U.S. patent application number 15/649194 was filed with the patent office on 2018-01-04 for method for the enrichment of rebaudioside b and/or rebaudioside d in stevia-derived glycoside compositions using adsorb-desorb chromatography with a macroporous neutral adsorbent resin.
This patent application is currently assigned to Cargill, Incorporated. The applicant listed for this patent is Cargill, Incorporated. Invention is credited to Robert Joshua Brower, III, Ting Liu Carlson, Aron Broman Erickson, Jenna Helgeson, Andrew Keith Ohmes, Troy Allen Rhonemus, Christopher Austin Tyler, Tongeu Vang.
Application Number | 20180002362 15/649194 |
Document ID | / |
Family ID | 46084425 |
Filed Date | 2018-01-04 |
United States Patent
Application |
20180002362 |
Kind Code |
A1 |
Brower, III; Robert Joshua ;
et al. |
January 4, 2018 |
METHOD FOR THE ENRICHMENT OF REBAUDIOSIDE B AND/OR REBAUDIOSIDE D
IN STEVIA-DERIVED GLYCOSIDE COMPOSITIONS USING ADSORB-DESORB
CHROMATOGRAPHY WITH A MACROPOROUS NEUTRAL ADSORBENT RESIN
Abstract
The invention relates to the use of adsorb/desorb chromatography
to prepare enriched compositions comprising rebaudioside B and/or
rebaudioside D. Compositions with enriched rebaudioside-B and/or
rebaudioside-D components may be prepared from Stevia-derived
glycoside compositions using an adsorb-desorb chromatography
process where the stationary phase of the chromatography bed
comprises a macroporous neutral adsorbent resin.
Inventors: |
Brower, III; Robert Joshua;
(Englewood, OH) ; Carlson; Ting Liu; (Marietta,
SC) ; Erickson; Aron Broman; (Albertville, MN)
; Helgeson; Jenna; (Otsego, MN) ; Ohmes; Andrew
Keith; (Jordan, MN) ; Rhonemus; Troy Allen;
(Plymouth, MN) ; Tyler; Christopher Austin;
(Minnetonka, MN) ; Vang; Tongeu; (Minnetonka,
MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cargill, Incorporated |
Wayzata |
MN |
US |
|
|
Assignee: |
Cargill, Incorporated
Wayzata
MN
|
Family ID: |
46084425 |
Appl. No.: |
15/649194 |
Filed: |
July 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13885113 |
Apr 17, 2014 |
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PCT/US2011/061386 |
Nov 18, 2011 |
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15649194 |
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61415548 |
Nov 19, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 15/256 20130101;
C07H 1/06 20130101; C07H 15/24 20130101; C07H 1/08 20130101 |
International
Class: |
C07H 1/06 20060101
C07H001/06; C07H 1/08 20060101 C07H001/08; C07H 15/24 20060101
C07H015/24; C07H 15/256 20060101 C07H015/256 |
Claims
1. A method of preparing an enriched composition comprising
rebaudioside B and rebaudioside D, the method comprising the steps
of: (A) contacting a macroporous neutral adsorbent resin with a
glycoside solution, which solution has a pH from about 8 to about
11 and comprises rebaudioside B, rebaudioside D, and at least one
other steviol glycoside, so that at least a portion of glycosides
in the glycoside solution are adsorbed onto the macroporous neutral
adsorbent resin; and (B) contacting that macroporous neutral
adsorbent resin with at least one elution solvent formulated to
selectively elute rebaudioside B and rebaudioside D from the
adsorbent and comprising about 10-50% w/w ethanol and about 50-90%
w/w water, thereby eluting a composition enriched, relative to the
glycoside solution, with rebaudioside B and rebaudioside D.
2. The method according to claim 1, wherein the pH of the glycoside
solution ranges from a pH of about 8 to a pH of about 10.
3. (canceled)
4. The method according to claim 2, wherein the at least one
elution solvent comprises about 20-35% w/w ethanol and about 65-80%
w/w water.
5. (canceled)
6. The method according to claim 1 wherein the method further
comprises, after step (B), eluting at least a portion of remaining
adsorbed glycosides from the macroporous neutral adsorbent
resin.
7. The method according to claim 1, wherein the method further
comprises, after step (B), eluting at least a portion of remaining
adsorbed glycosides from the macroporous neutral adsorbent resin
with a subsequent elution solvent that comprises about 36-100% w/w
ethanol and about 0-64% w/w water, and the composition eluted with
the subsequent elution solvent is enriched, relative to the steviol
glycoside solution, with rebaudioside A.
8. (canceled)
9. The method according to claim 25, wherein the at least one
elution solvent comprises about 20-35% w/w ethanol and about 65-80%
w/w water.
10. The method according to claim 25, wherein the at least one
elution solvent comprises about 25-33% w/w ethanol and about 67-75%
w/w water.
11. (canceled)
12. The method according to claim 25, wherein the pH of the
Stevia-derived glycoside solution ranges from a pH of about 5 to a
pH of about 6.
13. (canceled)
14. The method according to claim 27, wherein the pH of the
glycoside solution ranges from a pH of about 1 to a pH of about
3.
15. The method according to claim 27, wherein the at least one
first elution solvent comprises about 40-60% w/w ethanol and about
40-60% w/w water.
16. (canceled)
17. The method according to claim 15, wherein the first eluted
composition is enriched with rebaudioside D.
18-20. (canceled)
21. The method according to claim 15, wherein the first eluted
composition is enriched with rebaudioside A.
22. The method according to claim 21, wherein the second elution
solvent comprises from about 70-100% w/w ethanol and about 0-39%
w/w water.
23. The method according to claim 1, wherein the macroporous
neutral adsorbent resin comprises a macroporous resin of divinyl
benzene.
24. An enriched composition prepared according to the method of
claim 1.
25. A method of preparing an enriched composition comprising
rebaudioside D, the method comprising the steps of: (A) contacting
a macroporous neutral adsorbent resin with a glycoside solution,
which solution has a pH from about 4 to about 6 and comprises
rebaudioside D and at least one other steviol glycoside, so that at
least a portion of glycosides in the glycoside solution are
adsorbed onto the macroporous neutral adsorbent resin; and (B)
contacting that macroporous neutral adsorbent resin with at least
one elution solvent formulated to selectively elute rebaudioside D
from the adsorbent and comprising about 10-40% w/w ethanol and
about 60-90% w/w water, thereby eluting a composition enriched with
rebaudioside D.
26. The method according to claim 25, wherein the method further
comprises, after step (B), eluting at least a portion of remaining
adsorbed glycosides from the macroporous neutral adsorbent
resin.
27. A method of preparing an enriched composition comprising
rebaudioside B, the method comprising the steps of: (A) contacting
a macroporous neutral adsorbent resin with a glycoside solution,
which solution has a pH from about 1 to about 4 and comprises
rebaudioside B and at least one of rebaudioside A and rebaudioside
D, so that at least a portion of glycosides in the glycoside
solution are adsorbed onto the macroporous neutral adsorbent resin;
(B) contacting that macroporous neutral adsorbent resin with at
least one first elution solvent formulated to selectively elute
rebaudioside A, rebaudioside D, or a mixture thereof from the
adsorbent and comprising about 10-60% w/w ethanol and from about
40-80% w/w water, thereby eluting a first eluted composition with
rebaudioside A, rebaudioside D, or a mixture thereof; and,
subsequently, (C) contacting the macroporous neutral adsorbent
resin with at least one second elution solvent comprising about
60-100% w/w ethanol and about 0-40% w/w water, thereby eluting a
second eluted composition enriched, relative to the glycoside
solution, with rebaudioside B.
28. An enriched composition prepared according to the method of
claim 25.
29. An enriched composition prepared according to the method of
claim 27.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of the U.S. Provisional
Patent Application 61/415,548, filed Nov. 19, 2010, entitled METHOD
FOR THE ENRICHMENT OF REBAUDIOSIDE B AND/OR REBADIOSIDE D IN
STEVIA-DERIVED GLYCOSIDE COMPOSITIONS USING ADSORB-DESORB
CHROMATOGRAPHY WITH A MACROPOROUS NEUTRAL ADSORBENT RESIN, which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the use of adsorb/desorb
chromatography to prepare enriched compositions comprising
rebaudioside B and/or rebaudioside D (reb-B and/or reb-D). As
described herein, it has been discovered that enriched reb-B and/or
reb-D can be prepared from Stevia-derived glycoside compositions
using an adsorb-desorb chromatography process where the stationary
phase of the chromatography bed comprises a macroporous neutral
adsorbent resin. Examples of useful resins include "SP70" and
"SP710" (from Mitsubishi) and "FPX66" (from Dow).
SUMMARY OF THE INVENTION
[0003] In some embodiments, the invention provides a method of
preparing an enriched composition comprising at least one of reb-B,
reb-D, or a mixture thereof, the method comprising the steps
of:
(A) providing a pH adjusted Stevia-derived glycoside solution
comprising: (i) at least one glycoside selected from the group of
reb-B and reb-D; (ii) at least one other Stevia-derived glycoside;
(B) providing a macroporous neutral adsorbent resin; (C) contacting
the macroporous neutral adsorbent resin with the Stevia-derived
glycoside solution so that at least a portion of glycosides in the
Stevia-derived glycosides solution are adsorbed onto the adsorbent;
(D) providing at least one elution solvent comprising an ethanol
and water mixture formulated to selectively elute reb-B, reb-D, or
a mixture thereof from the adsorbent; (E) contacting the
macroporous neutral adsorbent resin with the ethanol and water
elution solvent in order to elute a composition enriched with at
least one glycoside selected from the group of reb-B and reb-D; and
(F) optionally, eluting at least a portion of the remaining
adsorbed glycosides from the macroporous neutral adsorbent
resin.
