CN102827891B - 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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- CN102827891B CN102827891B CN201210352461.6A CN201210352461A CN102827891B CN 102827891 B CN102827891 B CN 102827891B CN 201210352461 A CN201210352461 A CN 201210352461A CN 102827891 B CN102827891 B CN 102827891B
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- stevioside
- steviol
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- glucuroide
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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 compound, is specifically related to a kind of use and derives from aspergillus niger
(Aspergillus niger)'s
β-glucuroide temperature programming catalytic hydrolysis stevioside is prepared the method for steviol.
Background technology
Stevioside (13-[(2-O-
β-D-Glucopyranosyl-
β-D-glucopyranosyl) oxy] kaur-16-en-18-oic acid
β-D-glucopyranosyl ester, stevioside, hereinafter to be referred as St) be from sweet Stevia (
stevia rebaudianabertoni) tetracyclic diterpene compound extracting in is a kind of sweeting agent with rear bitter taste.In St molecule, contain three kinds of glycosidic links, through hydrolysis, can obtain steviol, isosteviol, stevia rebaudianum monoglycosides, steviolbioside, immature fruit of Juteleaf Raspberry glycosides or its mixture; Each component concentration of hydrolysate depends on hydrolysising condition: for example with mineral alkali catalytic hydrolysis St, obtain steviolbioside (Sharipova R, et al, 2009), with mineral acid catalytic hydrolysis St, obtain the mixture (patent No.: 200910025817.3) of steviol and isosteviol.Such as magnifying forever, wait and prepare steviol by chemical method two steps, first St, through sodium periodate or lead tetra-acetate oxidation, then obtains the steviol (patent No.: 200810242967.5) after basic hydrolysis, column chromatography purification.Avent etc. point out acidic hydrolysis St except obtaining steviol and isosteviol, also have other two kinds of isomerss (Avent A G, et al, 1990).With enzymatic hydrolysis St of the same race, also may obtain different products.If thereby by utilizing the high regioselectivity enzymatic process hydrolysis St of enzyme, can a step directly obtain high-quality single hydrolysate, can also form by changing hydrolysis reaction condition regulation and control product simultaneously, Milagre etc. point out that the product of pancreatin (Pancreatin) catalysis St hydrolysis reaction is isosteviol pH 4.0 times; And pH 7.0 times, product is steviol (Milagre H M S, et al, 2009).
Although enzyme catalysis advantage is numerous, poor heat stability becomes unavoidable problem in enzyme use procedure.Conventionally can pass through additional stablizer, the approach such as immobilization improve thermostability.Derive from aspergillus niger
β-glucuroide is 75
othe very fast inactivation of meeting under C, but simultaneously again at 70-75
ounder C, there is the highest catalytic hydrolysis efficiency.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
aspergillus niger's
βthe stability of-glucuroide effective time that has shortened hydrolysis reaction.
Summary of the invention
The object of this invention is to provide a kind of use
β-glucoside enzymatic hydrolysis stevioside is prepared the method for steviol.The aspergillus niger of deriving from filtering out for the present invention
β-glucuroide, hydrolysis St prepares steviol; By temperature programming, react, reduce the heat inactivation of enzyme.Production process environmental protection, catalytic efficiency is high, and separation and purification of products is simple, and purity is high.
Derive from aspergillus niger
βthe reaction equation that-glucoside enzyme catalysis stevioside is prepared steviol is as follows:
Object of the present invention can reach by the following technical solutions: the Folium Chrysanthemi extract of take stevioside St or containing St (simultaneously containing rebaudioside A (RA), dulcoside B (RC)) etc. is substrate, substrate is mixed with to the reaction solution of 150-300 g/L with deionized water or damping fluid; 55
ounder C, after constant temperature 0.5 h, add and to derive from aspergillus niger
β-glucuroide, reacts 2-6 h under equality of temperature, the reaction times determines with enzyme concentration, when preferably enzyme concentration is 800 U/g St, react 3 h (enzyme activity determination substrate used be 4-nitrophenols-
β-D-glucopyranoside pNPG); Then be warming up to 70-75
ounder C, react to stevioside transformation efficiency and no longer raise; Stevioside is hydrolyzed into steviol and separates out from reaction solution, and the composition of other qualities of stevia extract does not have or seldom reaction; Reaction solution is carried out to suction filtration, with 20% methanol aqueous solution recrystallization, can obtain steviol white products after washing filter residue with water.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 aspergillus niger (being purchased from Korea S biobean bio tech ltd), and its form is enzyme liquid.
