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Stevioside derivative prepared by stervioside biotransformation, preparation method and application thereof

A technology of biotransformation and stevioside, which is applied in the preparation of sugar derivatives, sugar derivatives, sugar derivatives, etc., can solve the problems of product impurity, non-specific position of enzyme-transferred glycosyl groups, and reduced sweetness, so as to achieve purification Easy, high product purity and low production cost

Active Publication Date: 2016-02-24
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 prepared by stervioside biotransformation, preparation method and application thereof
  • Stevioside derivative prepared by stervioside biotransformation, preparation method and application thereof
  • Stevioside derivative prepared by stervioside biotransformation, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Construction and induced expression of recombinant Escherichia coli

[0039] 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:

[0040] 1) Synthesize the required primer fragments, obtain the required UDP-glucosyltransferase M301 encoding DNA fragments by PCR amplification, and integrate them into the expression cassette of the pNYK expression vector through homologous recombination technology.

[0041] 2) Transform the recombinant plasmid into Escherichia coli to obtain the engineering bacterium J301 containing the target gene.

[0042] 3) Put 1 ml of engineering bacteria J301 in TB medium, shake at 250 rpm at 37°C until OD600=1.0, add final concentration of 0.1mMIPTG and shake at 25°C ...

Embodiment 2

[0045] Direct preparation of NY101 by enzymatic method using stevioside (stevioside) as substrate (Route 1)

[0046] The bacterial cell M301 precipitated in Example 1 was taken, the wet bacterial weight was 35 mg, the cells were resuspended in sterile water, and the cells were broken by ultrasonic waves in an ice bath, which was the crude enzyme solution used in the reaction. Precisely weigh the sample and prepare a 1.85mL system, in which the final concentration of stevioside (stevioside) is 2.70g / L, UDP-glucose is 2.70g / L, and 0.286g / L of MgCl 2 ; Then add crude enzyme solution, and add Tris-HCl buffer solution (0.1M, pH8.0) to the system of 1.85ml, start the reaction. Shake at 150 rpm for 24 hours on a constant temperature shaker at 37°C, and boil at 100°C to terminate the reaction. Centrifuge at 13000 g for 10 min, and take the supernatant as a sample. The content of NY101 in the reaction system was detected by LC-MS method. The experimental results show that the conver...

Embodiment 3

[0048] Determination of Optimum Metal Ion Concentration

[0049] Get 35 mg of the bacterium M301 precipitate in Example 1, transfer it to a 5ml centrifuge tube according to the method in Example 2, add stevioside with a final concentration of 2.70 g / L, UDP-glucose is 2.70 g / L and MgCl2 , 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 and 0.286g / L, respectively. After 24 hours of reaction, the samples were boiled and centrifuged, and the supernatant samples were analyzed by HPLC. where MgCl 2 When the concentration is 0.1g / L, the yield of NY101 is the highest.

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Abstract

The invention relates to a stevioside derivative prepared by stervioside biotransformation and a preparation method and an application thereof, and belongs to the field of food chemistry. According to the method, stervioside, which is used as a substrate, undergoes enzyme catalysis by the utilization of UDP-glucosyltransferase in the presence of a glucosyl donor under conditions of proper temperature, pH, salinity, substrate concentration and the like, so as to obtain a stevioside derivative NY101 with a brand-new structure. The method for preparing the stevioside derivative NY101 by stervioside biotransformation is simple to operate, is low-cost, and has high transformation efficiency. Transformation efficiency can be greater than 90%. Purification is easy, purity is high, and purity can be greater than 95%. The stevioside derivative NY101 can be used in the food and beverage industry and has huge potential application value.

Description

technical field [0001] The invention relates to a stevioside derivative prepared by biotransformation of stevioside, a preparation method and application thereof, and belongs 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 prov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H15/256C07H1/00C12P19/18A23L27/30A23L29/30A23L2/60A61K47/26
CPCY02P20/582C07H15/256A23L2/60A23V2002/00A61K47/26C07H1/00C12P19/18A23V2200/30A23V2250/262A23V2250/60
Inventor 朱惠霖丁雪峰张永正
Owner NANJING NUOYUN BIOLOGICAL TECH CO LTD
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