A method for enzymatic variable temperature high throughput preparation of glucosyl steviol glycosides

A technology based on stevioside and steviol glycoside, which is applied in the field of sweetener biosynthesis, can solve the problems of no rules to follow, no universal significance, and different effective stabilizers, so as to improve the quality of sweetness and ensure high efficiency and utilization, sweetness and mouthfeel

Active Publication Date: 2020-08-04
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] By comparing and summarizing the literature, it can be known that salt bridges, pH and organic solvents can change the activity and thermal stability of enzymes, but there is no rule to follow, and there is no universal significance—even the same enzyme in different The effective stabilizer in the reaction system is also different

Method used

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  • A method for enzymatic variable temperature high throughput preparation of glucosyl steviol glycosides
  • A method for enzymatic variable temperature high throughput preparation of glucosyl steviol glycosides
  • A method for enzymatic variable temperature high throughput preparation of glucosyl steviol glycosides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Preparation of cyclodextrin glucosyltransferase (cyclodextrin glucosyltransferase)

[0036] Geobacillus sp. strain (Geobacillus thermoglucosidasius, ATCC 43742) was used as the production strain, and the inoculation amount was 8% and then batch fermentation was carried out. The medium composition is: glucose 0.8%, lactose 0.05%, peptone 1.2%, yeast extract 2.4%, K 2 HPO 4 0.3%, KH 2 PO 4 0.98%, CaCl 2 0.28%. When the dissolved oxygen rises to 85%, start to feed the feed solution. The temperature is controlled at 33-37°C during the growth stage of the bacteria, and the dissolved oxygen is maintained at 25%-30%; 600 When it reaches 25, add 1% (mass volume fraction) glycine; when the bacteria OD 600 When it reaches 50, reduce the temperature to 23-27℃ and continuously add 0.2-0.4g·l -1 ·H -1 Lactose, while supplementing the feed liquid with a decreasing gradient; after 30 hours of fermentation, the fermentation was terminated, and the bacteria were removed by centrif...

Embodiment 2

[0037] Example 2 Preparation of glucosyl steviol glycosides with tapioca dextrin and rebaudioside A as raw materials

[0038] Add 100 kg of water into the jacketed reactor, and after heating to 85° C., 15 kg of tapioca dextrin and 15 kg of rebaudioside A (HPLC content 98%) are added successively, and stirred to dissolve. After being fully dissolved, quickly cool to 65°C, and add 5 mL of 1% calcium chloride and barium chloride (mass ratio 0.2:1) aqueous solution, 4 g of glycerol and 250 kU of the ring obtained in Example 1 into the jacketed reactor under stirring. A solution of dextrin glucosyltransferase. Reacted at 65°C for 3 hours, then heated to 75°C for 24 hours, and then heated to 80°C for 10 hours to complete the reaction. The product is directly spray-dried to obtain glucosyl steviol glycoside with a moisture content of 2.1%. The product can be directly used in products that do not require a total glycoside content of not less than 95% without decolorization. The content...

Embodiment 3

[0045] Example 3 Preparation of glucosyl steviol glycosides with β-cyclodextrin and rebaudioside A as raw materials

[0046] Add 100 kg of water into the jacketed reactor, and after heating to 82° C., add 10 kg of β-cyclodextrin and 25 kg of rebaudioside A (HPLC content 97%), and stir to dissolve. After being fully dissolved, quickly cool to 60°C, add 10mL of 1% calcium chloride and barium chloride (mass ratio 0.5:1) aqueous solution, 2g glycerol, 2g sorbitol and 1000kU into the jacketed reactor under stirring. 1 The resulting cyclodextrin glucosyltransferase solution. The reaction was carried out at 60°C for 3 hours, then the temperature was raised to 74°C for 15 hours, and then the temperature was raised to 80°C for 8 hours to complete the reaction. The product is spray-dried to obtain a glucose-based steviol glycoside with a moisture content of 2.0%. The product can be directly used in products that do not require a total glycoside content of not less than 95% without decolor...

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Abstract

The present invention relates to the technical field of sweetener biosynthesis, and provides a method for preparing glucose-based stevioside in enzymatic variable temperature and high throughput. According to the present invention, cyclodextrin glucosyltransferase derived from Geobacillus sp. is used as a catalyst, stevioside is used as a glycosyl receptor, dextrin or oligosaccharide is used as a glycosyl donor, and a calcium barium ion salt bridge is used as a main stabilizer, and glycerol is combined with to adjust the conformation and binding domain opening degree of enzyme, the variable temperature utilizes the transesterification and hydrolysis activity of amylase by stages to prepare the glucose-based stevioside in enzymatic variable temperature and high throughput. The technology disclosed by the present invention can improve the utilization rate of the enzyme, and obtain the glucose-based stevioside with excellent sweetness and good taste.

Description

Technical field [0001] The invention relates to an enzymatically variable temperature high-throughput method for preparing glucosyl steviol glycosides, belonging to the technical field of sweetener biosynthesis. Background technique [0002] Steviol glycosides are the general term for glycoside molecules in stevia extracts; they are composed of sweet hydrophilic sugar groups and hydrophobic steviol aglycones, and about 40 derivatives have been identified. Among them, Rebaudioside A (RA), Rebaudioside D and Rebaudioside M have better taste and mouthfeel, while Stevioside (St), Rebaudioside C and other low molecular weight steviol glycosides have certain It has a slight bitterness or licorice aftertaste, which reduces its quality as a natural sweetener. Steviol has a very poor taste and has a severe bitter aftertaste, which gives steviol glycosides a slightly bitter taste. Therefore, in addition to the bitterness impurities that have an impact on the taste and quality of steviol ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/56C12P19/18
CPCC12P19/18C12P19/56
Inventor 夏咏梅朱理平方云刘湘王海军
Owner JIANGNAN UNIV
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