US 11,653,679 B2
High-purity steviol glycosides
Avetik Markosyan, Yerevan (AM); Indra Prakash, Alpharetta, GA (US); Cyrille Jarrin, Muret (FR); Aurelien Badie, Labege (FR); Robert Ter Halle, Baziege (FR); Cynthia Curran, Atlanta, GA (US); and Daniel Auriol, Roques (FR)
Assigned to PURECIRCLE SDN BHD, Kuala Lumpur (MY)
Appl. No. 15/512,711
Filed by PureCircle Sdn Bhd, Kuala Lumpur (MY)
PCT Filed Aug. 21, 2015, PCT No. PCT/US2015/046354
§ 371(c)(1), (2) Date Sep. 27, 2017,
PCT Pub. No. WO2016/043926, PCT Pub. Date Mar. 24, 2016.
Application 15/512,711 is a continuation in part of application No. PCT/US2015/045906, filed on Aug. 19, 2015.
Claims priority of provisional application 62/061,359, filed on Oct. 8, 2014.
Claims priority of provisional application 62/062,288, filed on Oct. 10, 2014.
Claims priority of provisional application 62/064,630, filed on Oct. 16, 2014.
Claims priority of provisional application 62/082,446, filed on Nov. 20, 2014.
Claims priority of provisional application 62/097,387, filed on Dec. 29, 2014.
Claims priority of provisional application 62/185,964, filed on Jun. 29, 2015.
Claims priority of provisional application 62/118,132, filed on Feb. 19, 2015.
Claims priority of provisional application 62/052,544, filed on Sep. 19, 2014.
Prior Publication US 2017/0303565 A1, Oct. 26, 2017
Int. Cl. A23L 2/60 (2006.01); A23L 27/30 (2016.01); C07H 1/06 (2006.01); C07H 15/256 (2006.01); C12P 19/18 (2006.01); C12P 19/56 (2006.01)
CPC A23L 2/60 (2013.01) [A23L 27/36 (2016.08); C07H 1/06 (2013.01); C07H 15/256 (2013.01); C12P 19/18 (2013.01); C12P 19/56 (2013.01); C12Y 204/01 (2013.01); C12Y 302/01021 (2013.01); A23V 2002/00 (2013.01); Y02P 20/582 (2015.11)] 5 Claims
 
1. A method for producing a highly purified target steviol glycoside composition, comprising the steps of:
(a) providing a starting composition comprising steviol glycosides;
(b) providing a recombinant microorganism E. coli, the recombinant microorganism 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 (“UGT76G1”), an amino acid sequence having greater than 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 (“UGTSL2”) and an amino acid sequence having greater than 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 recombinant microorganism and the enzyme with β-glucosidase activity with a medium comprising the starting composition to produce a composition comprising a target steviol glycoside, wherein the target steviol glycoside is selected from the group consisting of rebaudioside A (“reb A”), rebaudioside D (“reb D”), rebaudioside D2 (“reb D2”), rebaudioside M (“reb M’), rebaudioside M2 (“reb M2”), rebaudioside I (“reb”’), and combinations thereof; and
(d) separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition.