Note: Descriptions are shown in the official language in which they were submitted.
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
MONATIN SWEETENED FOOD OR BEVERAGE WITH IMPROVED
SWEETENER PERFORMANCE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Patent Application 61/423,948,
filed
December 16, 2010, and which is hereby incorporated by reference in its
entirety.
FIELD
[0002] The present disclosure relates generally to a monatin-sweetened food or
beverage.
Aspects of the disclosure are particularly directed to a monatin-sweetened
food or a beverage
containing steviol glycosides, resulting in improved sweetener performance.
BACKGROUND
[0003] Monatin (2-hydroxy-2-(indo1-3-ylmethyl)-4-aminoglutaric acid) is a
naturally
occurring, high potency sweetener that was originally isolated from the plant
Sckrochiton
ilicifolius, found in the Transvaal Region of South Africa. Monatin has the
chemical
structure:
HO 1 0
NH2
2
3 OH
4
OH
0
4101 \
[0004] Because of various naming conventions, monatin is also known by a
number of
alternative chemical names, including but not limited to: 2-hydroxy-2-(indo1-3-
ylinethyl)-4-
aminogiutaric acid; 4-amino-2-hydroxy-2-(1H-indo1-3-ylmethyl)-pentanedioic
acid; 4-
hydroxy-4-(3-indolylmethyl)glutamic acid; and, 3-(1-amino-1,3-dicarboxy-3-
hydroxy-but-4-
ypindole.
[0005] Monatin has two chiral centers thus leading to four potential
stereoisomeric
configurations; the R,R configuration (the "R,R stereoisomer" or "R,R
monatin"); the S,S
configuration (the "S,S stereoisomer" or "S,S inonatin"); the R,S
configuration (the "R,S
1
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
stereoisomer" or "R,S monatin"); and the S,R configuration (the "S,R
stereoisomer" or "S,R
monatin").
[0006] Monatin has an excellent sweetness quality, and depending on a
particular
composition, monatin may be several hundred times sweeter than sucrose and in
some cases
thousands of times sweeter than sucrose. Monatin has four stereoisomeric
configurations
which exhibit differing levels of sweetness. The R,R
stereoisomer of monatin is about
2,000-3,000 times sweeter than sucrose by weight. Depending on the methods of
manufacture, purification, and intended uses, a monatin composition may
include a pure
stereoisomer or it may include a mixture of stereoisomers.
[0007] Monatin can be isolated from the bark of the roots of the plant
Seleroehiton
For example, the bark can be ground and extracted with water, filtered and
freeze dried to
obtain a dark brown, amorphous mass. The mass can be re-dissolved in water and
reacted
with a cation resin in the acid form, e.g., AG 50W-X8 in the HC1 form (Bio-Rad
Laboratories, Richmond, Calif.). The resin can be washed with water and the
compounds
bound to the resin eluted using an aqueous ammonia solution. The eluate can be
freeze dried
and subjected to aqueous gel filtration. See, e.g., U.S. Pat. No. 5,128,164,
which is hereby
incorporated by reference in its entirety.
Alternatively, monatin can be chemically
synthesized. See, e.g., the methods of Holzapfcl and Olivier, Synth. Commun.
23:2511
(1993); Holzapfel et al., Synth. Commun. 38:7025 (1994); U.S. Pat. No.
5,128,164; U.S. Pat.
No. 4,975,298; and U.S. Pat. No. 5,994,559, which are hereby incorporated by
reference in
their entirety.
[0008] Monatin can also be produced via enzymatic processes. Reference is made
to WO
2003/091396 A2, which discloses, inter alia, polypeptides, pathways, and
microorganisms for
in vivo and in vitro production of monatin. WO 2003/091396 A2 (see, e.g.,
Figures 1-3 and
11-13) and U.S. Patent Publication No. 2005/282260 describe the production of
monatin
from tryptophan through multi-step pathways involving biological conversions
with
polypeptides (proteins) or enzymes and are hereby incorporated by reference in
their entirety.
One pathway described involves converting tryptophan to indole-3-pyruvate ("1-
3-P"),
converting indole-3-pyruvate to 2-hydroxy 2-(indo1-3-ylmethyl)-4-keto glutaric
acid
(monatin precursor, "MP"), and converting MP to monatin, biologically, for
example, with
enzymes. This process may be used to make stereoisomer enriched monatin
compositions
including a monatin composition that comprises greater than 90% R,R monatin.
Stevia is a
natural sweetener, derived from the South American perennial Stevia
rebaudiana. The
2
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
sweetness in the stevia plant is attributed to the presence of high potency
sweet glycosides.
The two major glycosides found in the leaves and stems of the stevia plant are
stevioside and
rebaudioside A. Rebaudioside A is a desirable glycoside for use in non-caloric
sweeteners.
It is recognized that other steviol glycosides or a combination of steviol
glycosides may be
used as a sweetener. Various methods are available for the purification of
rebaudioside A
from crude rebaudioside A-containing stevia extracts.
[0009] The use of high-potency sweeteners allows for the formulation of
sweetened
beverages with zero, or at least significantly fewer, calories than standard
sweetened
beverages, an important health and wellness feature as many countries are
attempting to
address weight-related public health concerns. In particular, a naturally
occurring high-
potency sweetener with a pleasing, sugar-like taste profile, such as monatin,
is desirable.
However, at times various high-potency sweeteners, including monatin, may
present
formulation challenges due to varying chemical stability under common beverage
conditions,
such as low pH, exposure to light, presence of carbonation ("CO2"), presence
of oxygen
("02"), interaction with various flavor components, packaging that is
transparent to
ultraviolet ("UV") light or visible light or packaging that is to some extent
02 permeable.
