CN103146780A - Method of simultaneously liquefying and saccharifying cirn starch - Google Patents
Method of simultaneously liquefying and saccharifying cirn starch Download PDFInfo
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- CN103146780A CN103146780A CN2011104016772A CN201110401677A CN103146780A CN 103146780 A CN103146780 A CN 103146780A CN 2011104016772 A CN2011104016772 A CN 2011104016772A CN 201110401677 A CN201110401677 A CN 201110401677A CN 103146780 A CN103146780 A CN 103146780A
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Abstract
The invention relates to a method of simultaneously liquefying and saccharifying cirn starch. The method uses an extreme heat-stable amylase maltose with an optimum catalytic reaction temperature of 90-100 DEG C to substitute an Aspergillus niger glucoamylase with an optimum catalytic reaction temperature of 55-65 DEG C. Because the extreme heat-stable amylase maltose has similar optimum catalytic reaction temperature and optimum catalytic reaction pH with a Bacillus licheniformis alpha-amylase, the extreme heat-stable amylase maltose, together with Bacillus licheniformis alpha-amylase, can be added into a corn starch slurry preheated to 90-100 DEG C and with a concentration of 10-30 % (w/v), and then kept warm for a while at a same temperature to obtain the corn starch syrup with a total yield of glucose, maltose and isomaltose being more than 110 % (w/w). The method overcomes a problem that in traditional production technology of corn starch syrup, the Bacillus licheniformis alpha-amylase is firstly used to liquefy the corn starch under 90-100 DEG C and a pH of 5.0-6.0, and then the Aspergillus niger glucoamylase is used to saccharify the corn starch under 55-65 DEG C and a pH of 4.2-4.5, and operation steps of adjusting temperature and pH are thus needed, thereby improving production speed of the starch sugar.
Description
Technical field
This invention belongs to technical field of agricultural product process.
This invention relates to and a kind of W-Gum being liquefied and the method for saccharification simultaneously, refer in particular to: W-Gum is mixed with water, after high temperature preheating, add simultaneously α-amylase and saccharifying enzyme, continue to be incubated for some time under identical hot conditions, the complete enzymolysis of W-Gum is converted into the method for monose and disaccharide the most at last again.
Background technology
Corn starch sugar (such as: glucose and malto-oligosaccharide etc.) be the important source material of the industries such as medicine industry, foodstuffs industry, biochemical industry.A kind of two step method production technique that comprises liquefaction and saccharification operation is used in the production of corn starch sugar usually; That is: successively adopt starch liquefacation enzyme and diastatic enzyme, W-Gum is liquefied, carries out saccharification, and produce the technique of glucose and malto-oligosaccharide.
The starch liquefacation enzyme is two kinds of amylase different to the starch storage crop mode with diastatic enzyme.At present, commercial α-amylase is Bacillus licheniformis (Bacillus licheniformis) α-amylase, its the suitableeest catalytic condition is 90 ℃, pH 6.0, can be at random hydrolyzed starch intramolecular α-1 rapidly, 4 glycosidic links, produce limit dextrin and a small amount of maltose, cause the starch slurry viscosity to descend rapidly; Commercial saccharifying enzyme is aspergillus niger (Aspergillus niger) glucoamylase, the suitableeest catalytic condition of this saccharifying enzyme is 60 ℃, pH 4.2～4.5, can be hydrolyzed the α-1 of the non-reduced end of limit dextrin molecule, 4 glycosidic links or a-1,6 glycosidic links, produce glucose, cause the starch slurry reducing power to raise.Simultaneously, in order to accelerate saccharification, the final sugar yield of raising, also usually add the starch debranching enzyme---have a liking for sour Propiram bacillus (Bacillus acidopullulyticus) Pullulanase.
Because above-mentioned α-amylase and saccharifying enzyme have the suitableeest different catalytic reaction condition, thus need to use different temperature of reaction and pH in the process of the liquefaction of starch and saccharification, thereby increased production time and the production cost of β-amylose.