[0004] In certain embodiments of the present invention, the pH of
the Stevia-derived glycoside solution can be an alkaline pH. In
some of these embodiments, the pH of the pH adjusted Stevia-derived
glycoside solution can range from a pH of about 8 to a pH of about
11. In other embodiments, pH of the pH adjusted Stevia-derived
glycoside solution can range from a pH of about 8 to a pH of about
10. In some of these embodiments, the at least one elution solvent
comprises from about 20-35% w/w ethanol and from about 65-80% w/w
water. In other embodiments, the at least one elution solvent
comprises from about 25-33% w/w ethanol and from about 67-75% w/w
water. In these embodiments, the composition enriched with at least
one glycoside is enriched with reb-B and reb-D. In some of these
embodiments, at least a portion of the remaining adsorbed
glycosides are eluted from the macroporous neutral adsorbent resin
with a subsequent elution solvent wherein the subsequent elution
solvent comprises from about 36-100% w/w ethanol and about 0-64%
w/w water, and the composition eluted with the subsequent elution
solvent is enriched with reb-A.
[0005] In other particular embodiments of the present invention,
the pH of the Stevia-derived glycoside solution can be a slightly
acidic pH. In these embodiments, the pH of the pH adjusted
Stevia-derived glycoside solution can range from a pH of about 4 to
a pH of about 7. In some of these embodiments, the at least one
elution solvent comprises from about 20-35% w/w ethanol and from
about 65-80% w/w water. In other embodiments, the at least one
elution solvent comprises from about 25-33% w/w ethanol and from
about 67-75% w/w water. In these embodiments, the composition
enriched with at least one glycoside is enriched with reb-D. In
some of these embodiments, at least a portion of the remaining
adsorbed glycosides are eluted from the macroporous neutral
adsorbent resin with a subsequent elution solvent wherein the
subsequent elution solvent comprises from about 36-100% w/w ethanol
and about 0-64% w/w water, and the composition eluted with the
subsequent elution solvent is enriched with reb-A and reb-B.
[0006] In yet other particular aspects of the present invention,
the pH of the Stevia-derived glycoside solution can be a very
acidic pH. In these embodiments, the pH of the pH adjusted
Stevia-derived glycoside solution can range from a pH of about 1 to
a pH of about 4. In other embodiments, the pH of the pH adjusted
Stevia-derived glycoside solution can range from a pH of about 1 to
a pH of about 3. In some of these embodiments, the at least one
elution solvent comprises from about 40-65% w/w ethanol and from
about 35-60% w/w water. In other embodiments, the at least one
elution solvent comprises from about 40-60% w/w ethanol and from
about 40-60% w/w water. In these embodiments, the composition
enriched with at least one glycoside is enriched with reb-A and
reb-D. In some of these embodiments, at least a portion of the
remaining adsorbed glycosides are eluted from the macroporous
neutral adsorbent resin with a subsequent elution solvent wherein
the subsequent elution solvent comprises from about 61-100% w/w
ethanol and about 0-39% w/w water, and the composition eluted with
the subsequent elution solvent is enriched with reb-B.
[0007] In embodiments of the present invention, the macroporous
neutral adsorbent resin comprises a macroporous resin of divinyl
benzene. The present invention is also directed to enriched
compositions prepared according to the methods described
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A and 1B illustrate the structures of "rebaudioside
B" and "rebaudioside D."
[0009] FIG. 2 is a graph of the recovery of various glycosides for
each elution of Example 1.
[0010] FIG. 3 is a graph of the glycoside profile of the samples
eluted at different ethanol concentrations in Example 2.
[0011] FIG. 4 is a graph of the concentration of glycosides eluting
from the column during loading in Example 3.
[0012] FIG. 5 is a graph of the concentration of glycosides eluting
from the column during a wash with 15% ethanol and elution with
100% ethanol in Example 3.
[0013] FIG. 6 is a graph of the concentration of glycosides during
loading, washing, and eluting in Example 4.
[0014] FIG. 7 illustrates the mass of rebaudioside A, stevioside,
rebaudioside B, and rebaudioside D collected during elution in
Example 4.
[0015] FIG. 8 is a graph of the concentration of glycosides during
loading, washing, and eluting in Example 5.
[0016] FIG. 9 illustrates the mass of rebaudioside A, stevioside,
rebaudioside B, and rebaudioside D collected during elution in
Example 5.
DETAILED DESCRIPTION
[0017] Reb-B and reb-D have desirable sensory properties. For
example, reb-B is less bitter than reb-A and has slightly less
sweetness. Pure component sensory data indicate a reduction in
bitterness in reb-B, indicating that blends of reb-B and reb-A may
have less bitterness. Reb-D has similar or more intense sweetness
as compared to reb-A with substantially reduced bitterness. It is
anticipated that blends of reb-D, reb-B and reb-A will have
improved taste because of the reduced bitterness of reb-D and
reb-B.
[0018] The present invention provides for methods of enriching
reb-B and/or reb-D in order to utilize the desirable properties in
sweetener compositions.