These stability challenges can manifest themselves in a perceived loss of
sweetness,
discoloration, or undesirable flavors or aromas. High-potency sweeteners often
affect
sensory properties beyond simply maximum sweetness intensity. For example,
some high-
potency sweeteners cause a prolonged sweetness in the mouth, which is not
common with
caloric sweeteners. Finally, the combination of sweeteners may not behave in
predictable
ways with respect to sweetness intensity. Selection of appropriate blends of
sweeteners may
permit more cost-effective blends. The present disclosure addresses one or
more of these
commercially-relevant concerns.
SUMMARY
[0010] Provided herein are compositions containing monatin and steviol
glyclosides. In one
aspect, provided is a method of improving a sweetener performance in a food
product, the
method comprising: adding monatin or a salt thereof to the food product ; and
adding steviol
glycosides to the food product, wherein the steviol glycosides are added in an
amount
sufficient such that the food product exhibits at least one of a reduction in
discoloration and a
reduction in off-flavors after exposure to ultraviolet or visible light, as
compared to a
comparable food product containing monatin without steviol glycosides. In
some
3
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
embodiments, the food product comprises between about 10 ppm and about 50 ppm
of
monatin and between about 10 ppm and about 80 ppm of rebaudioside A. In some
embodiments, the food product contains less than about 0.5 ppm of 3-methyl
indole after 28
days storage at about 22 C in the continuous presence of fluorescent lighting
of about 43000
lux. In some embodiments, the food product retains at least about 65% of its
monatin
equivalents after 28 days storage at about 22 C in the continuous presence of
fluorescent
lighting of about 43000 lux, wherein monatin equivalents are defined as
monatin and monatin
lactone. In some embodiments, the food product does not exhibit any
appreciable
discoloration or appreciable off-flavors after 28 days storage at about 22 C
in the continuous
presence of fluorescent lighting of about 4,000 lux. In some embodiments, the
food product
exhibits decreased sweetness linger as compared to a comparable food product
containing
either monatin or steviol glycosides alone. In some embodiments, the food
product exhibits
greater sweetness than the sweetness expected from sweetness calculations made
using the
Beidler equation. In some embodiments, the food product is a beverage.
[0011] In a second aspect, provided is a method of making a stable beverage,
the method
comprising: adding monatin or a salt thereof to the beverage; and adding
steviol glycosides
to the beverage in an amount sufficient such that the beverage retains at
least about 65% of
its monatin equivalents after 28 days storage at about 22 C in the continuous
presence of
fluorescent lighting of about 4000 lux, wherein monatin equivalents are
defined as monatin
and monatin Intone. In some embodiments, the beverage retains at least about
80% of its
monatin equivalents after 28 days storage at about 22 C in the continuous
presence of
fluorescent lighting of about 4,000 lux. In some embodiments, the beverage is
a carbonated
beverage. In some embodiments, the beverage is a still beverage. In some
embodiments, the
beverage comprises about 10 ppm to about 80 ppm of rebaudioside A. In some
embodiments, the beverage comprises about 10 ppm to about 50 ppm of monatin.
In some
embodiments, the beverage exhibits decreased sweetness linger as compared to a
comparable
food product containing either monatin or steviol glycosides alone. In some
embodiments,
the beverage exhibits greater sweetness than the sweetness expected from
sweetness
calculations made using the Beidler equation.
[0012] In a third aspect, provided is a beverage comprising: about 10 ppm to
about 50 ppm
of monatin or a salt thereof; and about 10 ppm to about 80 ppm of steviol
glycosides, wherein
the beverage retains at least about 65% of its monatin equivalents after 28
days storage at
about 22 C in the continuous presence of fluorescent lighting of about 4,000
lux, wherein
4
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
monatin equivalents are defined as monatin and monatin lactone. In some
embodiments, the
monatin is at least about 90% R,R-monatin. In some embodiments, the monatin is
at least
about 95% R,R-monatin. In some embodiments, the beverage retains at least
about 80% of
its monatin equivalents after 28 days storage at about 22 C in the continuous
presence of
fluorescent lighting of about 4,000 lux. In some embodiments, the beverage is
a carbonated
beverage. In some embodiments, the steviol glycosides are at least about 95
wt%
rebaudioside A. In some embodiments, the steviol glycosides are chosen from
the group
consisting of rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D,
rebaudioside
E, rebaudioside F, stevioside, dulcoside A, steviolbioside, and a combination
thereof. In
some embodiments, the beverage exhibits decreased sweetness linger as compared
to a
comparable food product containing monatin without steviol glycosides. In
some
embodiments, the beverage exhibits greater sweetness than the sweetness
expected from
sweetness calculations made using the Beidler equation.
[0013] In a fourth aspect, provided is a beverage composition comprising:
about 10 ppm to
about 50 ppm of monatin or a salt thereof; and about 10 ppm to about 80 ppm of
steviol
glycosides, wherein the beverage composition does not exhibit any appreciable
odors or any
appreciable off-flavors after 28 days storage at 22 C in the continuous
presence of
fluorescent lighting of about 4,000 lux. In some embodiments, the beverage
composition is a
carbonated beverage. In some embodiments, the monatin comprises at least about
90% R,R-
monatin. In some embodiments, the steviol glycosides include rebaudioside A.
In some
embodiments, the steviol glycosides are at least about 80 wt% rebaudioside A.
In some
embodiments, the steviol glycosides are at least about 95 wt% rebaudioside A.
