CN103865906A - Method for preparing alpha-glucanase by vitue of chaetomium gracile fermentation under ultrasonic condition - Google Patents
Method for preparing alpha-glucanase by vitue of chaetomium gracile fermentation under ultrasonic condition Download PDFInfo
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- CN103865906A CN103865906A CN201410097002.7A CN201410097002A CN103865906A CN 103865906 A CN103865906 A CN 103865906A CN 201410097002 A CN201410097002 A CN 201410097002A CN 103865906 A CN103865906 A CN 103865906A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
- C12N9/2454—Dextranase (3.2.1.11)
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- C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
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- 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)
- C12Y302/01011—Dextranase (3.2.1.11)
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Abstract
The invention discloses a method for preparing an alpha-glucanase by virtue of chaetomium gracile fermentation under an ultrasonic condition. The method is characterized in that when the chaetomium gracile is cultured in a culture medium, an ultrasonic action having the frequency of 20-40KHZ and the power of 80-120W is applied by the frequency of 40-80 minutes each time for 40-80 seconds each time. Beyond the expectation of the technicist in the field, the method for preparing the alpha-glucanase by virtue of chaetomium gracile fermentation under the low-frequency and low-power ultrasonic condition increases the growth rate of cells, and meanwhile, the enzyme activity of the glucanase is improved by 215%.
Description
Technical field
The invention belongs to microorganism and biology techniques field, prepare the method for alpha-glucanase in particular to thin beautiful chaetomium fermentation under ultrasound condition.
Background technology
Dextran (dextran) claim again dextran, is a kind of polysaccharide, is present in the mucus that certain micro-organisms secretes in process of growth.Dextran has higher molecular weight, mainly by D-glucopyanosyl with α, 1 → 6 key connect, side chain point have 1 → 2,1 → 3,1 → 4 connect.Along with the difference of microbe species and growth conditions, glucan structure also has difference.In refined sugar production process, the microbes secretion invertases such as the Leuconostoc mesenteroides that sugarcane is carried secretly consume sucrose, generate a large amount of dextran, are commonly called as " sugarcane meal ".The accumulation of dextran is except causing the loss of sugar, also causes liquid glucose viscosity to increase, the series of problems such as specific rotation is higher, crystallization and filtration difficulty, improves to the scale operation in sugar refinery and sugar products quality and brings many disadvantageous effects.
Domestic a lot of scholars take many kinds of measures to reduce or eliminate the dextran in liquid glucose at present, are mainly divided into Physical, chemical method and biological process etc.Physical uses the methods such as clarification, membrane filtration, and its technical costs is higher, is difficult to extensive popularization.Chemical method mainly adds the antibiosis such as the sterilant such as chlorinated lime and penicillin, duomycin usually to suppress microbial growth.Comparatively speaking, chemical method is cheap, does not need extras, but the residual of pharmaceutical chemicals may work the mischief to human body, and established dextran is not had to the effect of degraded.Biological process is mainly to use enzymolysis process, adopts dextranase to degrade to dextran contained in syrup, and the dextran in can removing syrup rapidly and efficiently reaches and improves former sugar yield, reduces the objects such as liquid glucose viscosity.But the dextranase in the level of security source that current food is used is comparatively rare, has limited greatly the widespread use of biological process in sugar industry.Therefore, develop a kind of dextranase of safety source, become one of subject matter that current sugar industry faces.
Thin beautiful chaetomium is a kind of food safety bacterial strain of the U.S. and European Union's accreditation, in its fermenting process, can secrete alpha-glucanase.At present domestic less for the research that utilizes thin beautiful chaetomium fermentation to produce alpha-glucanase, main research concentrates on method and the step of utilizing thin beautiful chaetomium to prepare the thick enzyme of alpha-glucanase, and dextranase output is lower, is difficult to reach industrial applications level.
Therefore, how to improve output and activity that alpha-glucanase is prepared in thin beautiful chaetomium fermentation, reaching industrial applications level is those skilled in the art's technical problem urgently to be resolved hurrily.