[0019] The method of the invention may also be used to create
enriched compositions comprising reb-A by using the process to
remove at least a portion of the reb-B and/or reb-D from a
composition comprising reb-A.
[0020] In the method of the invention, the Stevia-derived glycoside
solution may be a solution prepared from Stevia leaf extract, or
the Stevia-derived glycoside solution may be the mother liquor of a
crystallization process. In some embodiments, the Stevia-derived
glycoside solution may be the product of the enrichment process of
the invention when multiple enrichment steps are desired. Examples
of glycosides that may be present in the Stevia-derived glycoside
solution include reb-A, reb-B, reb-C, reb-D, reb-F, stevioside,
dulcoside-A, steviolbioside, and rubusoside.
[0021] In some embodiments, the Stevia-derived glycoside solution
that is used as a starting material in the method of the invention
comprises steep water produced by steeping Stevia leaf in water at
a ratio of about 10:1 water to leaf to about 100:1 water to leaf,
more typically about 20:1 to about 50:1. The steeping is typically
conducted for about 1 to about 24 hours, more typically about 2
hours. The steep water typically contains about 0.1 to about 5.0 wt
% solids, more typically about 1 to about 2 wt % solids. Steep
water may also be produced by multiple extractions of the leaf in
counter-current or co-current extraction to improve the recovery of
steviol glycosides.
[0022] The solids present in the steep water typically range from
about 15 to about 30 wt % glycosides, more typically about 20 wt %
to about 25 wt % glycosides. The glycosides present vary depending
on the variety of Stevia leaf. Typically, reb-A and stevioside
comprise about 50% to about 80% of the glycosides present, with a
ratio of reb-A to stevioside of about 4:1 to about 1:4, more
typically about 2:1 to about 1:2.
[0023] In some embodiments, the Stevia-derived glycoside solution
can be concentrated prior to adsorption. The solution can be
concentrated to 10-50 wt % solids, more typically 20-40%. (g
solids/100 g solution)
[0024] In some embodiments, the Stevia-derived glycoside solution
that is used as a starting material in the method of the invention
comprises a major amount of reb-A, and one or more impurities
selected from reb-B and reb-D. Typically, the total amount of both
reb-B and reb-D in the impure reb-A composition can vary up to
about 6 wt %. For example, in some embodiments the impure reb-A
composition comprises about 90 wt % to about 96 wt % reb-A; about 1
wt % to 4 wt % reb-B; and about 1 wt % to about 4 wt % reb-D.
[0025] In some embodiments, the Stevia-derived glycoside solution
used as a starting material in the method of the invention
comprises a partially purified extract from the Stevia leaf
comprising about 10 wt % to about 90 wt % reb-A; about 0 wt % to
about 40 wt % stevioside; about 0 wt % to about 5 wt % reb-B; and
about 0 wt % to about 5 wt % reb-D. In other embodiments, the
Stevia-derived glycoside solution comprises about 80 wt % steviol
glycosides; about 40% to about 80 wt % reb-A; about 20 to about 40
wt % stevioside; about 1 to about 5 wt % reb-B, and about 1 to
about 5 wt % reb-D.
[0026] In many embodiments, the Stevia-derived glycoside solution
comprises a solvent. Solvent compositions for the Stevia-derived
glycoside solution typically comprise water or a mixture of a lower
alcohol (e.g., a C.sub.1-C.sub.3 alcohol) and water. Examples of
lower alcohols include methanol, ethanol, and propanol (e.g.,
n-propanol and i-propanol). Mixtures of two or more alcohols and
water may also be used. In many embodiments the solvent composition
comprises water only. In other embodiments, the solvent comprises
about 5 wt % to about 30 wt % lower alcohol and about 95 wt % to
about 70 wt % water, based on the total weight of the solvent
composition. More typically, the solvent composition comprises
about 5 wt % to about 20 wt % lower alcohol and about 95 wt % to
about 80 wt % water.
[0027] The Stevia-derived glycoside solution typically comprises
about 1 wt % to about 20 wt % dissolved solids and about 99 wt % to
about 80 wt % solvent. In some embodiments, the Stevia-derived
glycoside solution comprises about 2 wt % to about 5 wt % dissolved
solids and about 98 wt % to about 95 wt % solvent.
[0028] In the method of the invention, the stationary phase
adsorbent comprises a macroporous neutral adsorbent resin. Useful
stationary phase adsorbents include macroporous resins of divinyl
benzene, such as those commercially available under the trade
designations "SEPABEADS.RTM. SP70", "SEPABEADS.RTM. SP710",
"SEPABEADS.RTM. SP825", "SEPABEADS.RTM. SP850", "DIAION.TM. HP20",
and "DIAION.TM. HP21" (from Mitsubishi) and "AMBERLITE.TM. FPX66"
(from Dow). SP70 is reported to have a surface area of 800
m.sup.2/g, a pore radius of 70 Angstroms, and a mean particle
diameter of 0.45 mm. FPX66 is described as a macroreticular
aromatic polymer having a surface area of 700 m.sup.2/g, and a
particle size of 0.6 to 0.75 mm.