In some
embodiments, the steviol glycosides are chosen from the group consisting of
rebaudioside A,
rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside
F, stevioside,
dulcoside A, steviolbioside, and a combination thereof. In some embodiments,
the beverage
exhibits decreased sweetness linger as compared to a comparable food product
containing
monatin without steviol glycosides. In some embodiments, the beverage exhibits
greater
sweetness than the sweetness expected from sweetness calculations made using
the Beidler
equation.
[0014] In a fifth aspect, provided is a beverage composition comprising: about
10 ppm to
about 50 ppm of monatin or a salt thereof; and about 10 ppm to about 80 ppm of
steviol
glycosides, wherein the beverage does not exhibit any appreciable
discoloration after 28 days
storage at 22 C in the continuous presence of fluorescent lighting of about
4,000 lux. In
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
some embodiments, the beverage composition is a carbonated beverage. In
some
embodiments, the monatin comprises at least about 90% R,R-monatin. In some
embodiments, the steviol glycocides include rebaudioside A. In some
embodiments, the
steviol glycosides are at least about 80 wt% rebaudioside A. In some
embodiments, the
steviol glycosides are at least about 95 wt% rebaudioside A. In some
embodiments, the
steviol glycosides are chosen from the group consisting of rebaudioside A,
rebaudioside B,
rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, stevioside,
dulcoside A,
steviolbioside, and a combination thereof. In some embodiments, the beverage
exhibits
decreased sweetness linger as compared to a comparable food product containing
monatin
without steviol glycosides. In some embodiments, the beverage exhibits greater
sweetness
than the sweetness expected from sweetness calculations made using the Beidler
equation.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a sweetness intensity versus time curve for various blends of
monatin and 95w0/0
rebaudioside A, as compared to a monatin-only sample, a 95 wt% rebaudioside A-
only
sample, and a sucrose reference sample.
DETAILED DESCRIPTION
[0015] As stated above, monatin has two chiral centers leading to four
potential
stereoisomeric configurations. As used
herein, unless otherwise indicated, the term
"monatin" is used to refer to compositions including any combination of the
four
stereoisomers of monatin (or any of the salts thereof), including a single
isomeric form.
[0016] Unless otherwise stated, the term "monatin" includes any salt thereof.
As used herein,
unless otherwise stated, the term "monatin" is independent of the method by
which the
monatin was made, and thus encompasses monatin that was, for example,
synthesized in
whole or in part by biosynthetic pathway(s), purely synthetic means, or
isolated from a
natural source.
[0017] As used herein, "sucrose equivalence value", or "% SEV", is a measure
of sweetness.
The SEV is the concentration (% w/v) of a sucrose solution that gives a degree
of sweetness
that is perceived to be the same degree of sweetness as that of a test sample.
The % SEV of
an aqueous solution containing monatin is determined through comparison of the
monatin-
containing solution to a series of sucrose-containing solutions prepared in
the same medium.
The monatin-containing solution may also include a steviol glycoside, which
may contribute
sweetness to the solution. For purposes of this disclosure, an approximate %
SEV can be
6
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
calculated for a solution containing monatin and/or steviol glycosides, based
on the
concentration response curves for the monatin and/or steviol glycosides in the
composition.
[0018] Monatin-containing compositions are described herein as being "X" times
as sweet as
sucrose. The various stereoisomers of monatin are also described as being "X"
times sweeter
than sucrose. These values are determined from the % SEV of the compositions.
[0019] As used herein, the term "about" encompasses the range of experimental
error that
occurs in any measurement.
[0020] In some cases, a "comparable food product" is used herein to compare a
monatin-
containing food product to essentially the same monatin-containing food
product that also
includes steviol glycosides. Essentially the same food product means that the
food product
has essentially the same attributes, properties, and/or composition as the
food product it is
being compared to, and has been exposed to the same conditions as the food
product it is
being compared to. In some cases, a "comparable beverage" is used herein to
compare a
monatin- containing beverage to essentially the same monatin-containing
beverage that also
includes steviol glycosides. As used herein, "food", "food product",
"foodstuff", and "food
composition" include beverages.
[0021] In the process of formulating monatin into various beverage
compositions it has been
observed that monatin alone is insufficiently stable in extended trials when
found in the
combined presence of 02 and light (either visible and/or UV). What constitutes
appropriate
levels of stability varies by specific beverage application and market
requirements. A general
rule of thumb is that 13 weeks of storage stability (or "shelf stability")
under typical store
handling conditions is required for common cold-filled consumer beverage
applications. And
another general rule of thumb is that 26 to 52 weeks of storage stability (or
"shelf stability")
under typical handling conditions is required for common hot-filled consumer
beverage
applications. In general, storage stability is not readily quantifiable, but
is rather an
assessment of organoleptic taste, aroma perception, or appearance. Key
features of instability
can be identified and used to predict unacceptable levels of shelf stability.
In the case of
monatin, the levels of total indole-containing molecules (as an indole ring is
a substantial
portion of the monatin molecule) are a good indicator of the relative shelf
stability of various
formulations. For purposes of this report, total indole means the sum of
monatin, and its
lactone and lactam forms. It has also been found that monitoring "musty" off
flavors or
aromas by trained human sensory evaluators also is predictive of monatin's
shelf stability,
7
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Further, it is believed that a degradation product from monatin that
contributes to this
"musty" organoleptic property is 3-methyl indole. Limiting
the presence of these
components in the beverage in some instances can be used to ensure appropriate
shelf
stability. In particular, limitation of the presence of 3-methyl indole to
less than 0.5 ppb is
desirable in some embodiments.
[0022] In clear or lightly colored beverages, one may also quantitatively
observe yellow
discoloration. The prevention of discoloration is then predictive of
appropriate shelf stability.
[0023] It has been discovered that monatin is subject to degradation in the
combined
presence of dissolved 02 and light resulting in one or more of a loss of
sweetness, yellow
discoloration, and development of off-flavors or aromas. This light may be in
either the
visible or UV spectrum.
[0024] For purposes of this disclosure, an "off flavor" is defined as a
sensory experience of a
volatile compound(s) that may be detected orthonasally and/or retronasally. In
many
instances when an off flavor is referred to herein, the off flavor is a musty
off flavor.
However, it is recognized that other negative off flavors may be predictive of
monatin's shelf
stability. Off flavors and discoloration may be referred to herein in terms of
an "appreciable"
off flavor or an "appreciable" discoloration. For purposes of this disclosure,
an appreciable
off flavor is detectable nasally by sniffing or retronasally by tasting by a
trained human
sensory evaluator and an appreciable discoloration is detectable visually by a
trained human
sensory evaluator.
[0025] As stated above, a musty off flavor is believed to be, in part, from
formation of 3-
methyl indole from monatin. Thus instability may lead to a loss of monatin in
a sweetened
beverage composition, thus affecting the sweetness properties of the beverage.
[0026] The inventors unexpectedly observed that a combination of monatin and
steviol
glycosides in a beverage resulted in the beverage having an improved sweetener
performance, as compared to a beverage containing monatin without steviol
glycosides. As
described herein, the sweetener performance of a beverage may be defined in
terms of its
stability, or more specifically, in terms of the absence of off flavors and
yellow discoloration
after exposure to light over a certain period of time. Sweetener performance
may also be
described in terms of the preservation or retention of the monatin in the
beverage after
exposure to light over a certain period of time. Alternatively, sweetener
performance of a
beverage containing a blend of monatin and steviol glycosides may be described
herein in
8
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
terms of having a reduction in discoloration and/or a reduction in off flavors
over a certain
period of time as compared to a beverage containing monatin without steviol
glycosides. The
sweetener performance in a beverage may, in some cases, also be defined herein
in terms of
its sweetness linger, For purposes of this disclosure, sweetness linger is
detectable by tasting
by a trained human sensory evaluator. The sweetener performance in a beverage
may, in
some cases, also be defined in terms of its sweetness synergy. For purposes of
this
disclosure, sweetness synergy is detectable by tasting by a trained human
sensory evaluator.
[0027] The disclosure herein focuses on compositions containing a blend of
monatin and
steviol glycosides. The term "steviol glycosides" as used herein refers to any
of the
glycosides of the aglycone steviol (ent-13-hydroxykattr-16-en-19-oie-acid)
including, but not
limited to, stevioside, rebaudioside A, rebaudioside B, rebaudioside C,
rebaudioside D,
rebaudioside E, rebaudioside F, dulcosicle, rebusoside, steviohnonoside,
steviolbioside, and
19-0-I3 glucopyranosol-steviol. As used herein, the term "steviol glycosides"
also includes
isomers and derivatives of steviol glycosides. "Reb A", as used herein, refers
to a stevia leaf-
derived composition comprising greater than about 95% rebaudioside A. "RA80"
refers to a
stevia leaf-derived composition comprising greater than about 80% rebaudioside
A.
[0028] Several grades of steviol glycosides are available. Grades
having a high
rebaudioside A concentration contribute lower bitter off taste levels. For the
compositions
disclosed herein, in some embodiments, the steviol glycosides have a
rebaudioside A
concentration of about 30 to 99.5 wt. % relative to all steviol glycosides. In
some
embodiments, the steviol glycosides have a rebaudioside A concentration of
about 60 to 99.5
wt. % relative to all steviol glycosides. In other embodiments, the steviol
glycosides have a
rebaudioside A concentration of about 80 to 99.5 wt. % relative to all steviol
glycosides. In
other embodiments, the steviol glycosides have a rebaudioside A concentration
of about 95 to
99.5 wt. % relative to all steviol glycosides, and in yet other embodiments,
the steviol
glycosides have a rebaudioside A concentration of about 97 to 99.5 wt. %
relative to all
steviol glycosides. In some embodiments, the steviol glycosides in the blend
may be
essentially all or predominantly rebaudioside A (predominantly being defined
herein as
greater than 90% by weight). In some embodiments, the steviol glycosides may
include
rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside
E,
rebaudioside F, stevioside, duleoside A. steviolbioside, and a combination
thereof. In some
embodiments, the steviol glycosides may be predominantly stevioside. In
other
9
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
embodiments, the steviol glycosides may be predominantly rebaudioside D. In
yet other
embodiments, a mixture of any or all of the steviol glycosides may be used.
[0029] Methods to selectively extract one or more of the steviol glycosides,
for example,
rebaudioside A, have been previously disclosed in the art. For example,
Japanese Patent
63173531, which is hereby incorporated by reference in its entirety, describes
a method of
extracting sweet glycosides from the Stevia rebaudiana plant. This procedure
isolates a
mixture of sweet glycosides. Other techniques include those reported in
Japanese Publication
Numbers 56121454, 52062300, and 56121453 assigned to Ajinomoto Company, Inc.
which
are hereby incorporated by reference in their entirety.
[0030] Although the disclosure herein is focused on a beverage composition
containing
monatin and steviol glycosides, it is recognized that this disclosure is
applicable to food and
beverage compositions.
[0031] A composition may be described herein in terms of its total indole
concentration,
which is defined as monatin and its intramolecular rearrangement products ¨
monatin lactone
and monatin lactatn. A composition may also be described herein as retaining a
certain
percentage of monatin equivalents, which is defined herein as monatin plus
monatin lactone.
