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Preparation method of Rebaudioside D, product and application of Rebaudioside D

A technology of reaction and stevioside, which is applied in the field of preparation of rebaudioside D, can solve the problems of restricting the feasibility of rebaudioside D, high price, cumbersome process, etc., and achieve the goal of simple and efficient preparation method and increased yield Effect

Pending Publication Date: 2020-01-07
BINZHOU SANYUAN BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The content of rebaudioside D in stevia leaves is very small (less than 5%). The production of rebaudioside D by extraction requires a large amount of stevia raw materials. In addition, the process of enriching rebaudioside D is cumbersome. It needs to go through the column many times and desalt, decolorize, recrystallize, and generate a lot of waste water in the production process. The production cost is high and it is not suitable for industrialized large-scale production.
[0005] CN107446874A discloses a method for producing rebaudioside D using recombinant Escherichia coli, using biological methods to construct recombinant Escherichia coli, introducing the eugt11 gene into Escherichia coli to induce expression, obtaining recombinant protease, and catalyzing rebaudioside A (RA) Transformed into rebaudioside D (RD), but this method involves strain transformation, and the stability of the method needs to be improved
However, due to the extremely high price of UDP-glucose, the feasibility of industrialized preparation of rebaudioside D is almost completely limited, and the economy is poor and lacks market competitiveness.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Prepare an aqueous phase reaction system, the initial reaction concentration of stevioside is 2g / L, the initial reaction concentration of ethyl acetate is 80g / L, the initial reaction concentration of β-cyclodextrin is 60g / L, the initial reaction concentration of palmitoyltransferase The initial reaction concentration of β-cyclodextrin glycosyltransferase is 87000U / L and 42000U / L, and the reaction system is adjusted to pH=7 by using phosphate buffer.

[0044] The aqueous phase reaction system was placed in a constant temperature shaker, the reaction temperature was 35° C., and the reaction time was 24 hours. The aqueous solution containing rebaudioside D can be prepared, and the concentration of rebaudioside D is determined to be 1.57g / L by high performance liquid chromatography, and the conversion rate is 78.5%.

Embodiment 2

[0046] Prepare an aqueous phase reaction system, the initial reaction concentration of stevioside is 0.1g / L, the initial reaction concentration of ethyl acetate is 20g / L, the initial reaction concentration of β-cyclodextrin is 10g / L, and the initial reaction concentration of palmitoyltransferase The concentration is 57000U / L, the initial reaction concentration of β-cyclodextrin glycosyltransferase is 28000U / L, and the reaction system is adjusted to pH=6 by using phosphate buffer.

[0047] The aqueous phase reaction system was placed in a constant temperature shaker, the reaction temperature was 40° C., and the reaction time was 16 hours. The aqueous solution containing rebaudioside D can be prepared, and the concentration of rebaudioside D is determined to be 0.06 g / L by using high performance liquid chromatography, and the conversion rate is 60%.

Embodiment 3

[0049] Prepare an aqueous phase reaction system, the initial reaction concentration of stevioside is 3g / L, the initial reaction concentration of ethyl acetate is 120g / L, the initial reaction concentration of β-cyclodextrin is 90g / L, the initial reaction concentration of palmitoyltransferase The initial reaction concentration of β-cyclodextrin glycosyltransferase is 90000U / L and 46000U / L, and the reaction system is adjusted to pH=8 by using phosphate buffer.

[0050] The aqueous phase reaction system was placed in a constant temperature shaker, the reaction temperature was 37° C., and the reaction time was 24 hours. The aqueous solution containing rebaudioside D can be prepared, and the concentration of rebaudioside D is determined to be 2.57g / L by high performance liquid chromatography, and the conversion rate is 85.7%.

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Abstract

The invention provides a preparation method of Rebaudioside D, a product and application of the Rebaudioside D. The preparation method comprises the steps that steviol glycosides is taken as a raw material, ethyl acetate and beta-cyclodextrin are taken as glycosyl donors, a conversion reaction takes place under the catalysis of a complex enzyme, and the Rebaudioside D is generated. According to the preparation method of the Rebaudioside D, the product and application of the Rebaudioside D, the cheap and easily obtained ethyl acetate and beta-cyclodextrin are taken as the glycosyl donors to replace high-cost UDP-Glucose to generate the Rebaudioside D under the catalysis of the complex enzyme. The reaction opens a new way for the preparation of the Rebaudioside D, the product yield of the reaction is high, the preparation process is simple, and the reaction is applicable to industrial production and has broad application prospects.

Description

technical field [0001] The invention belongs to the technical field of biocatalytic conversion, and relates to a preparation method of rebaudioside D and its products and applications, in particular to a rebaudioside with ethyl acetate and β-cyclodextrin as glycosyl donors The preparation method of D and its products and applications. Background technique [0002] Steviol glycosides are natural sweeteners extracted from the leaves of Stevia rebaudiana, a herb of Compositae. It is a mixture of various glycosides, and different glycosides have great differences in taste and quality. Stevioside has high sweetness, low calorie, high stability, and potential curative effects such as anti-hyperglycemia, anti-hypertension, anti-inflammation, anti-tumor, anti-diarrhea, and immune regulation effects. It is used as a sweetener in food and Related research and economics in beverages have received extensive attention. High-purity stevia products have been officially approved as food ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/20
CPCC12P33/20
Inventor 李丽聂在建程保华李德春
Owner BINZHOU SANYUAN BIOLOGICAL TECH
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