CN107130008A - It is a kind of that the production method that the enzyme of general glycoside 80% modifies stevioside is obtained by organized enzyme deep reaction - Google Patents
It is a kind of that the production method that the enzyme of general glycoside 80% modifies stevioside is obtained by organized enzyme deep reaction Download PDFInfo
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- CN107130008A CN107130008A CN201710351178.4A CN201710351178A CN107130008A CN 107130008 A CN107130008 A CN 107130008A CN 201710351178 A CN201710351178 A CN 201710351178A CN 107130008 A CN107130008 A CN 107130008A
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- solution
- enzyme
- stevioside
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- starch
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/44—Preparation of O-glycosides, e.g. glucosides
- C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
Abstract
The invention discloses a kind of production method that the enzyme of general glycoside 80% modification stevioside is obtained by organized enzyme deep reaction, comprise the following steps：Prepared by solution, hydrolysis stevioside solution allocation is inactivated, filters and adsorbs, reclaim concentration, dry quality inspection, encapsulate, by by Starch Hydrolysis into tapioca starch, and in the presence of enzyme, glucosyl group on tapioca starch is transferred on stevioside, the sweet taste quality of stevioside is substantially improved, the present invention is hydrolyzed temperature by water-bath and strictly controlled, it is ensured that the high efficiency of hydrolase, the conversion ratio of raw material is improved, the production cost that enzyme modifies stevioside is reduced.
Description
Technical field
The enzyme of general glycoside 80% is obtained by organized enzyme deep reaction the present invention relates to stevioside technical field, more particularly to one kind
Modify the production method of stevioside.
Background technology
Stevioside has no toxic side effect, no carcinogenic substance, edible safety, and often eating can preventing hypertension, diabetes, obesity
It is a kind of preferable sweetener for substituting sucrose etc. illness, but existing stevioside production method existing defects, such as conversion ratio
It is low, and the stevioside extracted has strong rear bitter taste, limits the application of stevioside.
The content of the invention
The invention aims to solve shortcoming present in prior art, and the one kind proposed passes through organized enzyme depth
Reaction obtains the production method that the enzyme of general glycoside 80% modifies stevioside.
To achieve these goals, present invention employs following technical scheme：
It is a kind of that the production method that the enzyme of general glycoside 80% modifies stevioside is obtained by organized enzyme deep reaction, comprise the following steps：
S1：Prepared by solution, take a certain amount of pure water and starch to add tank body, tank body opening is sealed by sealed membrane,
Certain density starch solution is made after mixing；
S2：Solution is hydrolyzed, and the acid-base value to starch solution in S1 tank bodies is adjusted so that the pH value of starch solution is 4.5-
5.5, and increase simultaneously to tank body progress heating water bath, heating-up temperature is 55-60 DEG C, after solution temperature in tank body is stable, so
Alpha-amylase is added in backward tank body, tank body is sealed by sealed membrane, sustained response is kept for 4-6 hours, obtained solution
A；
S3：Stevioside solution allocation, a certain amount of stevioside is added into solution A, and the acid-base value of solution A is adjusted,
So that the pH value of solution A is 5.5-6.5, while it is 60-65 DEG C to adjust water bath heating temperature, treat that solution temperature is stable in tank body
Afterwards, CGTase enzymes are added into tank body, the enzyme activity of CGTase enzymes is 800U/mL, and tank body is sealed by sealed membrane, held
Continuous reaction is kept for 8-10 hours, obtained solution B, and CGTase enzymes：Stevioside：The rate of charge of pure water is 0.2：3：10；
S4：Inactivation, filtering, less than 3 are adjusted to by the pH value of solution B, and are kept for 30 minutes, are completed after inactivation, add alkali lye by PH
Value is adjusted to 7, after acid-base neutralization reaction is complete, is cooled to room temperature, and cellulose acetate miillpore filter is led to afterwards and is filtered, and mistake
Filter opening footpath is 2 microns, obtains solution C；
S5：Absorption, recovery, the solution C that S4 is obtained carry out sugared part absorption by large pore resin absorption column, and solution C is added completely
Afterwards, it is rinsed using pure water, flushing is more than 80% second with 2 times that amount of purified water is solution C volume, afterwards by concentration
Alcoholic solution is eluted, and ethanol consumption is 3 times of solution C volume and is divided into three additions, and collects pure water elution and second
Alcohol eluen, pure water elution and ethanol eluate are filtered by anion-cation exchange resin, obtain enzyme modification sweet tea
Synanthrin pregnant solution；
S6：Concentrate, dry, will be evaporated in enzyme modification stevioside pregnant solution addition Rotary Evaporators in concentration, evaporation process
Ethanol eluate is heated, heating-up temperature is 80 degree, and rotary rpm is 35 revs/min, and negative pressure is 0.05MP, treat that enzyme is modified
Stevioside pregnant solution volume stops concentration when reducing 50%, and discharges concentrate, then concentrate is spray-dried, and obtains enzyme
Modify stevia rebaudianum sugar crystal；
S7：Quality inspection, encapsulation, take each 5 milligrams of progress quality inspection of two parts of enzyme modification stevia rebaudianum sugar crystal in S6, envelope are weighed after quality inspection is qualified
Dress, deposits warehouse.
