Hydrolysis of steviol glycosides by beta-glucosidase

Patent metadata

Field	Value
Publication number	US-11414689-B2
Application number	US-201916725242-A
Country	US
Kind code	B2
Filing date	Dec 23, 2019
Priority date	Jun 30, 2017
Publication date	Aug 16, 2022
Grant date	Aug 16, 2022

Abstract

The present disclosure relates to the use of beta-glucosidase to enhance the production efficiency of desired steviol glycosides, such as rebaudioside M (reb M).

First claim

What is claimed is:

1. A method of altering the glycosylation of a steviol glycoside, said method comprising:
(a) exposing a first steviol glycoside to a Pichia sp. beta-glucosidase for sufficient time to generate a second steviol glycoside through the removal of at least one glucosyl group at the C19 position from said first steviol glycoside; and
(b) collecting said second steviol glycoside.

2. The method of claim 1 , wherein said first steviol glycoside is rubusoside and a glucosyl group is removed from the C19 position of said rubusoside to produce steviol-13-glucoside.

3. The method of claim 1 , wherein said first steviol glycoside is stevioside and a glucosyl group is removed from the C19 position of said stevioside to produce steviolbioside.

4. The method of claim 1 , wherein said first steviol glycoside is Reb E and a glucosyl group is removed from the C19 position of said Reb E to produce stevioside.

5. The method of claim 1 , wherein said first steviol glycoside is Reb I and a glucosyl group is removed from the C19 position of said Reb I to produce Reb A.

6. The method of claim 1 , wherein said first steviol glycoside is Reb A and a glucosyl group is removed from the C19 position of said Reb A to produce Reb B.

7. The method of claim 5 , wherein further a glucosyl group is removed from the C19 position of said Reb A to produce Reb B.

8. The method of claim 1 , wherein said first steviol glycoside is Reb D and two glucosyl groups are removed from the C19 position of said Reb D to produce Reb B.

9. The method of claim 1 , wherein said first steviol glycoside is Reb G and a glucosyl group at the C19 position and a glucosyl group at the C13 position are removed from said Reb G to produce steviol-13-glucoside.

10. The method of claim 9 , wherein further a glucosyl group is removed from the C13 position of said steviol-13-glucoside to produce steviol.

11. The method of claim 1 , wherein said second steviol glycoside is Reb B.

12. The method of claim 1 , wherein said first steviol glycoside Reb A.

13. The method of claim 1 , further comprising the use of beta-galactosidase or pectinase enzymes to increase the speed of enzymatic hydrolysis.

14. The method of claim 1 , wherein the beta-glucosidase has an amino acid sequence that has at least 90% identity to SEQ ID NO:1.

15. The method of claim 1 , wherein the beta-glucosidase has an amino acid sequence that is at least 95% identical to SEQ ID NO:3.

16. The method of claim 1 , wherein exposing said first steviol glycoside to the beta-glucosidase comprises exposing said first steviol glycoside to disrupted Pichia cells thereby releasing said Pichia sp. beta-glucosidase from the Pichia cells.

17. The method of claim 2 , wherein further a glucosyl group is removed from the C13 position of said steviol-13-glucoside to produce steviol.

18. The method of claim 4 , wherein further a glucosyl group is removed from the C19 position of said stevioside to produce steviolbioside.

Assignees

• Conagen Inc

Inventors

• Mao Guohong
• Vick Jacob Edward
• Batten Michael
• Yu Oliver

Classifications

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12P19/56 Primary

  having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin · CPC title

• A23L27/30

  Artificial sweetening agents · CPC title

• C07H15/256

  Polyterpene radicals · CPC title

• C12Y302/01023

  Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase · CPC title