[0029] In some embodiments, it is advantageous to adjust the pH of
the Stevia-derived glycoside solution to a pH ranging from a pH of
about 7 to a pH of about 11. In other embodiments, the pH of the
Stevia-derived glycoside solution can be adjusted to a pH ranging
from a pH of about 8 to a pH of about 11. In yet other embodiments,
the pH of the Stevia-derived glycoside solution can be adjusted to
a pH ranging from a pH of about 8 to a pH of about 10. In yet other
embodiments, the pH of the Stevia-derived glycoside solution can be
adjusted to pH of about 9. The pH is typically adjusted by the
addition of a base. Useful bases include sodium hydroxide,
potassium hydroxide, calcium oxide (lime), or calcium
hydroxide.
[0030] In other embodiments, the pH of the Stevia-derived glycoside
solution can be adjusted to a pH ranging from a pH of about 4 to a
pH of about 7. In yet other embodiments, the pH of the
Stevia-derived glycoside solution can be adjusted to a pH ranging
from a pH of about 5 to a pH of about 6. In yet other embodiments,
the pH of the Stevia-derived glycoside solution can be adjusted to
a pH ranging from a pH of about 1 to a pH of about 4. In yet other
embodiments, the pH of the Stevia-derived glycoside solution can be
adjusted to a pH ranging from a pH of about 1 to a pH of about 3.
In yet other embodiments, the pH of the Stevia-derived glycoside
solution can be adjusted to pH of about 2.
[0031] Adjusting the pH can be used to alter the binding
characteristics of reb-B on the adsorbent resin. For example,
increasing the pH can change reb-B from strongly binding to the
adsorbent resin to weakly binding to the adsorbent resin. Reducing
the pH can cause reb-B to strongly bind to the resin. By adjusting
the pH, the elution properties of reb-B may be altered to provide a
desired enrichment profile. A summary of the binding properties of
reb-B for various adsorbent resins is provided below.
TABLE-US-00001 Resin pH reb-B FPX66 5 Strong Binder SP710 9 Weak
Binder SP710 5 Strong Binder SP70 9 Weak Binder SP70 5 Strong
Binder
[0032] After feeding the Stevia-derived glycoside solution onto the
adsorbent, one or more elution solvents are then fed though the
adsorbent bed in order to elute one or more enriched fractions. In
many embodiments, the elution solvent is formulated to cause at
least a portion of the reb-B and/or reb-D that is adsorbed to be
released from the adsorbent and eluted from the bed. Useful elution
solvents for eluting reb-B and/or reb-D include, for example,
C.sub.1 to C.sub.3 alcohol/water solutions (e.g., ethanol/water
solutions).
[0033] The enrichment methods of the invention may be used to
prepare enriched compositions comprising reb-B, reb D, or both
reb-B and reb-D. In addition, the method may be used to remove
reb-B and/or reb-D from reb-A compositions thereby increasing the
relative purity of the reb-A. As used herein the term "enriched",
means that the amount of the enriched component is increased on a
dry solids basis as compared to the other glycosides present in the
starting composition. For example, in the enriched composition, the
amount of reb-B, reb-D, or both is increased on a dry solids basis
relative to the amount that was present in the Stevia-derived
glycoside composition that was used as the starting composition for
the enrichment process. For example, in some embodiments, the
purified reb-B and/or reb-D solution may comprise, on a glycoside
basis, about 25 to about 90 wt % reb-A, about 10 to about 50 wt %
reb-D; and 0 to about 20 wt % reb-B. In other embodiments, the
purified reb-B and/or reb-D solution comprises, on a glycoside
basis, about 25 to about 60 wt % reb-A, about 10 to about 50 wt %
reb-D, and about 5 to about 20 wt % reb-B.
[0034] In some embodiments of the invention the enrichment process
may be conducted multiple times in order to increase the final
purity of the enriched composition. For example, the enriched
composition from a first separation may be fed into a second
chromatography bed in order to further enrich the composition in
reb-B, reb-D, or mixtures thereof.
[0035] Elution solvents made up of various ratios of ethanol and
water can be used to preferentially elute compositions enriched in
reb-B, reb-D, or a combination of reb-B and reb-D. Applicants have
surprisingly discovered that, in combination with the deliberate pH
adjustment of the Stevia-derived glycoside solution, minor
modifications in the ratio of ethanol and water in elution solvents
can allow for elution of compositions enriched in reb-B, reb-D, or
a combination thereof.
[0036] Reb-B and Reb-D Enrichment
[0037] In certain embodiments, reb-B and reb-D can be
preferentially eluted. In these embodiments, the pH of the
Stevia-derived glycoside solution can be a pH ranging from a pH of
about 7 to a pH of about 11. In other embodiments, the pH of the
Stevia-derived glycoside solution can be adjusted to a pH ranging
from a pH of about 8 to a pH of about 11. In yet other embodiments,
the pH of the Stevia-derived glycoside solution can be adjusted to
a pH ranging from a pH of about 8 to a pH of about 10. In yet other
embodiments, the pH of the Stevia-derived glycoside solution can be
adjusted to a pH of about 9.