Said temperature of reaction is the temperature of temperature programming, first 55
ounder C, react 2-6 h, then at 70-75
ounder C, react to 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.
Described
βthe addition that-glucuroide is urged is for being 500-1000U/g stevioside.
beneficial effect of the present invention:this patent provides a kind of novel method of preparing steviol, has expanded the purposes of stevioside, greatly promotes its added value.Derive from aspergillus niger
β-glucuroide is foodstuffs industry zymin, and safety, efficient, selectivity is high.Different from additive method, in product, there is no isosteviol; And other stevioside derivatives 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, product separation purifying is simple.
Accompanying drawing explanation
The HPLC of product steviol (a) and total ion current figure (b) in Fig. 1 the present invention.
The one-level mass spectrum (a) of product steviol and second order ms (b) in Fig. 2 the present invention.
Product steviol in Fig. 3 the present invention
13cNMR.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1
15 g St (commercially available product, purity is 97%) are dispersed in to 55
oin the 100 mL deionized waters of C, put into 55
oin C shaking bath, jolt 0.5 h, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 800 U/g St; React to St transformation efficiency and no longer rise.St transformation efficiency is 99.0%.Suction filtration after cooling reaction solution, and use respectively mother liquor and deionized water wash filter cake, then can obtain steviol product with 20% methanol aqueous solution recrystallization.The HPLC purity of final product is greater than 98 %.Reaction needs 8 hours altogether.
Product is carried out to LC-MSMS and NMR analysis, the results are shown in accompanying drawing 1-3.The molecular ion peak of main composition in LC-MSMS (317.4, [M-H]
-) can corresponding steviol, the NMR of hydrolysate also can further confirm that product is steviol.
St transformation efficiency can be with reference to GB (GB8270-1999) by 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, containing 10% RA) are dissolved in to 55
oin the 100 mL deionized waters of C, put into 55
oin C shaking bath, jolt 0.5 h, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 600 U/g St; After reacting 3 h, rise to 75
oc continues to react to St transformation efficiency and no longer rises.With 20% methanol aqueous solution recrystallization, can obtain steviol product again.Reaction needs 3.5 hours altogether.
Embodiment 3
15 g St are dispersed in the acetate buffer solution of 100 mL pH 5.0 of 50 mM, put into 55
oin C shaking bath, jolt 0.5 h, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 700 U/g St; After reaction 3h, rise to 70
oc continues to react to St transformation efficiency and no longer rises.Reaction needs 4 hours altogether.With 20% methanol aqueous solution recrystallization, can obtain steviol product again.
Embodiment 4
30 g St crude products (50% St, 40% RA, 5% RC and other) are dissolved in 100 mL deionized waters, put into 55<sup TranNum="137">o</sup>in C shaking bath, jolt 0.5 h, shaking speed is 150 rpm; Add<i TranNum="138">β</i>-glucuroide 500 U/g St; React after 5 h, be warming up to 70<sup TranNum="139">o</sup>under C, react 1 h, by HPLC, analyzed and found that St is all converted into steviol, RA and RC transformation efficiency are all<5%.With 20% methanol aqueous solution recrystallization, can obtain steviol product again.
Embodiment 5
30 g St crude products (St 90%) are dissolved in 200 mL deionized waters, put into that shaking bath jolts and 55<sup TranNum="142">o</sup>constant temperature 0.5 h under C, shaking speed is 150 rpm; Add<i TranNum="143">β</i>-glucuroide 500 U/g St; React after 5 h, be warming up to 75<sup TranNum="144">o</sup>under C, react 5 h, by HPLC, analyzed and found that St is all converted into steviol, RA and RC transformation efficiency are all<5%.With 20% methanol aqueous solution recrystallization, can obtain steviol product again.