Summary of the invention
The object of the invention is to overcome the existing above-mentioned deficiency of prior art, provide a kind of under ultrasound condition thin beautiful chaetomium fermentation prepare the method for alpha-glucanase, improve output and the activity of alpha-glucanase.In order to realize object of the present invention, intend adopting following technical scheme:
One aspect of the present invention relates to the method that alpha-glucanase is prepared in thin beautiful chaetomium fermentation under ultrasound condition, it is characterized in that in the time that thin beautiful chaetomium is cultivated in substratum, the preferred 25-35KHZ of proportion 20-40KHZ(), power 80-120W(is preferably 90-110W) ultrasonication, frequency is 40-80 minute/time (being preferably 50-70 minute/time), 40-80 second each action time (be preferably 50-70 second).
In a preferred embodiment of the present invention, described fermentation culture based component and culture condition: 1.5% dextran, 1.0% yeast extract, 0.5% dipotassium hydrogen phosphate and/or potassium primary phosphate, 0.1% magnesium sulfate; With 1mol/L sodium hydroxide solution adjusting medium pH to 6.0, sterilizing adds a certain amount of sterilizing calcium carbonate in substratum, and the inoculum size with 2% is linked into thin beautiful chaetomium in substratum, 30 DEG C of constant temperature culture 3-4 days in the shaking table that is 200rpm in rotating speed.
In a preferred embodiment of the present invention, after supersound process, continuing that culture system is put into shaking table cultivates.
In a preferred embodiment of the present invention, after thin beautiful chaetomium is cultivated 12h, start ultrasonic.
Exceed those skilled in the art's rational expectation, the present invention, by under low frequency/small Power ultrasound condition, adopts thin beautiful chaetomium fermentation to prepare the method for alpha-glucanase, has improved the growth velocity of cell, and the enzyme work of dextranase has simultaneously improved 215%.
Brief description of the drawings
The impact of Fig. 1 supersound process on dry cell weight;
The impact of Fig. 2 supersound process on thalline autoflocculation phenomenon;
Fig. 3 ultrasound condition is processed the impact that thin beautiful chaetomium fermentative production alpha-glucanase enzyme is lived;
Embodiment
Below in conjunction with the drawings and specific embodiments, method of the present invention is described in more detail.
One, fermentation culture based component and culture condition: 1.5% dextran, 1.0% yeast extract, 0.5% dipotassium hydrogen phosphate (potassium primary phosphate), 0.1% magnesium sulfate.With 1mol/L sodium hydroxide solution adjusting medium pH to 6.0, sterilizing, in substratum, add a certain amount of calcium carbonate (sterilizing), the inoculum size with 2% is linked into thin beautiful hair shell in 25mL substratum, 30 DEG C of constant temperature culture 3-4 days in the shaking table that is 200rpm in rotating speed.
Supersound process condition: fermentation starts supersound process after 12h, frequency 30KHZ, power 100W, ultrasonication frequency is 1 hour/time, each action time 1min.Then put into shaking table and cultivate, culture condition is the same.
Two, the mensuration that alpha-glucanase enzyme is lived
Laboratory apparatus: spectrophotometer, thermostat water bath, electromagnetic oven, liquid-transfering gun, 25cm colorimetric cylinder
Experiment material: DNS reagent, alpha-glucanase, 2.0% dextran substrate
Dextran enzyme activity determination: get the dextran solution that 900 μ L massfractions are 2.0%, be placed in 50 DEG C of waters bath with thermostatic control and be incubated 5min, add immediately 100 μ L enzyme liquid, accurate response 10min, add immediately the DNS of 2mL with termination reaction, in boiling water bath 5min, rapidly that it is cooling, be settled to 25mL with distilled water, measure light absorption value in wavelength 540nm place.Try to achieve the amount of corresponding glucose from the regression equation of glucose typical curve, and convert out enzyme and live.Enzyme work is defined as: under these conditions, it is 1 Ge Meihuo unit that per minute discharges the required enzyme amount of 1 μ mol reducing sugar from dextran substrate, represents with U/mL.