[0032] As exemplified in the examples below, in some embodiments, the addition
of steviol
glycosides to a monatin-containing composition can result in the composition
retaining a
larger percentage of monatin equivalents after exposure to light as compared
to a sample
containing monatin without steviol glycosides. Due to the higher percentage of
monatin
retained, it is believed that there is less yellow discoloration and less
conversion to 3-methyl
indole, which may be a significant contributor to a musty off flavor. While
not being bound
by theory, it is believed that steviol glycosides may inhibit monatin
photooxidation.
[0033] In some embodiments, a food product (e.g., a beverage) including
monatin and steviol
glycosides may exhibit decreased sweetness linger as compared to a comparable
food product
containing monatin or steviol glycosides alone
[0034] In some embodiments, a food product (e.g., a beverage) including
monatin and steviol
glycosides may exhibit greater sweetness than the sweetness expected from
sweetness
calculations made using the Beidler equation, i.e,, sweetness synergy. The
concentration of
each sweetener correlating to a target sucrose equivalent value ("% SEV") may
be calculated
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
from the Beidler curve of concentration response data for each of the
individual sweeteners.
The standard form of the Beidler equation is:
(a * C)
Sweetness = ______________________________
(b + C)
where C is the concentration of the sweetener (in mg/L), and a and b are
coefficients
calculated from the empirical measurements. Monatin has a different Beidler
equation at
neutral pH as compared to acid pH.
[0035] A beverage composition disclosed herein can include a blend of monatin
and steviol
glycosides. In some embodiments, the amount of steviol glycosides is equal to
or greater
than an amount sufficient to cause the beverage composition to exhibit a
reduction in
discoloration and/or a reduction in off flavors after exposure to ultraviolet
and visible light, as
compared to a comparable beverage composition containing monatin without
steviol
glycosides. It is recognized that if steviol glycosides are added to the
monatin containing
composition, the resulting composition will have a higher sucrose equivalence
value ("%
SEV"). In order for the composition containing monatin and steviol glycosides
to have an
essentially equal % SEV to the monatin only containing composition, it is
recognized that a
smaller amount of monatin would be used in order to account for the sweetness
from the
steviol glycosides.
[0036] In some embodiments, the amount of steviol glycosides is equal to or
greater than an
amount sufficient such that the beverage retains at least about 65% of its
monatin equivalents
after 28 days stored at about 22 C in the continuous presence of fluorescent
lighting of about
4000 lux. In other embodiments, the amount of steviol glycosides is equal to
or greater than
an amount sufficient such that the beverage retains at least about 80% of its
monatin
equivalents under the same conditions. It is recognized that other conditions
may be used to
define a certain percent retention of monatin equivalents.
[0037] In some embodiments, beverage compositions containing monatin and
steviol
glycosides may comprise other stability enhancing features, such as, for
example,
antioxidants. Reference is made to PCT Publication Number WO 2010/138513,
titled
"SHELF STABLE MONATIN SWEETENED BEVERAGE," which is hereby incorporated
by reference in its entirety.
[0038] For a beverage composition having a blend of monatin and steviol
glycosides, in some
embodiments, monatin is present in an amount that ranges from about 0.0003 to
about I% of
11
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
the beverage composition (i.e., about 3 to about 10,000 ppm) (e.g., about
0.0005 to about
0.2%), including any particular value within that range (e.g., 0.0003%,
0.005%, 0.06% or
0.2% of the beverage composition). For example, a beverage composition may
comprise
about 0.0005 to about 0.005% (e.g., about 0.001 to about 0.0050%) of the R,R
monatin, or
about 0.005 to about 0.2% (e.g., about 0.01 to about 0.175%) of S,S monatin.
In some
embodiments, a beverage composition may comprise about 0.001% to about 0.0050%
(i.e.
about 10 to about 50 ppm), about 0.001% to about 0.0040% (i.e. about 10 to
about 40 ppm),
or about 0.001% to about 0.0035% (i.e. about 10 to about 35 ppm) of R,R
monatin and about
0.001% to about 0.008% (i.e. about 10 to about 80 ppm), about 0.0015% to about
0.0075%
(i.e. about 15 to about 75 ppm), or about 0.002% to about 0.007% (i.e. about
20 to about 70
ppm) of Reb A. In some embodiments, the rebaudioside A may be 95 wt%
rebaudioside A.
In other embodiments, beverages containing monatin and steviol glycosides may
have
amounts of monatin and/or amounts of steviol glycosides that are outside of
the ranges
disclosed herein.
[0039] It is recognized that an amount of monatin in a beverage composition
may vary as a
function, for example, of the desired sweetness intensity, the beverage
formulation, and the
stereoisomeric configuration of the monatin. Similarly, it is recognized that
the amounts of
steviol glycosides in a beverage composition may vary as a function, for
example, of the
profile of the steviol glycosides (i.e. rebaudioside A, rebaudioside D,
stevioside, other steviol
glycosides, and any combination thereof), the amount of monatin in the
beverage, the desired
stability profile, and the presence of any other stability enhancing features.
[0040] As used herein, "beverage composition" refers to a composition that is
drinkable as is
(i.e., does not need to be diluted, or is "ready-to-drink") or a liquid
concentrate or a dry
powder that can be diluted or mixed with additional liquid to form a drinkable
beverage.
Beverage compositions herein include carbonated and non-carbonated soft
drinks, coffee
beverages, tea beverages, dairy beverages, liquid concentrates, flavored
waters, enhanced
waters, fruit juice and fruit juice-flavored drinks, sport drinks, and alcohol
products.
Beverages herein include beverages formed from a powder. In some embodiments,
beverage
compositions comprising monatin and steviol glycosides include other
ingredients, such as,
for example, a flavoring, coloring, organic acids, inorganic acids,
preservatives, caffeine,
other sweeteners, and/or polyols.