Embodiment 6
20 g St are dissolved in 100 mL acetate buffer solutions (pH 4.6,50 mM), put into 55
oin C shaking bath, jolt 0.5 h, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 900 U/g St; After reacting 6 h, rise to 75
oc continues to react to St transformation efficiency and no longer rises.With 20% methanol aqueous solution recrystallization, can obtain steviol product again.
Embodiment 7
20 g St are dissolved in 100 mL phosphoric acid buffers (pH 6.0,50 mM), put into that shaking bath jolts and 55
oconstant temperature 0.5 h under C, shaking speed is 150 rpm; Add
β-glucuroide enzyme liquid 1000 U/g St; After reacting 6 h, rise to 75
oc continues to react to St transformation efficiency and no longer rises.With 20% methanol aqueous solution recrystallization, can obtain steviol product again.
Claims (5)
- One kind with β-glucoside enzymatic hydrolysis stevioside is prepared the method for steviol, it is characterized in that the qualities of stevia extract of take stevioside or containing stevioside is substrate, it is mixed with to the reaction solution of 150-300 g/L with deionized water or damping fluid; 55 ounder C, after constant temperature 0.5 h, add above-mentioned β-glucuroide, reacts 2-6 h under equality of temperature; Then be warming up to 70-75 ounder C, react to stevioside transformation efficiency and no longer raise; Stevioside is hydrolyzed into steviol and separates out from reaction solution, and the composition of other qualities of stevia extract does not have or seldom reaction; Reaction solution is carried out to suction filtration, wash filter residue with water, then can obtain steviol product with 20% methanol aqueous solution recrystallization filter residue.
- 2. use according to claim 1 β-glucoside enzymatic hydrolysis stevioside is prepared the method for steviol, it is characterized in that, said stevioside refers to the stevioside with following structure:。
- 3. use according to claim 1 β-glucoside enzymatic hydrolysis stevioside is prepared the method for steviol, 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. use according to claim 1 β-glucoside enzymatic hydrolysis stevioside is prepared the method for steviol, it is characterized in that: said β-glucuroide derives from aspergillus niger, and its form is enzyme liquid.
- 5. use according to claim 1 β-glucoside enzymatic hydrolysis stevioside is prepared the method for steviol, it is characterized in that: described in βthe addition that-glucuroide is urged is 500-1000U/g stevioside.
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| MX2016003540A (en) * | 2013-09-19 | 2016-12-08 | Purecircle Usa Inc | 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 |
| CN105255971B (en) * | 2015-10-27 | 2018-10-23 | 江南大学 | Two enzymes method prepares stevia rebaudianum monoglycosides |
| 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 |
| WO2017204284A1 (en) * | 2016-05-25 | 2017-11-30 | サントリーホールディングス株式会社 | Method for producing steviol and steviol glycoside by using aobgl11 homolog |
| 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 |
| CN108715876B (en) * | 2018-05-31 | 2021-08-24 | 东台市浩瑞生物科技有限公司 | Method for preparing low-grafting-number glucosyl stevioside |
| US11180788B2 (en) | 2018-05-31 | 2021-11-23 | Jiangnan University | Method for the preparation of lower graft degree glucosylated steviol glycosides |
| CN110564658B (en) * | 2019-09-06 | 2021-08-17 | 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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Non-Patent Citations (2)
| Title |
|---|
| Enzymatic determination of stevioside in Stevia Rebaudiana;Mizukami H. et al.;《Phytochemistry》;19820831;第21卷(第8期);第1927页右栏第1段，倒数第1段，第1928页右栏第2段 * |
| Mizukami H. et al..Enzymatic determination of stevioside in Stevia Rebaudiana.《Phytochemistry》.1982,第21卷(第8期),第1927页右栏第1段，倒数第1段，第1928页右栏第2段. |
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