High-purity steviol glycosides
Methods of preparing highly purified steviol glycosides, particularly rebaudiosides A, D and M are described. The methods include utilizing recombinant microorganisms for converting various staring compositions to target steviol glycosides. In addition, novel steviol glycosides reb D2, reb M2, and reb I are disclosed, as are methods of preparing the same. The highly purified rebaudiosides are useful as non-caloric sweetener in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.
1 . A method for producing a highly purified rebaudioside M composition, comprising the steps of:
(a) providing a starting composition comprising an organic compound with at least one carbon atom, wherein said starting composition is selected from the group consisting of 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, rebaudioside H2, rebaudioside H3, rebaudioside H4, rebaudioside H5, rebaudioside H6, rebaudioside K, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside M3, polyols, and combinations thereof;
(b) providing a biocatalyst, the biocatalyst containing uridine diphosphate glycosyltransferases (UDP-glycosyltransferases), wherein the UDP-glycosyltransferase comprises an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 11 uridine diphosphate-glycosyltransferase 76G1 (“UGT76G1”), an amino acid sequence having at least 95% amino-acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 11, the amino acid sequence set forth in SEQ ID NO: 9 uridine diphosphate-glycosyltransferase of Solanum lycoperiscum origin (“UGTSL2”) and an amino acid sequence having at least 95% amino-acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 9, and further providing an enzyme with β-glucosidase activity from Trichoderma reesei or Aspergillus niger;
(c) contacting the UDP-glycosyltransferases and the enzyme with β-glucosidase activity with a medium comprising the starting composition to produce a composition comprising at least rebaudioside M; and
(d) separating rebaudioside M from the medium to provide a highly purified rebaudioside M composition.
2 . The method of claim 1 , wherein rebaudioside M is separated from the medium using crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.
3 . The method of claim 1 , wherein the highly purified rebaudioside M composition comprises rebaudioside M in an amount of at least 95% by weight on a dry weight basis.
4 . The method of claim 1 , further comprising providing an enzyme with β-glucosidase activity for hydrolysis of rebaudioside D2 or rebaudioside M2.
5 . A highly purified rebaudioside M composition prepared according to the method of claim 1 , (i) comprising the rebaudioside M content in an amount of at least 95% by weight on a dry weight basis, or (ii) wherein the rebaudioside M is polymorphic.
6 . A consumable product comprising the highly purified rebaudioside M composition of claim 1 , wherein the product is selected from the group consisting of a food, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition, and a cosmetic composition.