[0038] After deliberately adjusting the pH, reb-B and reb-D can be
preferentially eluted through management of the ethanol/water ratio
of the elution solvent. In an exemplary embodiment, reb-B and reb-D
can be preferentially eluted using a solvent comprising about
10-50% w/w ethanol and about 50-90% w/w water. In other
embodiments, the elution solvent comprises about 10-40% w/w ethanol
and about 60-90% w/w water. In yet other embodiments, the elution
solvent comprises about 20-35% w/w ethanol and about 65-80% w/w
water. In yet other embodiments, the elution solvent comprises
about 25-33% w/w ethanol and about 67-75% w/w water. Typically, the
weight ratio of elution solvent to glycoside solution ranges from
about 20:1 to 1:100 (parts elution solvent: parts glycoside
solution), more typically ranging from about 10:1 to 1:20 (parts
elution solvent: parts glycoside solution). The ratio of solvents
depends strongly on the concentration of the glycosides in the
glycoside containing solution.
[0039] After eluting the reb-B and reb-D enriched composition, the
remaining adsorbed glycosides can be desorbed from the resin
utilizing an appropriate subsequent elution solvent. The subsequent
elution solvent is made up of a greater percentage of ethanol than
that used to preferentially elute reb-B and reb-D. The composition
eluted with this subsequent elution solvent will be enriched in
reb-A. In some embodiments, this subsequent elution solvent
comprises from about 36-100% w/w ethanol and 0-64% w/w water. This
subsequent elution solvent could also comprise for example, about
40-80% w/w ethanol and about 20-60% water.
[0040] Reb-D Enrichment
[0041] In other certain embodiments, reb-D can be preferentially
eluted. In these embodiments, the pH of the Stevia-derived
glycoside solution can be a pH ranging from a pH of about 4 to a pH
of about 7. In other embodiments, the pH of the Stevia-derived
glycoside solution can be adjusted to a pH ranging from a pH of
about 5 to a pH of about 7. In yet other embodiments, the pH of the
Stevia-derived glycoside solution can be adjusted to a pH ranging
from a pH of about 5 to a pH of about 6. After deliberately
adjusting the pH, reb-D can be preferentially eluted through
management of the ethanol/water ratio of the elution solvent. In
some embodiments, the elution solvent comprises about 10-40% w/w
ethanol and about 60-90% w/w water. In other embodiments, the
elution solvent comprises about 20-35% w/w ethanol and about 65-80%
w/w water. In yet other embodiments, the elution solvent comprises
about 25-33% w/w ethanol and about 67-75% w/w water. After eluting
the reb-D enriched composition, the remaining adsorbed glycosides
can be desorbed from the resin utilizing an appropriate subsequent
elution solvent. The subsequent elution solvent is made up of a
greater percentage of ethanol than that used to preferentially
elute reb-D. The composition eluted with this subsequent elution
solvent will be enriched in reb-A and reb-B. In some embodiments,
this subsequent elution solvent comprises from about 36-100% w/w
ethanol and 0-64% w/w water. This subsequent elution solvent could
also comprise for example, about 40-80% w/w ethanol and about
20-60% water.
[0042] Reb-B Enrichment
[0043] In certain other embodiments, an enriched reb-B composition
can be obtained. These embodiments can utilize a series of
elutions. In these embodiments, the pH of the Stevia-derived
glycoside solution can be a pH ranging from a pH of about 1 to a pH
of about 4. In other embodiments, the pH of the Stevia-derived
glycoside solution can be adjusted to a pH ranging from a pH of
about 1 to a pH of about 3. In yet other embodiments, the pH of the
Stevia-derived glycoside solution can be adjusted to a pH of about
2. In these embodiments, the pH of the Stevia-derived glycoside
solution is selected to cause reb-B to bind very strongly to the
resin.
[0044] In these embodiments, the bulk of Stevia-based glycosides
are first eluted. In some embodiments, the elution solvent
comprises about 30-70% w/w ethanol and about 30-70% w/w water. In
other embodiments, the elution solvent comprises about 40-60% w/w
ethanol and about 40-60% w/w water. In yet other embodiments, the
elution solvent comprises about 45-55% w/w ethanol and about 45-55%
w/w water. This eluted composition is enriched in reb-A and reb-D.
After eluting this reb-A and reb-D enriched composition, a reb-B
enriched composition can next be desorbed from the resin utilizing
an appropriate subsequent elution solvent. The subsequent elution
solvent is made up of a greater percentage of ethanol than that
used to elute the reb-A and reb-D enriched composition. In some
embodiments, the subsequent elution solvent comprises about 60-100%
w/w ethanol and 0-40% w/w water. In some embodiments, the
subsequent elution solvent comprises about 70-100% w/w ethanol and
0-30% w/w water. The composition eluted with this subsequent
elution solvent will be enriched in and reb-B.