1. the impact of supersound process on thin beautiful chaetomium thalli growth situation:
The present invention has investigated the impact of supersound process on thin beautiful chaetomium thalli growth situation, result confirmation, and low frequency ultrasound processing can obviously improve dry cell weight (Fig. 1).
2. the impact of supersound process on thin beautiful chaetomium autoflocculation phenomenon:
The present invention has investigated low frequency ultrasound and has processed the impact (Fig. 2) on thin beautiful chaetomium autoflocculation phenomenon, confirms that low frequency ultrasound processing can effectively reduce thalline autoflocculation phenomenon.
3. the impact that supersound process is lived on alpha-glucanase enzyme:
Experimental comparison ultrasound condition and common shaking table while cultivating, thin beautiful chaetomium fermentative production alpha-glucanase activity change (Fig. 3).
Result of study confirms, in thin beautiful chaetomium fermenting process, uses low frequency ultrasound technical finesse fermented liquid, can significantly improve thalline dispersiveness, reduces the generation of autoflocculation phenomenon.Meanwhile, the more common shaking table of fermentation broth enzyme activity is cultivated and is improved 215%.The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (3)
1. under ultrasound condition, the method for alpha-glucanase is prepared in thin beautiful chaetomium fermentation, it is characterized in that in the time that thin beautiful chaetomium is cultivated in substratum, the preferred 25-35KHZ of proportion 20-40KHZ(), power 80-120W(is preferably 90-110W) ultrasonication, frequency is 40-80 minute/time (being preferably 50-70 minute/time), 40-80 second each action time (be preferably 50-70 second).
2. method according to claim 1, described fermentation culture based component and culture condition: 1.5% dextran, 1.0% yeast extract, 0.5% dipotassium hydrogen phosphate and/or potassium primary phosphate, 0.1% magnesium sulfate; With 1mol/L sodium hydroxide solution adjusting medium pH to 6.0, sterilizing adds a certain amount of sterilizing calcium carbonate in substratum, and the inoculum size with 2% is linked into thin beautiful chaetomium in substratum, 30 DEG C of constant temperature culture 3-4 days in the shaking table that is 200rpm in rotating speed.
3. method according to claim 2, continues after supersound process that culture system is put into shaking table and cultivates.
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| CN201410097002.7A CN103865906A (en) | 2014-03-17 | 2014-03-17 | Method for preparing alpha-glucanase by vitue of chaetomium gracile fermentation under ultrasonic condition |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441410A (en) * | 2015-12-31 | 2016-03-30 | 宁夏乙征生物工程有限公司 | Production method of rhamnosidase |
| CN109486796A (en) * | 2018-12-06 | 2019-03-19 | 中诺生物科技发展江苏有限公司 | A method of preparing dextranase enzyme preparation |
-
2014
- 2014-03-17 CN CN201410097002.7A patent/CN103865906A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| MOHANAD BASHARI等: "Influence of low ultrasound intensity on the degradation of dextran catalyzed by dextranase", 《ULTRASONICS SONOCHEMISTRY》 * |
| 戴传云等: "低功率超声波对微生物发酵的影响", 《重庆大学学报》 * |
| 罗登林等: "超声对黑曲霉产菊粉酶的影响", 《声学技术》 * |
| 麻少莹等: "α-葡聚糖酶发酵工艺及其酶活性影响因素研究", 《食品科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441410A (en) * | 2015-12-31 | 2016-03-30 | 宁夏乙征生物工程有限公司 | Production method of rhamnosidase |
| CN109486796A (en) * | 2018-12-06 | 2019-03-19 | 中诺生物科技发展江苏有限公司 | A method of preparing dextranase enzyme preparation |
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Application publication date: 20140618 |