[0041] In some embodiments, the monatin and steviol glycosides containing
beverage
compositions comprise monatin that consists essentially of S,S or R,R monatin.
In other
12
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
embodiments, the compositions contain predominantly S,S or R,R monatin.
"Predominantly"
means that of the monatin stereoisomers present in the composition, the
monatin contains
greater than 90% of a particular stereoisomer. In some embodiments, the
compositions are
substantially free of S,S or R,R monatin. "Substantially free" means that of
the monatin
stereoisomers present in the composition, the composition contains less than
2% of a
particular stereoisomer. In another aspect, a beverage composition includes
a
stereoisomerically-enriched monatin mixture. "Stereoisomerically-enriched
monatin mixture"
means that the mixture contains more than one monatin stereoisomer and at
least 60% of the
monatin stereoisomers in the mixture is a particular stereoisomer, such as
R,R, S,S, S,R or
R,S. In other embodiments, the mixture contains greater than about 65%, 70%,
75%, 80%,
85%, 90%, 95%, 98%, or 99% of a particular monatin stereoisomer. In another
embodiment,
a beverage composition comprises a stereoisomerically-enriched R,R or S,S
monatin.
"Stereoisomerically-enriched" R,R monatin means that the monatin comprises at
least 60%
R,R monatin. "Stereoisomerically-enriched" S,S monatin means that the monatin
comprises
at least 60% S,S monatin. In other embodiments, "stereoisomerically-enriched"
monatin
comprises greater than about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of
R,R or
S,S monatin.
[0042] It should be understood that beverages and other beverage products in
accordance
vvith this disclosure may have any of numerous different specific formulations
or
constitutions. The formulation of a beverage product in accordance with this
disclosure can
vary to a certain extent, depending upon such factors as the product's
intended market
segment, its desired nutritional characteristics, flavor profile and the like.
For example, it
will generally be an option to add further ingredients to the formulation of a
particular
beverage embodiment, including any of the beverage formulations described
below.
Additional (i.e., more and/or other) sweeteners may be added, flavorings,
electrolytes,
vitamins, fruit juices or other fruit products, tastants, masking agents and
the like, flavor
enhancers, and/or carbonation typically can be added to any such formulations
to vary the
taste, mouthfeel, nutritional characteristics, etc. In general, a beverage in
accordance with
this disclosure typically comprises at least water, sweetener, acidulant, and
flavoring.
Exemplary flavorings which may be suitable for at least certain formulations
in accordance
with this disclosure include, without limitation, cola flavoring, citrus
flavoring, root beer
flavoring, and spice flavorings. Carbonation, in the form of carbon dioxide,
may be added
for effervescence. Natural and synthetic preservatives can be added if
desired, depending
13
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
upon the other ingredients, production technique, desired shelf life, etc.
Optionally, caffeine
can be added. Certain exemplary embodiments of the beverages disclosed herein
are, without
limitation, cola-flavored carbonated beverages, characteristically containing
carbonated
water, sweetener, kola nut extract, and/or other cola flavoring, caramel
coloring, and
optionally other ingredients. Additional and alternative suitable ingredients
will be
recognized by those skilled in the art given the benefit of this disclosure.
[0043] Some embodiments may be considered still beverages, i.e., beverages
which are not
carbonated. Common examples include, without limitation, coffee beverages, tea
beverages,
dairy beverages, flavored waters, enhanced waters, non-carbonated soft drinks,
fruit juice and
fruit juice-flavored drinks, sport drinks, and alcohol products other than
beer and
champagnes.
[0044] In other embodiments, the beverage may contain dissolved carbon dioxide
("CO2") in
amounts sufficient to provide effervescence. Common examples include, without
limitation,
carbonated soft drinks, beer and champagnes. Such carbonated beverages
typically have
carbon dioxide concentrations of about 1.6 volumes CO2 per volume of beverage
to about 4.2
volumes CO2 per volume of beverage. Carbon dioxide is typically introduced
into a beverage
by either fermentation (as in the case of beer and champagnes) or dissolving
the carbon
dioxide into the beverage under pressure (as in the case of carbonated
beverages). Specific
methods of beverage carbonation are well known to those skilled in the art.
[0045] In carbonated beverages, the process of carbonation results in a
removal or
displacement of dissolved oxygen from the beverage. In some embodiments, the
dissolved
oxygen levels may be reduced or controlled for stability purposes, as
disclosed in WO
2010/138513, which is hereby incorporated by reference in its entirety.
14
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
EXAMPLES
[0046] Aspects of certain embodiments in accordance with aspects of the
invention are
illustrated in the following examples. The materials and methods described in
these
examples are illustrative and not intended to be limiting.
[0047] Three lots of monatin were used in the following examples: Lot #5837-
72RRSYN
monopotassium salt; Lot #25001119 (90.3% 2R34R- monatin potassium/sodium
salt); Lot
#25001392 (90.4% 2R,4R- monatin sodium salt). Monatin concentration is
expressed on a
pure monatin salt basis in the examples unless otherwise specified. Lot #5837-
72RRSYN
was used in Example 11. Lot #25001119 was used in Examples 1 through 6 and 8
through
10. Lot #25001392 was used in Examples 7 and 12 through 16.
[0048] The Beidler equation for monatin in water is % SEV = 26.7 *
[monatin]/(69.6
[monatin]). The Beidler equation for Reb A in water at room temperature is %
SEV =
11.26*[Reb Al/(253.7 -4-_[Reb A]). These equations were used in all examples
unless
otherwise specified.