[0045] In embodiments of the present invention where the pH is
adjusted so that reb-B is very strongly bound to the resin,
utilization of multiple elutions along with the judicious
management of the ethanol/water ratio in each elution can allow for
compositions enriched in reb-D, reb-A, and reb-B to be eluted
separately. In this embodiment, reb-D can be eluted from the column
first. In some embodiments, the elution solvent to elute the reb-D
enriched composition comprises about 10-40% w/w ethanol and about
60-90% w/w water. In other embodiments, the elution solvent to
elute the reb-D enriched composition comprises about 20-35% w/w
ethanol and about 65-80% w/w water. In yet other embodiments, the
elution solvent to elute the reb-D enriched composition comprises
about 25-33% w/w ethanol and about 67-75% w/w water.
[0046] After eluting the reb-D enriched composition, a reb-A
enriched composition can next be eluted utilizing an appropriate
subsequent elution solvent. In some embodiments, this subsequent
elution solvent comprises about 40-60% w/w ethanol and about 40-60%
w/w water. In other embodiments, this subsequent elution solvent
comprises about 45-55% w/w ethanol and about 45-55% w/w water.
[0047] After eluting the reb-A enriched composition, a reb-B
enriched composition can be eluted utilizing an appropriate further
subsequent elution solvent. In some embodiments, this further
subsequent elution solvent comprises about 60-100% w/w ethanol and
about 0-40% w/w water. In other embodiments, this further
subsequent elution solvent comprises about 65-90% w/w ethanol and
about 10-35% w/w water. In yet other embodiments, this further
subsequent elution solvent comprises about 70-85% w/w ethanol and
about 15-30% w/w water.
[0048] The reb-D rich fraction obtained from the present invention
can, on a glycoside basis, contain about 10 wt % to about 50 wt %
reb-D and essentially no reb-B. The reb-B rich fraction obtained
from the present invention can, on a glycoside basis, contain about
1 wt % to about 5 wt % reb-B, and essentially no reb-D.
[0049] The invention will now be described with reference to the
following non-limiting examples.
EXAMPLES
[0050] The following abbreviations are used in the Examples.
RD or Reb-D: rebaudioside-D RA or Reb-A: rebaudioside-A Sty:
stevioside RC or Reb-C: rebaudioside-C RF or Reb-F: rebaudioside-F
RB or Reb-B: rebaudioside-B SB: steviolbioside 208: isomer of
rebaudioside-A.
SP710: Diaion Sepabead SP710
[0051] EtOH: ethanol
Example 1
[0052] Stevia leaf was extracted with room temperature water and an
extract was obtained with 0.58% dissolved solids and a pH of 5.8. A
volume of 370 ml of the steepwater was passed through a 25-ml
column packed with SP710 resin held at 50.degree. C. The column was
further washed with 148 ml of water. The material passing through
during loading and the initial water wash were pooled for analysis
and denoted as the pass fraction. The column was sequentially
eluted with 50 ml of increasing concentrations of ethanol from 20
to 60% (w/w) ethanol and 40 to 80% (w/w) water and rinsed with 35
ml of water in between each ethanol elution. Each of the water
rinses and the ethanol eluents at each of the various
concentrations were collected, mixed and dried for analysis. FIG. 2
is the recovery of various glycosides for each elution.
Approximately 50% of the mass was not adsorbed onto the column and
of the adsorbed mass, most of it was eluted from the column using a
concentration of 40% (w/w) ethanol and 60% (w/w) water. Reb-D
elutes off the column prior to reb-A and stevioside, with the
highest concentration at about 30% ethanol.
[0053] The 30% (w/w) ethanol and 70% (w/w) water eluted fraction is
enriched with reb-D as compared to the starting leaf extract.
TABLE-US-00002 % glycoside RA Stv RC RB SB RD Feed 36.10 55.32 7.11
0.00 0.00 1.47 30% 32.56 40.65 3.65 0.08 0.57 22.49 ethanol
[0054] Applicants note that while this and other examples are
conducted at elevated temperatures, the methods described herein
can be conducted at ambient temperatures.
Example 2
[0055] Stevia leaf was extracted with room temperature water,
treated with ferric chloride and lime, and filtered to generate a
steepwater containing 0.79% solids and had a pH of 9.5. A volume of
400 ml was passed through a 25-ml SP710 column held at 50.degree.
C. at 1.6 bed volume/hour (BV/hr). The column was washed with 75 ml
of water and combined with the material that passed through the
column during the initial loading, and denoted as the pass
fraction. Steviol glycosides were eluted off the column with 50 ml
of increasing concentrations of ethanol and water from 20 to 70%
(w/w) ethanol and 30 to 80% (w/w) water. 50 ml of water was used to
rinse off the column in between each ethanol elution and the water
rinse was combined with the ethanol fraction at each concentration
before drying. FIG. 3 is the glycoside profile of the samples
eluted at different ethanol concentrations. At this pH, reb-D,
reb-B and steviolbioside elute off the column primarily at 30%
(w/w) ethanol and 70% (w/w) water before the bulk of the reb-A and
stevioside.
