CN102827891A - Method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase - Google Patents
Method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase Download PDFInfo
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- CN102827891A CN102827891A CN2012103524616A CN201210352461A CN102827891A CN 102827891 A CN102827891 A CN 102827891A CN 2012103524616 A CN2012103524616 A CN 2012103524616A CN 201210352461 A CN201210352461 A CN 201210352461A CN 102827891 A CN102827891 A CN 102827891A
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- stevia rebaudianum
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Abstract
The invention discloses a method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase, belonging to the field of biosynthesis technology of organic compounds. The method takes the beta-glucosidase derived from aspergillus niger as a catalyst, and can be used for preparing the steviol by carrying out catalytic hydrolysis on the stevioside in single stevioside or stevia rebaudiana extract through temperature programming; the method has the advantages of being high in conversion rate and selectivity, simple in separation and the like; and the enzyme activity loss can be reduced by the temperature programming reaction, so that the utilization rate of enzyme can be improved. The invention provides a new method for preparing the steviol, which is efficient and environment-friendly.
Description
Technical field
Invention belongs to the biosynthesis technology field of organic cpds, is specifically related to a kind of usefulness and derives from black mold
(Aspergillus niger) β-glucuroide temperature programming catalytic hydrolysis stevioside prepares the method for stevia rebaudianum alcohol.
Background technology
Stevioside (13-[(2-O-
β-D-Glucopyranosyl-
β-D-glucopyranosyl) oxy] kaur-16-en-18-oic acid
β-D-glucopyranosyl ester, stevioside is hereinafter to be referred as St) be from sweet Stevia (
Stevia rebaudianaBertoni) the tetracyclic diterpene compounds that extracts in is a kind of sweeting agent with back bitter taste.Contain three kinds of glycosidic links in the St molecule, can obtain stevia rebaudianum alcohol, NSC 231875, stevia rebaudianum monoglycosides, stevia rebaudianum disaccharide glycosides, immature fruit of Juteleaf Raspberry glycosides or its mixture through hydrolysis; Each component concentration of hydrolysate depends on hydrolysising condition: for example obtain stevia rebaudianum disaccharide glycosides (Sharipova R with mineral alkali catalytic hydrolysis St; Et al; 2009), then obtain the mixture (patent No.: 200910025817.3) of the pure and mild NSC 231875 of stevia rebaudianum with mineral acid catalytic hydrolysis St.For example magnify and wait forever through chemical method two steps preparation stevia rebaudianum alcohol, at first St obtains the stevia rebaudianum alcohol (patent No.: 200810242967.5) then through sodium periodate or lead tetra-acetate oxidation behind basic hydrolysis, column chromatography purification.Avent etc. point out acidic hydrolysis St except that obtaining the pure and mild NSC 231875 of stevia rebaudianum, also have other two kinds of isomerss (Avent A G, et al, 1990).Also possibly obtain different products with enzymatic hydrolysis St of the same race.If thereby through utilizing the high regioselectivity enzymatic process hydrolysis St of enzyme; Can a step directly obtain high-quality single hydrolysate; Can also form through changing hydrolysis reaction condition regulation and control product simultaneously; Milagre etc. point out that the product of pancreatin (Pancreatin) catalysis St hydrolysis reaction is a NSC 231875 pH 4.0 times; And pH 7.0 times, product is a stevia rebaudianum alcohol (Milagre H M S, et al, 2009).
Though the enzyme catalysis advantage is numerous, poor heat stability becomes inevitable problem in the enzyme use.Usually can be through adding stablizer, approach such as immobilization improve thermostability.Derive from black mold
β-glucuroide is 75
oC is the very fast inactivation of meeting down, but simultaneously again at 70-75
oHas the highest catalytic hydrolysis efficient under the C.Therefore the present invention adopts the mode of temperature programming reaction can effectively reduce the heat inactivation of enzyme and take into account hydrolysis reaction speed, and avoids immobilized enzyme and the product of separating out to lump together, reduces the difficulty of purifying, and raising derives from
Black mold βThe stability of-glucuroide also effectively shortens the time of accomplishing hydrolysis reaction.
Summary of the invention
The purpose of this invention is to provide a kind of usefulness
β-glucoside enzymatic hydrolysis stevioside prepares the method for stevia rebaudianum alcohol.The present invention is with the black mold of deriving from that filters out
β-glucuroide, hydrolysis St prepares stevia rebaudianum alcohol; Through the temperature programming reaction, reduce the heat inactivation of enzyme.The production process environmental protection, catalytic efficiency (is high, and separation and purification of products is simple, and purity is high.