Example 1
[0049] Lemon-lime flavored carbonated beverages were stored in the dark and
evaluated
over a seven week period. Solutions of about 9 mM citric acid/trisodium
citrate (pH of
approximately 3.0) in high purity water were sweetened to about 8.0% SEV (the
calculated %
SEV of each sample is shown in Table 1 below) with various amounts of monatin
and/or Reb
A, depending on the intended blend, and then flavored with lemon-lime flavor;
and preserved
with approximately 150 ppm sodium benzoate. The solutions were carbonated in
10 liter
batch sizes using an Armfield carbonator, by adding approximately 3.5 liters
of carbon
dioxide ("CO2") per liter of solution before being transferred to twelve ounce
clear, glass
bottles. Caps were placed on the bottles after filling. The bottles were
stored in a dark oven
maintained at a temperature of approximately 38 C. Multiple bottles of each
sample were
created since a bottle was sacrificed when the measurements below were taken.
[0050] Total indole (monatin and its intramolecular rearrangement products,
monatin lactone
and monatin lactam) concentrations were quantified by with a Waters Alliance
2690 HPLC at
40 C using a Zorbax Eclipse XDB-C18 column (Agilent Technologies, Inc., Santa
Clara CA)
with UV detection at 280 nm by a photodiode array detector. The compounds were
eluted by
a gradient mobile phase consisting of (A) 0.3% formic acid in water with 10 mM
ammonium
formate and (B) 1:1 methanol and acetonitrile (v:v) with 0.3% formic acid and
10 mM
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
ammonium formate. The integrated peak areas for the three compounds were
expressed in
terms of monatin concentration (mg/L) based on a standard curve prepared for
monatin.
Thus, the extinction coefficient for monatin was applied to estimate
concentrations of
monatin lactone and monatin lactain since standards for these compounds were
unavailable.
[0051] Total indole concentrations are shown in Table 1 for each of the
blends. The percent
of the original indole concentration is shown in parentheses. Table 2 shows
monatin
equivalents, which is defined for purposes herein as monatin and monatin
lactone; the percent
of the original monatin equivalents is shown in parentheses.
Table 1. Total Indole Concentrations for Various Blends of Monatin and Reb A
Stored
in the Dark
Sample
1 2 3 4 5
Monatin (%
SEV) 7.9 6.3 4.8 3.4 1.7
Reb A(% SEV) 0.0 1.6 3.2 4.8 6.4
Total (% SEV) 7.9 8.0 8.2 8.1
Reb A (ppm) 0 35 81 144 236
Time (Weeks) Total Indole (ppm)
0.0 35.6 (100.0) 23.6 (100.0) 15.8 (100.0) 10.0 (100.0)
4.5 (100.0)
2.0 35.6 (100.0) 23.6 (100.0) 15.8 (100.0) 10.0 (100.0)
4.5 (100.0)
4.0 32.8 (92.1) 22.2 (94.1) 14.9 (94.3) 9.4 (94.0)
_ 4.1 (91.1)
7.3 33.0 (92.7) 23.9 (101.3) 16.0 (101.3) _ 9.3 (93.0)
4.4 (97.8)
Table 2. Monatin Equivalents Concentration for Various Blends of Monatin and
Reb A
Stored in the Dark
Sample
Time (Weeks) 1 2 3 4 5
0.0 35.6(100.0) 23.6 (100.0) 15.8 (100.0) 10.0 (100.0)
4.5 (100.0)
2.0 31.2 (87.6) 20.6 (87.3) 13.8 (87.3) 8.8 (88.0)
3.9 (86.7)
4.0 25.1 (70.5) 16.6 (70.3) 11.1(70.3) 7.1 (71.0) _
2.9 (64.4)
7.3 20.3 (57.0) 14.8 (62.7) 9.7 (61.4) 5.6 (56.0)
2.7 (60.0)
16
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 3. Lactarn Concentration for Various Blends of Monatin and Reb A Stored
in the
Dark
Sample
Time (Weeks) 1 2 3 4 5
0.0 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0
(0.0)
2.0 4.4 (12.4) 2.9 (12.3) 1.9 (12.0) 1.2 (12.0) 0.6
(13.3)
4.0 7.7 (23.5) 5.6 (25.2) 3.8 (25.5) 2.3 (24.5) 1.2
(29.7)
7.3 12.7 (38.5) 9.2 (38.5) 6.3 (39.4) 3.6 (38.7) 1.7
(38.6)
[0052] Table 2 shows that there is a decrease in monatin equivalents (monatin
plus monatin
lactone) over time. The decrease in monatin equivalents may be attributed to
the formation
of monatin lactarn from monatin. The presence of Reb A in samples 2 through 5
did not
affect the proportion of monatin converted to monatin lactam.
Example 2
[0053] Compositions from Example I were freshly prepared and exposed to light.
The
solutions were prepared using the same steps described above for Example 1.
The bottles
containing the solutions were exposed to light in a light box (approximately
4,000 lux light
intensity) continuously (24 hours per day), using a rotating carousel to
provide even
illumination. The source of light was provided by ultra-violet ("UV") light
wavelength
transmitting bulbs and standard fluorescent bulbs. The heat produced by the
lights warmed
the samples to a temperature of about 22 C. Multiple bottles of each sample
were created
since each bottle was sacrificed when the measurements below were taken. The
samples
were analyzed for total indole concentration and monatin equivalents, using
the methodology
disclosed in Example 1 above.
