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Method for preparing rebaudioside M through enzyme method

A technology for enzymatic preparation and rebaudioside, applied in the field of biological preparation of rebaudioside M, can solve the problems of high production cost, low substrate concentration, insufficient process optimization, etc. Purity-enhancing effect

Active Publication Date: 2014-04-30
PEPSICO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the process in this patent is not optimized enough, the highest conversion rate is only about 80%, and the purity is only 95%, the substrate concentration is low, the production cost is high, and it is not suitable for industrial production

Method used

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  • Method for preparing rebaudioside M through enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Preparation of recombinant Escherichia coli cells containing UGT-A

[0049]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 the BL21 (DE3) strain was transformed to obtain the recombinant strain GQ-A.

[0050] 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) When the OD600 value reached 0.6-0.8, a final concentration of 0.4 mM IPTG was added and cultured overnight at 20°C with shaking. After the induction, the cells we...

Embodiment 2

[0051] Embodiment 2: Preparation of UGT-A freeze-dried powder

[0052] 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

[0053] Embodiment 3: Prepare the recombinant escherichia coli cell containing UGT-B

[0054] 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 the BL21 (DE3) strain was transformed to obtain the recombinant strain GQ-B.

[0055] 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) When the OD600 value reached 0.6-0.8, a final concentration of 0.4 mM IPTG was added and cultured overnight at 20°C with shaking. After the induction, the cells ...

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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 sucrose and UDP under the catalytic action of a mixture of UDP-glucosyltransferase and sucrose synthetase or recombinant cells containing the UDP-glucosyltransferase and the sucrose synthetase, wherein the reaction is performed in a water-phase system having a pH value of 5.0-9.0 at 20-60 DEG C. 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] Sugar substitutes can be divided into four categories, which have different effects on human health. The first category is natural sweeteners such as fruit juice, honey, and maple syrup, which are similar in calorie and carbohydrate content to table sugar. The second category is artificial sweeteners, such as aspartame and saccharin, which have no calories and are considered non-nutritive sweeteners. The third category is sugar alcohols, such as xylitol and sorbitol, which mainly come from vegetables and fruits. The fourth category is sweet substances extracted from natural plants, such as stevioside extracted from stevia. The sweetness of artificial sweeteners, sugar alcohols and extracted sweeteners is many times higher than that of sucrose, and some can reach hundreds of times, so the a...

Claims

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Application Information

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IPC IPC(8): C12P19/56
Inventor 陶军华李国庆梁晓亮托马斯·李格雷戈瑞·叶普侯茂奇陶鼎合
Owner PEPSICO INC
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