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Stevioside derivative and application of stevioside derivative

A technology for daily use and medicine, which is applied in the field of stevioside derivatives and its applications. It can solve the problems of non-specific glycosyl transfer by enzymes, reduced sweetness, and impure products, and achieve high product purity, low production cost, The effect of easy purification

Inactive Publication Date: 2016-07-20
NANJING NUOYUN BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that stevia can be modified by cyclodextrin glycosyltransferase to improve its taste and taste, but the position of the enzyme to transfer glycosyl groups is not specific, the product is not pure, and its sweetness is also seriously reduced

Method used

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  • Stevioside derivative and application of stevioside derivative
  • Stevioside derivative and application of stevioside derivative
  • Stevioside derivative and application of stevioside derivative

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Construction and induced expression of recombinant Escherichia coli

[0019] The recombinant Escherichia coli expression strain containing the target gene is obtained by using molecular biology and genetic engineering techniques, and then the recombinant Escherichia coli is fermented and cultured to induce expression to prepare recombinant cells containing the target protein. The specific steps are as follows:

[0020] 1) Synthesize the required primer fragments, obtain the required UDP-glucosyltransferase M303 encoding DNA fragments by PCR amplification, and integrate them into the expression cassette of the pNYK expression vector through homologous recombination technology.

[0021] 2) Transform the recombinant plasmid into Escherichia coli to obtain the engineering bacterium J303 containing the target gene.

[0022] 3) Put 1ml of engineering bacteria J303 in TB medium, shake at 250rpm at 37°C until OD600=1.0, add final concentration of 0.1mMIPTG and shake at 25°C fo...

Embodiment 2

[0024] Preparation of Compound (IV) Using Compound (I) as Substrate

[0025]

[0026] Take 444 mg of wet bacteria M303, resuspend the cells with sterile water, and ultrasonically break the cells in an ice bath, which is the crude enzyme solution used in the reaction. Accurately weigh the sample and prepare a 1.85ml system, wherein the final concentration of the substrate compound (I) is 2.0g / L, UDP-glucose is 2.70g / L, and 0.286g / L of MgCl is added 2 ; Then add the crude enzyme solution, and add Tris-HCl buffer (pH8.0) with a final concentration of 0.1M until the system is 1.85ml, and start the reaction. Shake at 150 rpm for 38 hours at 25°C, and boil at 100°C to terminate the reaction. Centrifuge at 13000 g for 10 min, take the supernatant as a sample, use macroporous resin to perform primary purification on the sample, and use LC-MS method to detect the purity of the purified compound (IV).

Embodiment 3

[0028] Determination of Optimum Metal Ion Concentration

[0029] Get the bacterium M303444g in Example 1, according to the method in Example 2, transfer in the 5ml centrifuge tube, add the compound (I) that final concentration is 2.0g / L, UDP-glucose is 2.70g / L and MgCl 2 , and added 0.1M Tris-HCl buffer (pH8.0). According to the above method, take a parallel sample, add MgCl 2 The final concentrations were 0, 0.1, and 0.286 g / L, respectively. After 38 hours of reaction, the samples were boiled and centrifuged, and the supernatant samples were analyzed by HPLC. where MgCl 2 When it is 0.286, the yield of compound (Ⅳ) is the highest. ·

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Abstract

The invention relates to a stevioside derivative and application of the stevioside derivative and belongs to the technical field of food chemistry. The stevioside derivative takes a stevioside derivative compound (I) as a substrate so that the substrate is reacted in the presence of a glucosyl donor and under a catalytic effect of UDP (Uridine Diphosphate)-glucosyl transferase to generate a stevioside derivative compound (IV). The stevioside derivative has the advantages of green and safe production process, low production cost, short period and capability of extremely improving the competitiveness of a product. The stevioside derivative has the advantages of simplicity in operation, high conversion efficiency and easiness of purification, so that the purity of the obtained product is high and can be used for food and beverage industries; the product has an important application value.

Description

technical field [0001] The invention relates to a stevioside derivative and application thereof, belonging to the technical field of food chemical industry. Background technique [0002] Stevioside is a new type of natural sweetener extracted from the leaves and stems of the Compositae herb Stevia rebaudiana. It is a natural, green and health-care functional food. It has the characteristics of high and low calorie. Its sweetness is 200-300 times that of sucrose, and its calorific value is only 1 / 300 of that of sucrose. It is an ideal sweetener that can replace sucrose. As human beings pay more attention to health and green, stevioside is called by food scientists as the most promising sweetener in the future world. [0003] Stevioside is considered by the international medical community as a good nutritional supplement and health medicine for human beings. A large number of scientific experiments have proved that stevioside is beneficial to the regulation of blood sugar an...

Claims

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Application Information

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IPC IPC(8): C07H15/256C12P19/56C12P19/18A61K31/704A61P3/10A61P3/04A61P9/10A61P17/02A61K8/60A23L33/10A23L33/125A23L2/60A23L2/52A61Q19/00A61Q19/08
CPCC07H15/256A23L2/52A23L2/60A23V2002/00A61K8/602A61K31/704A61Q19/00A61Q19/08C12P19/18C12P19/56A23V2200/326A23V2200/328A23V2200/332A23V2250/60
Inventor 朱惠霖丁雪峰张永正
Owner NANJING NUOYUN BIOLOGICAL TECH CO LTD
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