Derive from black mold
βThe reaction equation that-glucoside enzyme catalysis stevioside prepares stevia rebaudianum alcohol is following:
The object of the invention can reach through following technical scheme: with stevioside St or the Folium Chrysanthemi extract (containing Lai Baodi glycosides A (RA), Lai Baodi glycosides C (RC) simultaneously) etc. that contains St is substrate, substrate is mixed with the reaction solution of 150-300 g/L with deionized water or damping fluid; 55
oAdd behind constant temperature 0.5 h under the C and to derive from black mold
β-glucuroide, reaction 2-6 h under the equality of temperature, the reaction times decide with enzyme concentration, react when preferably enzyme concentration is 800 U/g St 3 h (the used substrate of enzyme activity determination be the 4-nitrophenols-
β-D-glucopyranoside pNPG); Be warming up to 70-75 then
oReact under the C to the stevioside transformation efficiency and no longer raise; Stevioside is hydrolyzed into stevia rebaudianum alcohol and from reaction solution, separates out, and the composition of other qualities of stevia extract does not have or seldom reaction; Reaction solution is carried out suction filtration, with obtaining stevia rebaudianum alcohol white products with 20% methanol aqueous solution recrystallization behind the water washing filter residue.Said damping fluid is the acetate buffer solution of pH 4.0-5.4 or the phosphoric acid buffer of pH 6.0.
Said
β-glucuroide derives from black mold (purchasing the bio tech ltd in Korea S biobean), and its form is an enzyme liquid.
Said temperature of reaction is the temperature of temperature programming, promptly earlier 55
oC is reaction 2-6 h down, then at 70-75
oReact under the C to the St transformation efficiency and no longer raise.
Said damping fluid is acetate buffer solution or pH 6.0 phosphoric acid buffers of the pH 4.0-5.4 of 50 mM.
Said
βThe addition that-glucuroide is urged is for being the 500-1000U/g stevioside.
Beneficial effect of the present invention:This patent provides a kind of novel method for preparing stevia rebaudianum alcohol, has expanded the purposes of stevioside, promotes its added value greatly.Derive from black mold
β-glucuroide is that zymin is used in foodstuffs industry, safety, efficient, selectivity is high.Different with additive method is not have NSC 231875 in the product; And other stevioside verivates are not participated in hydrolysis.Adopt the mode of temperature programming reaction can effectively reduce the heat inactivation of enzyme and take into account hydrolysis reaction speed, thereby improve the utilization ratio of enzyme.Reaction conditions is gentle, productive rate is high, the product separation and purification is simple.
Description of drawings
The HPLC (a) and the total ion current figure (b) of product stevia rebaudianum alcohol among Fig. 1 the present invention.
The one-level mass spectrum (a) and the second order ms (b) of product stevia rebaudianum alcohol among Fig. 2 the present invention.
Product stevia rebaudianum alcohol among Fig. 3 the present invention
13CNMR.
Embodiment
Below in conjunction with embodiment the present invention is elaborated.
Embodiment 1
15 g St (commercially available article, purity are 97%) are dispersed in 55
oIn the 100 mL deionized waters of C, put into 55
oJolt 0.5 h in the C shaking bath, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 800 U/g St; React to the St transformation efficiency and no longer rise.The St transformation efficiency is 99.0%.Suction filtration behind the cooling reaction liquid, and use mother liquor and deionized water wash filter cake respectively, can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization again.The HPLC purity of final product is greater than 98 %.Reaction needs 8 hours altogether.
Product is carried out LC-MSMS and NMR analysis, and the result sees accompanying drawing 1-3.The molecular ion peak of main composition among the LC-MSMS (317.4, [M-H]
-) can be corresponding stevia rebaudianum alcohol, the NMR of hydrolysate can confirm further that also product is a stevia rebaudianum alcohol.
The St transformation efficiency can be with reference to GB (GB8270-1999) through HPLC external standard method (formula 1).
X
t: t is the transformation efficiency of St constantly;
C
0: the concentration (g/L) of initial St;
C
t: t is the concentration (g/L) of St constantly;
Embodiment 2
20 g, 90% St (stevia extract-containing liquid contains 10% RA) is dissolved in 55
oIn the 100 mL deionized waters of C, put into 55
oJolt 0.5 h in the C shaking bath, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 600 U/g St; Rise to 75 after reacting 3 h
oC continues to react to the St transformation efficiency and no longer rises.Can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization again.Reaction needs 3.5 hours altogether.
15 g St are dispersed in the acetate buffer solution of 100 mL pH 5.0 of 50 mM, put into 55
oJolt 0.5 h in the C shaking bath, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 700 U/g St; Rise to 70 behind the reaction 3h
oC continues to react to the St transformation efficiency and no longer rises.Reaction needs 4 hours altogether.Can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization again.
