Process for producing high-purity rubusoside

1. A process for preparing Rubusoside from steviol glycosides of Stevia rebaudiana comprising the steps of:

A) providing a solution comprising Stevioside;

B) adding 2000 fungal β-glucanase (FBG) units of an enzyme composition having fungal β-glucanase activity to the solution comprising Stevioside to obtain a reaction mixture; and

C) incubating the reaction mixture at 28-50° C. for 12-48 hours to facilitate transformation of Stevioside to Rubusoside to obtain a reaction mixture containing Rubusoside.

2. The process of claim 1 wherein in step A) the concentration of Stevioside is 1-50% (wt/vol).

3. The process of claim 1 wherein in step C) the molar yield of Rubusoside from Stevioside is 1-100%.

4. The process of claim 1 further comprising:

D) at least one purification technique employed on the reaction mixture containing Rubusoside, selected from the group comprising ion exchange treatment, macroporous adsorbent treatment, membrane process, chromatographic separation, and crystallization from various solvent systems to obtain a purified Rubusoside.

5. The process of claim 1 further comprising:

D) recovery and purification of Rubusoside comprising the steps of:

a) providing the reaction mixture containing Rubusoside;

b) contacting the reaction mixture containing Rubusoside with macroporous adsorbent resin;

c) eluting the adsorbed Rubusoside with aqueous alcohol to obtain an eluate;

d) removing the alcohol from the eluate and drying the eluate to yield the purified Rubusoside.

6. The process of claim 1 further comprising:

D) recovery and purification of Rubusoside comprising the steps of:

a) providing a dried reaction mixture containing Rubusoside;

b) dissolving the dried reaction mixture in a first aqueous alcoholic solution to obtain a first mixture;

c) inducing crystallization in the first mixture by temperature gradient treatment;

d) filtering the first mixture to obtain a first precipitate and a first filtrate;

e) dissolving the first precipitate in a second aqueous alcoholic solution to obtain a second mixture;

f) adding activated carbon to the second mixture and filtering it to obtain a decolorized second mixture;

g) inducing crystallization in the decolorized second mixture by temperature gradient treatment;

h) filtering the decolorized second mixture to obtain a second precipitate and a second filtrate;

i) suspending the second precipitate in a third aqueous alcoholic solution to obtain a third mixture;

j) filtering the third mixture to obtain a third precipitate and a third filtrate; and

k) a drying the third precipitate to yield the purified Rubusoside.

7. The process of claim 6 wherein the first aqueous alcoholic solution in step b) is a methanol-water solution, with 75-99% methanol.

8. The process of claim 6 wherein the second aqueous alcoholic solution in step e) is a methanol-water solution with 70-90% methanol.

9. The process of claim 6 wherein the third aqueous alcoholic solution in step i) is a methanol-water solution with 90-99% methanol.

10. The process of claim 6 wherein steps c) and g) inducing crystallization comprises increasing the mixture temperature to 65-70° C. then gradually cooling it to 0-40° C., at a rate of 1-20° C. per hour with continuous mild agitation.

11. The process of claim 6 wherein the aqueous alcoholic solution comprises one or more organic solvents selected from the group consisting of methanol, ethanol, 1-propanol, and isopropanol.

12. The process of claim 1 , wherein the purified Rubusoside has a purity greater than 95% on a dry basis.

13. The process of claim 1 , wherein the purified Rubusoside has a purity greater than 98% on a dry basis.