Simultaneously W-Gum is carried out liquefying-saccharifying or makes the starch of liquefaction can continue the technique of saccharification under identical condition in order to set up a kind of single stage method, glucose industry in the urgent need to the suitableeest a kind of catalyzed reaction temperature 90 ℃ of left and right, the suitableeest catalyzed reaction pH is at the saccharifying enzyme of slightly acidic scope.At present, generally believing and adopt genetic engineering technique to improve thermostability, the clonal expression thermophilic microorganism glucoamylase of aspergillus niger (Aspergillus niger) glucoamylase, is two approach that address the above problem.
Extreme Pyrococcus furiosus is the microorganism that a class can be grown in hot environment more than 80 ℃; Recently research discovery, the extreme Pyrococcus furiosus of many kinds has the thermostability glucose amylase gene.
gene recombination is had a liking for bitter bacterium (Picrophilus torridus) glucoamylase at 35-65 ℃, has activity in pH 4.5-6.5 scope, but its suitableeest hydrolysis substrate is trisaccharide maltose (Schepers, B.Thiemann, V.Antranikian, G.Characterization of a novel glucoamylase from the thermoacidophilic archaeon Picrophilus torridus heterologously expressed in E.coli.Engineering in life sciences.2006, 6 (3): 311-317.), hydrolytic activity to starch is low, and fail for mashing industry.
the suitableeest catalyzed reaction temperature of gene recombination thermoplasma acidophilum bacterium (Thermoplasma acidophilum) glucoamylase is only 75 ℃, the suitableeest catalyzed reaction pH is 5.0, can be in 5 hours be glucose (Dock with the amylopectin potato Partial Conversion of 0.5% (w/v), C.Hess, M.Antranikian, G.A thermoactive glucoamylase with biotechnological relevance from the thermoacidophilic euryarchaeon Thermoplasma acidophilum.Applied microbiology and biotechnology.2008, 78 (1): 105-114.).
The gene recombination glucoamylase (SSGA) of sulfolobus solfataricus (Sulfolobus solfataricus) and the suitableeest hydrolysis substrate of gene recombination glycogen debranching enzyme (SSGDE) are respectively trisaccharide maltose and amylopectin; These two kinds of enzyme requires are used in conjunction with, and can be 5.5～6.5 at pH, temperature is the starch of the condition second line of a couplet Heshui solution liquefaction of 75～80 ℃, and obtain the glucose (KR 20040025179, KR101014802) of high yield.
Because the suitableeest catalyzed reaction temperature of these extreme Pyrococcus furiosus glucoamylases is lower or low to the hydrolytic activity of starch, can not be under the condition more than 90 ℃, use together with Bacillus licheniformis (Bacillus licheniformis) α-amylase, W-Gum is liquefied and saccharification simultaneously.
Summary of the invention
This invention discloses a kind ofly carries out High-temperature Liquefaction and saccharification to W-Gum simultaneously, and a step prepares the method for β-amylose; Namely, with bacillus licheniformis alpha-amylase and a kind of archeobacteria maltogenic amylase (maltogenic amylase, MAase, EC 3.2.1.133) join simultaneously in the corn starch liquid of high temperature preheating, proceed to be incubated hydrolysis reaction, the complete enzymolysis of W-Gum is converted into glucose, maltose and isomaltose (Figure of description 1) the most at last.
The starch liquefacation enzyme that this invention is related, it is commercial Bacillus licheniformis (Bacillus licheniformis) α-amylase, the method of determination of activity is the method for reporting in document " Bemfeld; P.Amylase alpha and beta.Methods in enzymology.1955,1:149-158. ".
The diastatic enzyme that this invention is related, belong to glucoside hydrolase 13 family, by building genetic engineering bacterium, express the maltogenic amylase gene of Pyrococcus furiosus, and the suitableeest catalyzed reaction temperature that obtains is stablized maltogenic amylase at 90-100 ℃, the suitableeest catalyzed reaction pH in the gene recombination extreme heat of 5.0-5.5 scope; The method of its preparation, purifying and determination of activity is the method for reporting in document " Li; D.et al.Overexpression and characterization of an extremely thermostable maltogenic amylase; 100 ℃ of with an optimal temperature of; from the hyperthermophilic archaeon Staphylothermus marinus.New biotechnology.2010,27 (4): 300-307. ".