Embodiment 4
30 g St bullions (50% St, 40% RA, 5% RC and other) are dissolved in the 100 mL deionized waters, put into 55
oJolt 0.5 h in the C shaking bath, shaking speed is 150 rpm; Add
β-glucuroide 500 U/g St; After reacting 5 h, be warming up to 70
oC is reaction 1 h down, analyzes through HPLC and finds that St all is converted into stevia rebaudianum alcohol, and RA and RC transformation efficiency are equal<5%.Can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization again.
Embodiment 5
30 g St bullions (St 90%) are dissolved in the 200 mL deionized waters, put into that shaking bath jolts and 55
oConstant temperature 0.5 h under the C, shaking speed is 150 rpm; Add
β-glucuroide 500 U/g St; After reacting 5 h, be warming up to 75
oC is reaction 5 h down, analyze through HPLC and find that St all is converted into stevia rebaudianum alcohol, and RA and RC transformation efficiency are equal<5%.Can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization again.
Embodiment 6
20 g St are dissolved in the 100 mL acetate buffer solutions (pH 4.6,50 mM), put into 55
oJolt 0.5 h in the C shaking bath, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 900 U/g St; Rise to 75 after reacting 6 h
oC continues to react to the St transformation efficiency and no longer rises.Can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization again.
Embodiment 7
20 g St are dissolved in the 100 mL phosphoric acid buffers (pH 6.0,50 mM), put into that shaking bath jolts and 55
oConstant temperature 0.5 h under the C, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 1000 U/g St; Rise to 75 after reacting 6 h
oC continues to react to the St transformation efficiency and no longer rises.Can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization again.
Claims (5)
- One kind with β-glucoside enzymatic hydrolysis stevioside prepares the method for stevia rebaudianum alcohol, it is characterized in that with stevioside or the qualities of stevia extract that contains stevioside be substrate, and it is mixed with the reaction solution of 150-300 g/L with deionized water or damping fluid; 55 oAdd above-mentioned under the C behind constant temperature 0.5 h β-glucuroide, reaction 2-6 h under the equality of temperature; Be warming up to 70-75 then oReact under the C to the stevioside transformation efficiency and no longer raise; Stevioside is hydrolyzed into stevia rebaudianum alcohol and from reaction solution, separates out, and the composition of other qualities of stevia extract does not have or seldom reaction; Reaction solution is carried out suction filtration, use the water washing filter residue, can obtain stevia rebaudianum alcohol product with 20% methanol aqueous solution recrystallization filter residue again.
- 2. usefulness according to claim 1 β-glucoside enzymatic hydrolysis stevioside prepares the method for stevia rebaudianum alcohol, it is characterized in that said stevioside is meant the stevioside with following structure:。
- 3. usefulness according to claim 1 β-glucoside enzymatic hydrolysis stevioside prepares the method for stevia rebaudianum alcohol, and it is characterized in that: said damping fluid is acetate buffer solution or pH 6.0 phosphoric acid buffers of the pH 4.0-5.4 of 50 mM.
- 4. usefulness according to claim 1 β-glucoside enzymatic hydrolysis stevioside prepares the method for stevia rebaudianum alcohol, it is characterized in that: said β-glucuroide derives from black mold, and its form is an enzyme liquid.
- 5. usefulness according to claim 1 β-glucoside enzymatic hydrolysis stevioside prepares the method for stevia rebaudianum alcohol, it is characterized in that: said βThe addition that-glucuroide is urged is the 500-1000U/g stevioside.