It is preferred that, acid-base value regulation acid solution in appeal step：Mass fraction is 20% citric acid solution, alkali lye：Quality
Fraction is 30% sodium hydroxide solution.
It is preferred that, in S1 during starch dissolution, liquid in tank body is persistently stirred using magnetic stirring apparatus, and
Starch：The rate of charge of pure water is 1：10.
It is preferred that, the mass ratio of the added stevioside of general glycoside 80% and starch is 1 in S3:1-1:Between 3.
A kind of production method that the enzyme of general glycoside 80% modification stevioside is obtained by organized enzyme deep reaction that the present invention is provided,
By the way that into tapioca starch, and in the presence of CGTase enzymes, Starch Hydrolysis is transferred into stevioside by the glucosyl group on tapioca starch
On, the sweet taste quality of stevioside is substantially improved, the present invention is hydrolyzed temperature by water-bath and strictly controlled, it is ensured that hydrolase
High efficiency, improve the conversion ratio of raw material, reduce enzyme modify stevioside production cost.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Embodiment 1
It is a kind of that the production method that the enzyme of general glycoside 80% modifies stevioside is obtained by organized enzyme deep reaction, comprise the following steps：
S1：Prepared by solution, take a certain amount of pure water and starch to add tank body, tank body opening is sealed by sealed membrane,
Certain density starch solution is made after mixing；
S2：Solution is hydrolyzed, and the acid-base value to starch solution in S1 tank bodies is adjusted so that the pH value of starch solution is 4.5, and
Increase simultaneously carries out heating water bath to tank body, and heating-up temperature is 55 DEG C, after solution temperature in tank body is stable, then into tank body
Alpha-amylase is added, tank body is sealed by sealed membrane, sustained response is kept for 4 hours, obtained solution A；
S3：Stevioside solution allocation, appropriate stevioside is added into solution A, and the acid-base value of solution A is adjusted, and is made
The pH value for obtaining solution A is 5.5, while it is 60 DEG C to adjust water bath heating temperature, after solution temperature in tank body is stable, into tank body
CGTase enzymes are added, the enzyme activity of CGTase enzymes is 800U/mL, and tank body is sealed by sealed membrane, and sustained response keeps 8
Hour, obtained solution B, and CGTase enzymes：Stevioside：The rate of charge of pure water is 0.2：3：10；
S4：Inactivation, filtering, less than 3 are adjusted to by the pH value of solution B, and are kept for 30 minutes, are completed after inactivation, add alkali lye by PH
Value is adjusted to 7, after acid-base neutralization reaction is complete, is cooled to room temperature, and cellulose acetate miillpore filter is led to afterwards and is filtered, and mistake
Filter opening footpath is 2 microns, obtains solution C；
S5：Absorption, recovery, the solution C that S4 is obtained carry out sugared part absorption by large pore resin absorption column, and solution C is added completely
Afterwards, it is rinsed using pure water, flushing is more than 80% second with 2 times that amount of purified water is solution C volume, afterwards by concentration
Alcoholic solution is eluted, and ethanol consumption is 3 times of solution C volume and is divided into three additions, and collects pure water elution and second
Alcohol eluen, pure water elution and ethanol eluate are filtered by anion-cation exchange resin, obtain enzyme modification sweet tea
Synanthrin pregnant solution；
S6：Concentrate, dry, will be evaporated in enzyme modification stevioside pregnant solution addition Rotary Evaporators in concentration, evaporation process
Ethanol eluate is heated, heating-up temperature is 80 degree, and rotary rpm is 35 revs/min, and negative pressure is 0.05MP, treat that enzyme is modified
Stevioside pregnant solution volume stops concentration when reducing 50%, and discharges concentrate, then concentrate is spray-dried, and obtains enzyme
Modify stevia rebaudianum sugar crystal；
S7：Quality inspection, encapsulation, take each 5 milligrams of progress quality inspection of two parts of enzyme modification stevia rebaudianum sugar crystal in S6, envelope are weighed after quality inspection is qualified
Dress, deposits warehouse.