Liquefaction simultaneously disclosed in this invention and saccharification W-Gum prepare the method for β-amylose, comprise the following steps:
(1) W-Gum is added to the water, concentration is that 10-30% (w/v), pH scope are 5.0-6.0;
(2) be heated to 90-100 ℃;
(3) adding enzyme activity is that the extreme heat of 50-150U/g starch is stablized maltogenic amylase and enzyme activity is the bacillus licheniformis alpha-amylase of 50-150U/g starch, mixing;
(4) at 90-100 ℃, enzymolysis 3-7hr;
(5) naturally cool to room temperature, obtain take glucose, maltose and isomaltose as main component, overall yield is greater than the corn starch syrup of 110% (w/w).
The detection method of corn starch sugar involved in the present invention is the high performance anion exchange chromatography method, and testing conditions is:
The sugar that adopts high-efficiency anion exchange Electrochemical Detection chromatographic instrument (the ICS-3000 type is worn peace, the U.S.) to measure in above-mentioned starch syrup forms and content, anion-exchange column: CarboPac
TMPA10 (4mm * 250mm); Leacheate: 18mmol/LNaOH; Flow velocity: 1.0mL/min; Sample size: 25 μ L; Column temperature: 30 ℃; Pulsed amperometric detects, starch syrup high performance anion exchange chromatography figure that must be as shown in Figure of description 2.Adopt the peak area calibration curve method, three chromatographic peaks are carried out quantitatively ((saccharic amount/starch quality) * 100%) being defined as productive rate.
This invention uses the Pyrococcus furiosus maltogenic amylase to replace the aspergillus niger glucoamylase, and share with bacillus licheniformis alpha-amylase, under identical temperature and identical pH value, W-Gum is liquefied and saccharification (Figure of description 1) simultaneously, save the operation steps that needs to change temperature and regulate pH before saccharification in the two step method, after liquefaction, improved the production rate of β-amylose.
Description of drawings
Fig. 1 is the operational flowchart of while liquefying-saccharifying W-Gum.BLA is Bacillus licheniformis (Bacillus licheniformis) α-amylase; SMMA is that botryoidalis thermophile bacteria (Staphylothermus marinus) maltogenic amylase is given birth in the sea.
Fig. 2 is that Bacillus licheniformis (Bacillus licheniformis) α-amylase is given birth to botryoidalis thermophile bacteria (Staphylothermus marinus) the maltogenic amylase high performance anion exchange chromatography figure of liquefying-saccharifying W-Gum gained syrup simultaneously with the sea.In figure, retention time 3.717min is the glucose chromatographic peak, and in figure, retention time 6.217min is the isomaltose chromatographic peak, and in figure, retention time 11.767min is the maltose chromatographic peak.
Embodiment
With 20mg W-Gum (great achievement industry group, Changchun City) mixed with 200 μ L damping fluids (50mM pH5.5 acetic acid-sodium acetate), 90 ℃ of preheatings after 3 minutes, adding enzyme activity is Bacillus licheniformis (Bacillus licheniformis) α-amylase (BLA of 10U
120, Novi letter, Denmark) and enzyme activity be that botryoidalis thermophile bacteria (Staphylothermus marinus) maltogenic amylase (SMMA) is given birth in the sea of 5U, 90 ℃ be incubated 5h after, naturally cool to room temperature, obtain starch syrup.Adopt the high performance anion exchange chromatography instrument to measure the moiety of starch syrup, calculate: the productive rate of glucose is 38.92%, the productive rate of maltose is 39.87%, the productive rate of isomaltose is 31.83%, overall yield is 110.62%.
