urea, 2～5% ammonium sulfate, 2～5% potassium hydrogenphosphates in the fermention medium, surplus is a water; Incubation time is 3-7 days;
(II) 10%-80% with nutrient solution emits, and replenishes the fresh fermention medium of equal volume simultaneously and cultivates, and incubation time is 12-48 hour; Circulation is carried out step (II) 10～15 times, puts jar.
2. preparation method as claimed in claim 1 is characterized in that, said step (I) and culture temperature (II) are 25-32 ℃.
3. like the preparation method of claim 1 or 2, it is characterized in that, in step (I) before, also comprise seed activation and cultivation.
4. preparation method as claimed in claim 3 is characterized in that, said seed activation and cultivation may further comprise the steps:
1) slant culture: under aseptic technique, original seed is inserted in the PDA test tube slant substratum, cultivated 4～8 days for 30-32 ℃;
2) shaking culture: under aseptic condition, slant strains inserted hold in the triangular flask of seed culture medium, put on the bottle swingging machine shaking culture 24～48 hours, the temperature of cultivation is 30-32 ℃;
3) enlarged culturing: then the shake-flask seed seeding tank of transferring continue was cultivated 24～48 hours, culture temperature is 30-32 ℃.
5. preparation method as claimed in claim 4 is characterized in that, said seed culture medium contains 3～5% glucose, 2～4% steeping waters, 0.5～1% urea, 2～4% ammonium sulfate, 1～3% potassium hydrogenphosphate, 0.5～1% sal epsom, and surplus is a water.
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| CN102392004A (en) * | 2011-10-27 | 2012-03-28 | 安徽丰原发酵技术工程研究有限公司 | Composite enzyme of cellulase, xylanase and cellobiase, as well as preparation method thereof |
| CN110713998B (en) * | 2019-11-29 | 2021-11-02 | 江南大学 | Preparation method and application of arabinoxylan degrading enzyme system |
| CN114015559B (en) * | 2021-09-08 | 2023-06-23 | 安徽华恒生物科技股份有限公司 | Efficient valine semi-continuous fermentation method and complete equipment thereof |
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Non-Patent Citations (3)
| Title |
|---|
| R. P. TENGERDY,等.Liquid Fluidized Bed Starter Culture.《Applied Biochemistry and Blotechnology》.1991,第27卷195-204. * |
| 傅力 等.里氏木霉高产纤维素酶菌株的选育及产酶培养基的优化.《食品与机械》.2009,第25卷(第3期),10-13. * |
| 勇强, 等..分批添料半连续发酵制备纤维素酶.《林产化学与工业》.1998,第18卷(第2期),73-77. * |
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