Appeal acid-base value regulation acid solution in step：Mass fraction is 20% citric acid solution, alkali lye：Mass fraction is
30% sodium hydroxide solution.
In S1 during starch dissolution, liquid in tank body is persistently stirred using magnetic stirring apparatus, and starch：It is pure
The rate of charge of water purification is 1：10.
The mass ratio of the added stevioside of general glycoside 80% and starch is 1 in S3:1.1 between.
Embodiment 2
It is a kind of that the production method that the enzyme of general glycoside 80% modifies stevioside is obtained by organized enzyme deep reaction, comprise the following steps：
S1：Prepared by solution, take a certain amount of pure water and starch to add tank body, tank body opening is sealed by sealed membrane,
Certain density starch solution is made after mixing；
S2：Solution is hydrolyzed, and the acid-base value to starch solution in S1 tank bodies is adjusted so that the pH value of starch solution is 5, and together
Shi Zengjia carries out heating water bath to tank body, and heating-up temperature is 58 DEG C, after solution temperature in tank body is stable, is then added into tank body
Enter alpha-amylase, tank body is sealed by sealed membrane, sustained response is kept for 5 hours, obtained solution A；
S3：Stevioside solution allocation, a certain amount of stevioside is added into solution A, and the acid-base value of solution A is adjusted,
So that the pH value of solution A is 6, while it is 63 DEG C to adjust water bath heating temperature, after solution temperature in tank body is stable, into tank body
CGTase enzymes are added, the enzyme activity of CGTase enzymes is 800U/mL, and tank body is sealed by sealed membrane, and sustained response keeps 9
Hour, obtained solution B, and CGTase enzymes：Stevioside：The rate of charge of pure water is 0.2：3：10；
S4：Inactivation, filtering, less than 3 are adjusted to by the pH value of solution B, and are kept for 30 minutes, are completed after inactivation, add alkali lye by PH
Value is adjusted to 7, after acid-base neutralization reaction is complete, is cooled to room temperature, and cellulose acetate miillpore filter is led to afterwards and is filtered, and mistake
Filter opening footpath is 2 microns, obtains solution C；
S5：Absorption, recovery, the solution C that S4 is obtained carry out sugared part absorption by large pore resin absorption column, and solution C is added completely
Afterwards, it is rinsed using pure water, flushing is more than 80% second with 2 times that amount of purified water is solution C volume, afterwards by concentration
Alcoholic solution is eluted, and ethanol consumption is 3 times of solution C volume and is divided into three additions, and collects pure water elution and second
Alcohol eluen, pure water elution and ethanol eluate are filtered by anion-cation exchange resin, obtain enzyme modification sweet tea
Synanthrin pregnant solution；
S6：Concentrate, dry, will be evaporated in enzyme modification stevioside pregnant solution addition Rotary Evaporators in concentration, evaporation process
Ethanol eluate is heated, heating-up temperature is 80 degree, and rotary rpm is 35 revs/min, and negative pressure is 0.05MP, treat that enzyme is modified
Stevioside pregnant solution volume stops concentration when reducing 50%, and discharges concentrate, then concentrate is spray-dried, and obtains enzyme
Modify stevia rebaudianum sugar crystal；
S7：Quality inspection, encapsulation, take each 5 milligrams of progress quality inspection of two parts of enzyme modification stevia rebaudianum sugar crystal in S6, envelope are weighed after quality inspection is qualified
Dress, deposits warehouse.
