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Method for preparing rebaudioside M through enzyme method

A technology for enzymatic preparation and rebaudioside, which is applied in the field of biological preparation of rebaudioside M, can solve problems such as no commercial production of rebaudioside M, and achieves shortening production cycle, expanding application scope, and producing The effect of cycle shortening

Active Publication Date: 2013-11-20
PEPSICO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no commercial production of rebaudioside M at present

Method used

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  • Method for preparing rebaudioside M through enzyme method
  • Method for preparing rebaudioside M through enzyme method
  • Method for preparing rebaudioside M through enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Preparation of recombinant Escherichia coli cells containing UGT-A

[0046]According to Sequence 1 and Sequence 2, the UGT-A gene fragment was synthesized, NdeI and BamHI restriction sites were added to both ends, and then connected into pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.). The UGT gene fragment was digested with restriction endonucleases NdeI and BamHI, the purified fragment was recovered, and T4 ligase was added to connect the fragment into the corresponding restriction site of pET30a, and transformed into BL21 (DE3) strain.

[0047] Inoculate UGT strains into 4ml liquid LB medium with 1% ratio, shake culture at 37°C (200rpm) overnight, transfer overnight culture to 50ml liquid LB medium with 1% inoculum, shake culture at 37°C (200rpm) to OD 600 When the value reaches 0.6-0.8, add final concentration of 0.4mM IPTG and culture overnight at 20°C with shaking. After the induction, the cells were collected by centrifugation (8,000 rpm, 10 ...

Embodiment 2

[0048] Embodiment 2: Preparation of UGT-A freeze-dried powder

[0049] The recombinant cells of UGT-A prepared in Example 1 were ultrasonically disrupted in an ice bath, the disrupted solution was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and freeze-dried for 24 hours to obtain a freeze-dried powder of UGT-A.

Embodiment 3

[0050] Embodiment 3: Prepare the recombinant escherichia coli cell containing UGT-B

[0051] According to Sequence 3 and Sequence 4, the UGT-B gene fragment was synthesized, NdeI and BamHI restriction sites were added to both ends, and it was connected into pUC57 vector (Suzhou Jinweizhi Biotechnology Co., Ltd.). The UGT gene fragment was digested with restriction endonucleases NdeI and BamHI, the purified fragment was recovered, and T4 ligase was added to connect the fragment into the corresponding restriction site of pET30a, and transformed into BL21 (DE3) strain.

[0052] Inoculate UGT strains into 4ml liquid LB medium with 1% ratio, shake culture at 37°C (200rpm) overnight, transfer overnight culture to 50ml liquid LB medium with 1% inoculum, shake culture at 37°C (200rpm) to OD 600 When the value reaches 0.6-0.8, add final concentration of 0.4mM IPTG and culture overnight at 20°C with shaking. After the induction, the cells were collected by centrifugation (8,000 rpm, 1...

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PUM

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Abstract

The invention relates to a method for preparing rebaudioside M through an enzyme method. According to the method, rebaudioside A or rebaudioside D is used as a substrate, and the substrate reacts to generate the rebaudioside M in the presence of a glucosyl donor under the catalytic action of UDP-glucosyltransferase and / or recombinant cells containing the UDP-glucosyltransferase. The method for preparing rebaudioside M through an enzyme method has important application value; and compared with the existing technology of extracting rebaudioside M from stevia rebaudian leaves, the method provided by the invention obviously shortens the production cycle, improves the productivity and lowers the cost, and can provide products having higher purity. Thus, the method can be used in the food and beverage industry in a more economical manner.

Description

technical field [0001] The invention relates to a preparation method of rebaudioside M, in particular to a biological preparation method of rebaudioside M. Background technique [0002] Sweeteners are a class of food additives widely used in food, beverage and confectionary production. It can be added during the production of food, or it can be properly diluted as a substitute for sucrose in home baking. Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, honey, etc., and artificial sweeteners such as aspartame and saccharin. Stevioside is a kind of natural sweetener extracted from plant stevia, which has been widely used in food and beverages. The stevia extract contains a variety of steviosides including rebaudioside. Naturally extracted steviosides vary greatly in different batches and require subsequent purification. The current commercialized product rebaudioside A contains some other steviosides such as rebaudioside C, D and F, etc. The...

Claims

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Application Information

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IPC IPC(8): C12P19/56C12R1/185C12R1/865A23L27/30
CPCC12N9/1051A23L27/36C12P19/56C12Y204/01017C12P19/18A23V2002/00
Inventor 陶军华李国庆梁晓亮托马斯·李格雷戈瑞·叶普
Owner PEPSICO INC
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