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High-purity steviol glycosides

A kind of steviol, highly advanced technology, applied in confectionery, chewing gum, sugar derivatives, etc., can solve problems such as unsuitable for commercial use

Pending Publication Date: 2021-02-12
PURECIRCLE USA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although methods are known for the preparation of steviol glycosides from Stevia rebaudiana, most of these methods are not suitable for commercial use

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0535] Protein sequences of engineered enzymes used in biocatalytic methods

[0536] SEQ ID 1:

[0537] >SuSy_At, variant PM1-54-2-E05 (engineered sucrose synthase; wild-type gene source: Arabidopsis)

[0538]

[0539] SEQ ID 2:

[0540] > UGTS12 variant 0234 (engineered glucosyltransferase; wild-type gene source: tomato)

[0541]

[0542] SEQ ID 3:

[0543] >UGT76G1 variant 0042 (engineered glucosyltransferase; wild-type gene source: Stevia rebaudiana)

[0544]

[0545] SEQ ID 4:

[0546] >UDP-glycosyltransferase 91C1 (UGlyT91C1; wild-type gene source: japonica rice; NCBI reference sequence: XP_015629141.1)

[0547]

Embodiment 2

[0549] Expression and formulation of the SuSy At variant of SEQ ID 1

[0550] The gene encoding the SuSy_At variant of SEQ ID 1 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0551] In ZYM505 medium supplemented with kanamycin (50mg / l) at 37°C (F.William Studier, Protein Expression and Purification ("Protein Expression and Purification"), Volume 41, 2005, No. 207-234 page) to culture cells. The expression of the gene was induced by IPTG (0.2 mM) in the log phase, and the expression was performed at 30° C. and 200 rpm for 16-18 hours.

[0552] Cells were harvested by centrifugation (3220×g, 20min, 4°C) and washed with cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl 2 , DNA nuclease 20U / mL, lysozyme 0.5mg / mL) resuspended cells, reaching 200 optical density (measured at 600nm (OD 600 )). Cells were then disrupted by sonication and the crude extract was sep...

Embodiment 3

[0555] Expression and formulation of the UGTS12 variant of SEQ ID 2

[0556] The gene encoding the UGTS12 variant of SEQ ID 2 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0557] In ZYM505 medium supplemented with kanamycin (50mg / l) at 37°C (F.William Studier, Protein Expression and Purification ("Protein Expression and Purification"), Volume 41, 2005, No. 207-234 page) to culture cells. The expression of the gene was induced by IPTG (0.1 mM) in the log phase, and the expression was performed at 30° C. and 200 rpm for 16-18 hours.

[0558] Cells were harvested by centrifugation (3220×g, 20min, 4°C) and washed with cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl 2 , DNA nuclease 20U / mL, lysozyme 0.5mg / mL) resuspended cells, reaching 200 optical density (measured at 600nm (OD 600 )). Cells were then disrupted by sonication and the crude extract was separ...

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Abstract

Methods of preparing highly purified steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, steviolbioside E, rubusoside, dulcoside A, dulcoside C, dulcoside D, stevioside A, stevioside B, stevioside C, stevioside G, stevioside H rebaudioside B2, rebaudioside A4, rebaudioside C, rebaudioside C3, rebaudioside C4,rebaudioside C5, rebaudioside C6, rebaudioside E3, rebaudioside E4, rebaudioside E5, rebaudioside E6, rebaudioside E7, rebaudioside D5, rebaudioside D6, rebaudioside D7, rebaudioside D8, rebaudiosideH2, rebaudioside H3, rebaudioside H4, rebaudioside H5, rebaudioside H6, rebaudioside K, rebaudioside N2, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside M3 and / or rebaudioside 04 are described. The methods include utilizing enzyme preparations and recombinant microorganisms for converting various staring compositions to target steviol glycosides. The highly purified steviol glycosides are useful as non-caloric sweetener, flavor enhancer, sweetness enhancer, and foaming suppressor in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

Description

technical field [0001] The present invention relates to methods for preparing steviol glycoside-containing compositions, including highly purified steviol glycoside compositions. [0002] sequence listing [0003] The text file entitled "PC_78PROV.txt," created on June 8, 2018, having 19 kilobytes of data and filed concurrently herewith, is hereby incorporated by reference into this application in its entirety. Background technique [0004] High intensity sweeteners have a sweetness level many times greater than that of sucrose. They are essentially non-caloric and are commonly used in diets and low-calorie products, including food and beverages. High-intensity sweeteners do not trigger a glycemic response, making them suitable for use in products aimed at diabetics and others interested in controlling their carbohydrate intake. [0005] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23L27/30A23L2/60A23G4/10A23C9/13A24B15/40C07H15/256C12P19/56
CPCC07H1/00A23G4/10A23L2/60A23G9/32A23G9/34A23G4/06A23G3/38A23G3/42A23G3/36A23G1/32A23G1/40C12P19/56C07H15/256A23L33/21A23L27/36A23V2002/00A23V2200/322A23V2250/258C12N9/1051C12R2001/19
Inventor 阿韦季克·马尔科西亚沙拉瓦南·A·L·拉曼达赫穆罕默德·阿夫扎尔本哈西姆克里斯蒂娜·查汗海尔·尼扎姆本纳威周绍银
Owner PURECIRCLE USA
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