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Cited By (11)
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| CN104725443A (en) * | 2013-12-19 | 2015-06-24 | 江南大学 | Method for reaction separation purification of rebaudioside A |
| CN105255971A (en) * | 2015-10-27 | 2016-01-20 | 江南大学 | Double-enzyme method for preparing steviolmonoside |
| CN105861573A (en) * | 2016-06-02 | 2016-08-17 | 广西大学 | Method for preparing steviol through two-enzyme method |
| CN105899087A (en) * | 2013-09-19 | 2016-08-24 | 谱赛科美国股份有限公司 | Glucosylated steviol glycoside as a flavor modifier |
| WO2017073716A1 (en) * | 2015-10-30 | 2017-05-04 | サントリーホールディングス株式会社 | Method for producing steviol and steviol glycoside using aobgl3 homolog |
| WO2017073717A1 (en) * | 2015-10-30 | 2017-05-04 | サントリーホールディングス株式会社 | Method for producing steviol and steviol glycoside using aobgl1 homolog |
| CN107188800A (en) * | 2017-07-19 | 2017-09-22 | 江南大学 | Stevia rebaudianum alcohol crystals, its preparation method and application with crystal formation A forms |
| CN109154012A (en) * | 2016-05-25 | 2019-01-04 | 三得利控股株式会社 | Use the steviol of AOBGL11 autoploid and the manufacturing method of steviol glycoside |
| WO2019227532A1 (en) * | 2018-05-31 | 2019-12-05 | 东台市浩瑞生物科技有限公司 | Method for preparing glucosyl steviol glycoside having small number of grafts |
| CN110564658A (en) * | 2019-09-06 | 2019-12-13 | 广西大学 | Escherichia coli engineering bacterium and method for producing steviol through whole-cell catalysis of escherichia coli engineering bacterium |
| US11180788B2 (en) | 2018-05-31 | 2021-11-23 | Jiangnan University | Method for the preparation of lower graft degree glucosylated steviol glycosides |
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| MIZUKAMI H. ET AL.: "Enzymatic determination of stevioside in Stevia Rebaudiana", 《PHYTOCHEMISTRY》 * |
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| CN105899087A (en) * | 2013-09-19 | 2016-08-24 | 谱赛科美国股份有限公司 | Glucosylated steviol glycoside as a flavor modifier |
| CN104725443B (en) * | 2013-12-19 | 2018-04-06 | 江南大学 | A kind of method that rebaudioside A is purified by Reaction Separation |
| CN104725443A (en) * | 2013-12-19 | 2015-06-24 | 江南大学 | Method for reaction separation purification of rebaudioside A |
| CN105255971A (en) * | 2015-10-27 | 2016-01-20 | 江南大学 | Double-enzyme method for preparing steviolmonoside |
| CN105255971B (en) * | 2015-10-27 | 2018-10-23 | 江南大学 | Two enzymes method prepares stevia rebaudianum monoglycosides |
| US20180327798A1 (en) * | 2015-10-30 | 2018-11-15 | Suntory Holdings Limited | Method for producing steviol and steviol glycoside using aobgl1 homolog |
| US11268118B2 (en) * | 2015-10-30 | 2022-03-08 | Suntory Holdings Limited | Method for producing steviol and steviol glycoside using AOBGL1 homolog |
| WO2017073717A1 (en) * | 2015-10-30 | 2017-05-04 | サントリーホールディングス株式会社 | Method for producing steviol and steviol glycoside using aobgl1 homolog |
| CN108350475A (en) * | 2015-10-30 | 2018-07-31 | 三得利控股株式会社 | Use the steviol glycoside of AOBGL1 autoploids and the manufacturing method of steviol |
| WO2017073716A1 (en) * | 2015-10-30 | 2017-05-04 | サントリーホールディングス株式会社 | Method for producing steviol and steviol glycoside using aobgl3 homolog |
| US10415071B2 (en) | 2015-10-30 | 2019-09-17 | Suntory Holdings Limited | Method for producing steviol and steviol glycoside using AOBGL3 homolog |
| CN108350475B (en) * | 2015-10-30 | 2022-12-13 | 三得利控股株式会社 | Steviol glycoside using AOBGL1 homologue and method for producing steviol |
| CN109154012A (en) * | 2016-05-25 | 2019-01-04 | 三得利控股株式会社 | Use the steviol of AOBGL11 autoploid and the manufacturing method of steviol glycoside |
| US20190203246A1 (en) * | 2016-05-25 | 2019-07-04 | Suntory Holdings Limited | Method for producing steviol and steviol glycoside by using aobgl11 homolog |
| CN109154012B (en) * | 2016-05-25 | 2022-04-15 | 三得利控股株式会社 | Method for producing steviol and steviol glycoside Using AOBGL11 homolog |
| US10988790B2 (en) * | 2016-05-25 | 2021-04-27 | Suntory Holdings Limited | Method for producing steviol and steviol glycoside by using AOBGL11 homolog |
| CN105861573A (en) * | 2016-06-02 | 2016-08-17 | 广西大学 | Method for preparing steviol through two-enzyme method |
| CN105861573B (en) * | 2016-06-02 | 2020-07-07 | 广西大学 | Method for preparing steviol by double-enzyme method |
| CN107188800B (en) * | 2017-07-19 | 2021-05-28 | 江南大学 | Steviol crystal in crystal form A, and preparation method and application thereof |
| CN107188800A (en) * | 2017-07-19 | 2017-09-22 | 江南大学 | Stevia rebaudianum alcohol crystals, its preparation method and application with crystal formation A forms |
| US11180788B2 (en) | 2018-05-31 | 2021-11-23 | Jiangnan University | Method for the preparation of lower graft degree glucosylated steviol glycosides |
| WO2019227532A1 (en) * | 2018-05-31 | 2019-12-05 | 东台市浩瑞生物科技有限公司 | Method for preparing glucosyl steviol glycoside having small number of grafts |
| CN110564658A (en) * | 2019-09-06 | 2019-12-13 | 广西大学 | Escherichia coli engineering bacterium and method for producing steviol through whole-cell catalysis of escherichia coli engineering bacterium |
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