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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  • 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

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

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

[0047] Inoculate UGT strains into 4ml liquid LB medium at a ratio of 1%, culture with shaking at 37°C (200rpm) overnight, transfer the overnight culture to 50ml liquid LB medium with 1% inoculation volume, and culture with shaking 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 centrifug...

Example Embodiment

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

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

Example Embodiment

[0050] Example 3: Preparation of recombinant E. coli cells containing UGT-B

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

[0052] Inoculate UGT strains into 4ml liquid LB medium at a ratio of 1%, culture with shaking at 37°C (200rpm) overnight, transfer the overnight culture to 50ml liquid LB medium with 1% inoculation volume, and culture with shaking 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 induction, the cells were collected by centrifugation (8...

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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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