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Method for preparing rubusoside by catalytically hydrolyzing stevioside with beta-galactosidase

A technology of galactosidase and steviol glycosides, which is applied in the field of biosynthesis of organic compounds, can solve problems such as inability to prove steviolbiosides, lack of structural characterization, and difficulty in product separation, and achieve expanded applications, high selectivity, and product separation and purification simple effect

Active Publication Date: 2011-11-23
DONGTAI HIRYE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the product is only characterized by analytical means such as mass spectrometry, lacks structural characterization, and cannot prove whether the obtained substance is rubusoside or its isomer steviolbioside, and the reaction time needs 48 h, and the substrate concentration is as low as 10g / L , the product is difficult to isolate, and the safety of this genus is questionable

Method used

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  • Method for preparing rubusoside by catalytically hydrolyzing stevioside with beta-galactosidase
  • Method for preparing rubusoside by catalytically hydrolyzing stevioside with beta-galactosidase
  • Method for preparing rubusoside by catalytically hydrolyzing stevioside with beta-galactosidase

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Effect test

Embodiment 1

[0025] Dissolve 20g St in 100mL deionized water, shake in a water bath shaker and shake at 50 o C under constant temperature for 0.5h, the shaker speed is 150rpm; add β -Galactosidase enzyme solution 14000U / g St; after 4 hours of reaction, the conversion rate of St does not increase. St conversion was 99%. The reaction solution was boiled for 10 minutes to inactivate the enzyme, and after centrifugation, the supernatant was concentrated and dried. Add 2g of crude product to 20mL of methanol; continue to stir and heat to reflux; gradually add methanol dropwise until the solution is clear, the total amount of methanol added is about 160mL; leave to cool naturally to precipitate crystals, filter and dry the crystals; repeat the above steps for two times After recrystallization, 1.2 g of a product with a content greater than 97% can be obtained at last. Select column chromatography silica gel (200-300 mesh), with chloroform, methanol and water as eluent 5:5:1 ( v / v / v ), accor...

Embodiment 2

[0027] First, the enzyme was immobilized, AB-8 resin 0.1g, 0.5mL enzyme solution was dissolved in phosphate buffer solution of pH 6 to prepare a 20mL system, and the temperature was 4 o C fixed for 5h, after immobilization β - Galactosidase in 5% CaCl 2 The solution was preserved, and the enzyme activity was determined to be 20000U / g by FCCIV method. Dissolve 1g St in 100mL deionized water, put it in a water bath shaker and shake it at 50 o C for 0.5h at constant temperature, the shaker speed is 150rpm; add immobilized β -Galactosidase reaction 10000U / g St; regular sampling for HPLC analysis, until the St conversion rate no longer changes ( Figure 5 ). The reaction solution mainly contains rubusoside and glucose hydrolyzed. Reclaim the immobilized enzyme, concentrate and dry the gained rubusoside crude product (80% rubusoside, 20% glucose, w / w ) can be used in food and other fields. The specific characterization of the product is described in the technical scheme part...

Embodiment 3

[0029] Dissolve 1g St in 100mL deionized water, put it in a water bath shaker and shake it at 50 o C for 0.5h at constant temperature, the shaker speed is 150rpm; add immobilized β - Galactosidase 10000U / g St; react for 5 hours until the St conversion rate no longer increases. The immobilized enzyme is recovered and purified by recrystallization to obtain rubusoside with a content greater than 97%. The specific characterization of the product is described in the technical scheme part.

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Abstract

The invention relates to a method for preparing rubusoside by catalytically hydrolyzing stevioside with beta-galactosidase, belonging to the technical field of biosynthesis of organic compounds. In the invention, beta-galactosidase is utilized to selectively and catalytically hydrolyzing stevioside obtained from stevia so as to prepare the rubusoside. Thus, the invention has the advantages of high conversion rate, high speed, high selectivity, simple separation and clearing process and the like. The invention provides a new efficient and environment-friendly method for preparing rubusoside.

Description

technical field [0001] The invention belongs to the technical field of biosynthesis of organic compounds, in particular to a β - A method for preparing rubusoside by catalyzing the hydrolysis of steviol glycoside by galactosidase. Background technique [0002] Rubusoside (also known as rubusoside or sweet tea element, Rubusoside, Ru) is Guangxi sweet tea ( Rubus usavissmius. The main sweetness ingredient of S Lee) has the characteristics of high sweetness, low calorie value, natural and non-toxic, and has a good health care effect on people with diabetes or "three highs". However, the planting area of ​​sweet tea is small and the output is low. At present, there are only a handful of companies in Guangxi engaged in the production of rubusoside. The highest purity of the product is only about 70%, and the composition is complex, including steviol and steviol monoglycoside. [0003] Stevioside (13-[(2-O- β -D-Glucopyranosyl- β -D-glucopyranosyl)oxy]kaur-16-en-18-oic acid ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/56
Inventor 夏咏梅方云万会达
Owner DONGTAI HIRYE BIOTECH CO LTD
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