Claims (4)
1. method of liquefying-saccharifying W-Gum simultaneously is characterized in that adopting following steps:
(1) W-Gum is added to the water, concentration is that 10-30% (w/v), pH scope are 5.0-6.0;
(2) be heated to 90-100 ℃;
(3) adding enzyme activity is that the extreme heat of 50-150U/g starch is stablized maltogenic amylase and enzyme activity is the bacillus licheniformis alpha-amylase of 50-150U/g starch, mixing;
(4) at 90-100 ℃, enzymolysis 3-7hr;
(5) naturally cool to room temperature, obtain take glucose, maltose and isomaltose as main component, overall yield is greater than 110% (w/w) corn starch syrup.
2. the method for a kind of while liquefying-saccharifying W-Gum according to claim 1, is characterized in that it is that botryoidalis thermophile bacteria (Staphylothermus marinus) maltogenic amylase is given birth in the sea that the extreme heat of using is stablized maltogenic amylase.
3. the method for a kind of while liquefying-saccharifying W-Gum according to claim 1 is characterized in that extreme heat is stablized in maltogenic amylase (saccharifying enzyme) joins 90-100 ℃ together with bacillus licheniformis alpha-amylase (α-amylase) corn starch liquid.
4. the method for a kind of while liquefying-saccharifying W-Gum according to claim 1, the chief component composition that it is characterized in that the product β-amylose is glucose, maltose and isomaltose.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108823265A (en) * | 2018-07-21 | 2018-11-16 | 安徽启慧信息科技有限公司 | A kind of preparation method and its special enzyme preparation of superelevation starch grape slurry |
| CN109371078A (en) * | 2018-10-18 | 2019-02-22 | 山东福田药业有限公司 | A kind of high-purity malt sugar preparation process |
| CN112941056A (en) * | 2021-02-24 | 2021-06-11 | 长春大学 | Starch pullulanase mutant and application thereof |
| CN114480528A (en) * | 2021-12-28 | 2022-05-13 | 滨州中裕食品有限公司 | Method for hydrolyzing starch in food slurry and application thereof |
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| CN1587421A (en) * | 2004-08-10 | 2005-03-02 | 华南理工大学 | Method for producing high purity malt sugar product |
| CN101724669A (en) * | 2009-11-30 | 2010-06-09 | 山东省鲁洲食品集团有限公司 | Method for producing moderately inverted syrup by synergistic saccharification with multiple enzymes |
| CN101838674A (en) * | 2009-03-19 | 2010-09-22 | 上海融氏企业有限公司 | Production method of starch sugar |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1587421A (en) * | 2004-08-10 | 2005-03-02 | 华南理工大学 | Method for producing high purity malt sugar product |
| CN101838674A (en) * | 2009-03-19 | 2010-09-22 | 上海融氏企业有限公司 | Production method of starch sugar |
| CN101724669A (en) * | 2009-11-30 | 2010-06-09 | 山东省鲁洲食品集团有限公司 | Method for producing moderately inverted syrup by synergistic saccharification with multiple enzymes |
Non-Patent Citations (1)
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| EMMANUEL LÉVEˆSQUE, ET AL.: "Thermophilic archaeal amylolytic enzymes", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108823265A (en) * | 2018-07-21 | 2018-11-16 | 安徽启慧信息科技有限公司 | A kind of preparation method and its special enzyme preparation of superelevation starch grape slurry |
| CN109371078A (en) * | 2018-10-18 | 2019-02-22 | 山东福田药业有限公司 | A kind of high-purity malt sugar preparation process |
| CN109371078B (en) * | 2018-10-18 | 2022-03-11 | 山东福田药业有限公司 | Preparation process of high-purity maltose |
| CN112941056A (en) * | 2021-02-24 | 2021-06-11 | 长春大学 | Starch pullulanase mutant and application thereof |
| CN112941056B (en) * | 2021-02-24 | 2022-11-18 | 长春大学 | Starch pullulanase mutant and application thereof |
| CN114480528A (en) * | 2021-12-28 | 2022-05-13 | 滨州中裕食品有限公司 | Method for hydrolyzing starch in food slurry and application thereof |
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Application publication date: 20130612 |