Appeal acid-base value regulation acid solution in step：Mass fraction is 20% citric acid solution, alkali lye：Mass fraction is
30% sodium hydroxide solution.
In S1 during starch dissolution, liquid in tank body is persistently stirred using magnetic stirring apparatus, and starch：It is pure
The rate of charge of water purification is 1.5：10.
The mass ratio of the added stevioside of general glycoside 80% and starch is 1 in S3:1.
A kind of production method that the enzyme of general glycoside 80% modification stevioside is obtained by organized enzyme deep reaction that the present invention is provided,
By the way that into tapioca starch, and in the presence of CGTase enzymes, Starch Hydrolysis is transferred into stevioside by the glucosyl group on tapioca starch
On, the sweet taste quality of stevioside is substantially improved, the present invention is hydrolyzed temperature by water-bath and strictly controlled, it is ensured that hydrolase
High efficiency, improve the conversion ratio of raw material, reduce enzyme modify stevioside production cost.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Claims (4)
1. a kind of obtain the production method that the enzyme of general glycoside 80% modifies stevioside by organized enzyme deep reaction, it is characterised in that：Including
Following steps：
S1：Prepared by solution, take a certain amount of pure water and starch to add tank body, tank body opening is sealed by sealed membrane,
Certain density starch solution is made after mixing；
S2：Solution is hydrolyzed, and the acid-base value to starch solution in S1 tank bodies is adjusted so that the pH value of starch solution is 4.5-
5.5, and increase simultaneously to tank body progress heating water bath, heating-up temperature is 55-60 DEG C, after solution temperature in tank body is stable, so
Alpha-amylase is added in backward tank body, tank body is sealed by sealed membrane, sustained response is kept for 4-6 hours, obtained solution
A；
S3：Stevioside solution allocation, the appropriate stevioside of general glycoside 80% is added into solution A, and the acid-base value of solution A is carried out
Regulation so that the pH value of solution A is 5.5-6.5, while it is 60-65 DEG C to adjust water bath heating temperature, treats solution temperature in tank body
After stable, CGTase enzymes added into tank body, the enzyme activity of CGTase enzymes is 800U/mL, tank body is carried out by sealed membrane close
Envelope, sustained response is kept for 8-10 hours, obtained solution B, and CGTase enzymes：Stevioside：The rate of charge of pure water is 0.2：3：10；
S4：Inactivation, filtering, less than 3 are adjusted to by the pH value of solution B, and are kept for 30 minutes, are completed after inactivation, add alkali lye by PH
Value is adjusted to 7, after acid-base neutralization reaction is complete, is cooled to room temperature, and cellulose acetate miillpore filter is led to afterwards and is filtered, and mistake
Filter opening footpath is 2 microns, obtains solution C；
S5：Absorption, recovery, the solution C that S4 is obtained carry out sugared part absorption by large pore resin absorption column, and solution C is added completely
Afterwards, it is rinsed using pure water, flushing is more than 80% second with 2 times that amount of purified water is solution C volume, afterwards by concentration
Alcoholic solution is eluted, and ethanol consumption is 3 times of solution C volume and is divided into three additions, and collects pure water elution and second
Alcohol eluen, pure water elution and ethanol eluate are filtered by anion-cation exchange resin, obtain enzyme modification sweet tea
Synanthrin pregnant solution；
S6：Concentrate, dry, will be evaporated in enzyme modification stevioside pregnant solution addition Rotary Evaporators in concentration, evaporation process
Ethanol eluate is heated, heating-up temperature is 80 degree, and rotary rpm is 35 revs/min, and negative pressure is 0.05MP, treat that enzyme is modified
Stevioside pregnant solution volume stops concentration when reducing 50%, and discharges concentrate, then concentrate is spray-dried, and obtains enzyme
Modify stevia rebaudianum sugar crystal；
S7：Quality inspection, encapsulation, take each 5 milligrams of progress quality inspection of two parts of enzyme modification stevia rebaudianum sugar crystal in S6, envelope are weighed after quality inspection is qualified
Dress, deposits warehouse.