[0056] The 30% (w/w) ethanol and 70% (w/w) water eluted fraction is
enriched with reb-D, reb-B, and steviolbioside as compared to
feed.
TABLE-US-00003 % glycoside RA Stv RC RD RB SB Fe/lime 38.9 52.7 7.4
0.41 0.32 0.31 30% ethanol 31.7 25.3 1.5 31.6 4.2 5.7 eluent
Example 3
[0057] A 150 ml glass column filled with SP70 resin was
equilibrated with two bed volumes of a 15 wt % ethanol/85 wt %
water solution. A 2 wt % solution was prepared using 15 wt %
ethanol/85% water solution and a stevia extract lot containing
83.9% reb-A, 2.1% reb-D, 4.0% reb-B, and 1.6% Stevioside. The
solution was run over the column for approximately 18.5 bed
volumes, and then eluted for 2 bed volumes with a 15 wt %
ethanol/85 wt % water solution, and desorbed for 3 bed volumes with
100 wt % ethanol. Loading, elution, and desorption were conducted
at 55.degree. C. at a flow rate of 2 BV/hr. Material was collected
in about 50 ml samples, dried in a vacuum oven at 80.degree. C.,
and analyzed by HPLC to identify the concentration of glycosides in
each sample.
[0058] FIG. 4 is the concentration of glycosides eluting from the
column during loading. FIG. 5 is the concentration of glycosides
eluting from the column during wash with a 15% ethanol/85 wt %
water solution and elution with 100% ethanol. By isolating the
effluent from 10-15 BV, the material will be enriched in RD. The
pooled extract would have the following composition:
TABLE-US-00004 RebA 79.75% Stevioside 2.14% RebC 0.37% RebD 11.00%
RebB 3.52%
Example 4
[0059] A 160 ml column filled with SP70 resin was equilibrated with
water, and then loaded with 1440 ml of a water solution containing
5 g of stevia extract per 100 g, with pH adjusted to 2.0 by
addition of citric acid. The column was loaded at 2.5 BV/hr while
held at 55.degree. C. The column was then initially washed with 1.5
BV of water, and then eluted over 4 BV with a linear gradient of
ethanol, starting at 0% and ending at 100% ethanol. Finally, the
column was eluted for 0.5 BV with 100% ethanol The stevia extract
consisted of 45.6% RA, 31.8% Stev, 1.3% RF, 9.4% RC, 1.5 RD, 2.1%
RB, 0.8% Dulcoside A, and 1.3% Steviolbioside. FIG. 6 is the
concentration of glycosides during loading, washing, and eluting.
FIG. 7 shows the mass of RA, Stev, RB and RD collected during
elution. RD is primarily eluted at 30% (w/w) ethanol and 70% water;
RA and Stev at 50% (w/w) ethanol and 50% water; and RB at 70% (w/w)
ethanol and 30% water. Collecting the 30% ethanol eluant, 50%
ethanol eluant, and 70% ethanol eluants separately allows for the
separation of the extract into RD enriched, RA/Stev enriched, and
RB enriched streams. Alternatively, collecting the glycosides that
breakthrough the column during loading, from 3.5 to 5.5 BV, leads
to a product enriched in RB and RD.
Example 5
[0060] A 120 ml column filled with SP70 resin was equilibrated with
water, and then loaded with 1025 ml of a water solution containing
5 g of stevia extract per 100 g, with pH adjusted to 9.0 by
addition of potassium hydroxide. The column was loaded at 2.5 BV/hr
while held at 55.degree. C. The column was then initially washed
with 1.5 BV of water, and then eluted with 4 BV with a linear
gradient of ethanol, starting at 0% and ending at 100% ethanol.
Finally, the column was eluted with 4 BV of 100% ethanol. The
stevia extract consisted of 45.6% RA, 31.8% Stev, 1.3% RF, 9.4% RC,
1.5 RD, 2.1% RB, 0.8% Dulcoside A, and 1.3% Steviolbioside. FIG. 8
is the concentration of glycosides during loading, washing, and
eluting. FIG. 9 shows the mass of RA, Stev, RB and RD collected
during elution. RB and RD are primarily eluted at 30% (w/w) ethanol
and 70% (w/w) water; RA and Stev at 50% (w/w) ethanol and 50% (w/w)
water. Collecting the 30% ethanol eluant and 50% ethanol eluant
separately allows for the separation of the extract into RB/RD
enriched and RA/Stev enriched streams.
[0061] Other embodiments of this invention will be apparent to
those skilled in the art upon consideration of this specification
or from practice of the invention disclosed herein. Various
omissions, modifications, and changes to the principles and
embodiments described herein may be made by one skilled in the art
without departing from the true scope and spirit of the invention
which is indicated by the following claims.
* * * * *