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Preparing method for sweetening agent

A compound, glucose-based technology, applied in the field of sweetener preparation, can solve the problems of reduced sweetness, non-specific positions of enzymatically transferred glycosyl groups, impure products, etc., and achieves low production cost, high product purity, and easy purification. Effect

Inactive Publication Date: 2016-07-27
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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  • Preparing method for sweetening agent
  • Preparing method for sweetening agent
  • Preparing method for sweetening agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Construction and induced expression of recombinant Escherichia coli

[0025] 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:

[0026] 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.

[0027] 2) Transform the recombinant plasmid into Escherichia coli to obtain the engineering bacterium J303 containing the target gene.

[0028] 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

[0030] Preparation of Compound (Ⅲ) Using Compound (I) as Substrate

[0031]

[0032] Take 330 mg of wet bacteria M30, 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 crude enzyme solution, and add Tris-HCl buffer solution (pH8.0) to the system of 1.85ml, start the reaction. Shake at 150 rpm for 30 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 initial purification on the sample, and use LC-MS method to detect the purity of the purified compound (Ⅲ).

Embodiment 3

[0034] Determination of Optimum Metal Ion Concentration

[0035] Get the bacterium M30330mg 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, 1, and 0.286 g / L, respectively. After 30 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 (Ⅲ) is the highest. ·

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Abstract

The invention relates to a preparing method for a sweetening agent and belongs to the technical field of food chemistry.The preparing method comprises the steps that a stevioside derivative compound (I) serves as a substrate, and the substrate reacts under the existence of a glucosyl donor and under the catalytic action of UDP-glucosyltransferase to generate a stevioside derivative compound (III).The production process is environmentally friendly and safe, the production cost is low, the production cycle is short, and thus the competitive power of the product is greatly improved.The preparing method is easy and convenient to implement and high in conversion rate, purification is easy, the obtained product is high in purity and can be applied to the food and drink industry, and thus significant application value is achieved.

Description

technical field [0001] The invention relates to a preparation method of a sweetener, which 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 proved that stevioside is beneficial to the regulation of blood sugar and blood ...

Claims

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Application Information

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IPC IPC(8): C12P19/56
CPCC12P19/56
Inventor 朱惠霖丁雪峰张永正
Owner NANJING NUOYUN BIOLOGICAL TECH CO LTD
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