2. a kind of production that the enzyme of general glycoside 80% modification stevioside is obtained by organized enzyme deep reaction told according to claim 1
Method, it is characterised in that：Appeal acid-base value regulation acid solution in step：Mass fraction is 20% citric acid solution, alkali lye：Matter
Measure the sodium hydroxide solution that fraction is 30%.
3. a kind of production that the enzyme of general glycoside 80% modification stevioside is obtained by organized enzyme deep reaction told according to claim 1
Method, it is characterised in that：In S1 during starch dissolution, liquid in tank body is persistently stirred using magnetic stirring apparatus,
And starch：The rate of charge of pure water is 1：10.
4. a kind of production that the enzyme of general glycoside 80% modification stevioside is obtained by organized enzyme deep reaction told according to claim 1
Method, it is characterised in that：The mass ratio of the added stevioside of general glycoside 80% and starch is 1 in S3:1-1:Between 3.
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| CN201710351178.4A CN107130008A (en) | 2017-05-18 | 2017-05-18 | It is a kind of that the production method that the enzyme of general glycoside 80% modifies stevioside is obtained by organized enzyme deep reaction |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710351178.4A CN107130008A (en) | 2017-05-18 | 2017-05-18 | It is a kind of that the production method that the enzyme of general glycoside 80% modifies stevioside is obtained by organized enzyme deep reaction |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109293718A (en) * | 2018-10-08 | 2019-02-01 | 山东奥晶生物科技有限公司 | A kind of preparation method of stevia rebaudianum carbohydrase modification stevioside |
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| US20150037462A1 (en) * | 2011-02-17 | 2015-02-05 | Purecircle Usa Inc. | Glucosyl stevia composition |
| CN104388498A (en) * | 2014-10-14 | 2015-03-04 | 曲阜圣仁制药有限公司 | Method for producing total glycoside 80% enzymes for improving stevioside taste quality |
| US20150157045A1 (en) * | 2011-02-17 | 2015-06-11 | Purecircle Usa Inc. | Glucosyl stevia composition |
| KR101535427B1 (en) * | 2014-11-25 | 2015-07-14 | 주식회사 대평 | Method for production of sweet-improved enzymatically modified stevia sweetener |
| CN104894193A (en) * | 2015-05-12 | 2015-09-09 | 曲阜圣仁制药有限公司 | Method for producing total glycosides 95% enzyme modification stevioside |
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2017
- 2017-05-18 CN CN201710351178.4A patent/CN107130008A/en active Pending
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| US20150037462A1 (en) * | 2011-02-17 | 2015-02-05 | Purecircle Usa Inc. | Glucosyl stevia composition |
| US20150157045A1 (en) * | 2011-02-17 | 2015-06-11 | Purecircle Usa Inc. | Glucosyl stevia composition |
| CN104388498A (en) * | 2014-10-14 | 2015-03-04 | 曲阜圣仁制药有限公司 | Method for producing total glycoside 80% enzymes for improving stevioside taste quality |
| KR101535427B1 (en) * | 2014-11-25 | 2015-07-14 | 주식회사 대평 | Method for production of sweet-improved enzymatically modified stevia sweetener |
| CN104894193A (en) * | 2015-05-12 | 2015-09-09 | 曲阜圣仁制药有限公司 | Method for producing total glycosides 95% enzyme modification stevioside |
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| CN109293718A (en) * | 2018-10-08 | 2019-02-01 | 山东奥晶生物科技有限公司 | A kind of preparation method of stevia rebaudianum carbohydrase modification stevioside |
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