17
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
Table 4. Total Ind le Concentrations for Various Blends of Monatin and Reb A
Exposed to Light
Sample
1 2 3 4 5
Monatin
(% SEV) 7.9 6.3 4.8 3.4 1.7
Reb A (% SEV) 0.0 1.6 3.2 4.8 6.4
Total (% SEV) 7.9 7.9 8.0 8.2 8.1
Reb A (ppm) 0 35 81 144 236
Time (Weeks) Total lndole (ppm)
35.6
0.0 (100.0) 23.6(100.0) 15.8(100.0) 10.0(100.0)
4.5(100.0)
2.3 32.7 (92.0) 23.1 (97.9) 18.1 (114.3) 9.3 (93.7)
4.2 (93.0)
4.4 26.9 (75.7) 20.4 (86.4) 11.8 (74.8) 8.2 (82.2)
3.7 (82.5)
7.3 22.5 (63.2) 20.6 (87.5) 7.9 (50.1) 7.9 (79.7)
3.7 (81.8)
Table 5. Monatin Equivalents for Various Blends of Monatin
and Reb A Exposed to Light
Sample
Time (Weeks) _ 1 2 3 4 5
35.6
0.0 (100.0) 23.6 (100.0) 15.8 (100.0) 10.0
(100.0) 4.5 (100.0)
2.3 31.8(89.3) 22.4(94.9) 18.0(113.9) _ 9.1 (91.0)
_ 4.1 (91.1)
4.4 25.7 (72.2) 19.3 (81.8) 11.4(72.2) 7.8 (78.0)
3.5 (77.8)
7.3 21.0 (59.0) 19.3 (81.8) 7.2 (45.6) 7.4 (74.0)
3.7 (82.2)
[0054] Tables 4 and 5 show that each of the blends of monatin and Reb A, with
the exception
of sample 3, had a smaller loss of monatin over time, as compared to sample 1
having
monatin alone. Sample 3, which contained an amount of Reb A in between the
amount in
samples 2 and 4, showed the largest loss of monatin over time compared to all
samples,
including sample 1. Although a specific explanation is not provided, it is
believed that there
was a deficiency in sample 3, for example, an error in preparation of the
sample.
[0055] At the same points in time that data was collected for Tables 4 and 5,
the samples
were also evaluated for discoloration by comparing each sample to a control
solution stored
in the same environment but covered in foil and thus protected from light. The
evaluator
gave the sample a rating between 0 and 9, and the results are shown in Table 6
below.
Similarly, the same evaluator observed the samples for any off flavors (using
the same
18
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
control used for discoloration), using a scale of 0 and 9, and the results are
shown in Table 7
below.
Table 6. Discoloration of Blends Exposed to Light
_
Sample 1 2 3 4 5
Time " 'Yellow Color Intensity Rating
(Weeks) bo ppm
b35 ppm b81 ppm b144 ppm b236
ppm =
0.0 0 0 0 0 0
2.3 1 0 0 0 0
4.4 1 0 0 0 0
_.
7.3 No Sample 1 2 1 0
_
aThe intensity of color was rated on a scale from 0 to 9 where 0 = none, 1 =
trace, 2 = faint, 3 = slight,
4 ¨ mild, 5 = moderate, 6 ¨ definite, 7 = strong, 8 ¨ very strong, and 9 =
extreme.
blnitial Reb A concentration.
Table 7. Musty Off-flavor of Blends Exposed to Light
,
Sample 1 2 3 4 5
.
Time 'Musty Off-Flavor
Intensity Rating
(Weeks) bo ppm
b35 ppm b81 ppm b144 ppm b236 ppm
0 0 0 0 0 0
2.3 0 . 0 0 0 0
4.4 0 0 0 0 0
, . .
7.3 No Sample 0 2 0 0
'The intensity of musty off-flavor was rated on a scale from 0 to 9 where 0 =
none, I ¨ trace, 2 ¨ faint,
3 = slight, 4 = mild, 5 = moderate, 6 ¨ definite, 7 ¨ strong, 8 ¨ very strong,
and 9 ¨ extreme.
blnitial Reb A concentration.
[0056] Discoloration was observed in sample 1 (no Reb A) at 2.3 weeks and 4.4
weeks.
However, for samples 2 through 4 having a blend of monatin and Reb A,
discoloration was
not observed until 7.3 weeks. None of the samples exhibited a musty off flavor
except for
sample 3 at 7.3 weeks.
19
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Example 3
[0057] Solutions containing blends of monatin and Reb A were exposed to light
and
underwent the same evaluations as were done for the samples in Example 2. In
contrast to
Example 23 the solutions were exposed to sunlight rather than fluorescent
light. More
specifically, the samples were stored on a windowsill of a north facing window
and at a
temperature ranging between about 20 and about 25 degrees Celsius. The
solutions were
prepared in the same manner as those of Examples 1 and 2 above.
[0058] Table 8 shows the various blends of monatin and Reb A. The amount of
monatin
varied at 13.1 ppm, 17.6 ppm, 21.2 ppm, 25.4 ppm and 31.6 ppm. Except for the
sample
containing 31.6 ppm monatin, the samples were created by making a composition
and
dividing it into two equal sized samples, with the first sample containing
only monatin and
the second sample containing monatin and Reb A. For example, sample 1
contained 13.1
ppm of monatin for a % SEV of 4.2%; sample 2 contained 13.1 ppm of monatin and
73.2
ppm of Reb A for a % SEV of 7.2%. This sample preparation method reduced the
potential
for a formulation error and improved the validity of the resulting comparative
data between
monatin alone and its corresponding monatin/Reb A blend.
[0059] Samples 2, 4, 6, and 8 had % SEVs of 7.2%1 7.4%, 7.3%, and 7.3%
respectively, and
contained a blend of monatin and Reb A. These samples were compared to sample
9 having
a % SEV of 7.4% without any Reb A.
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
Table 8. Blends of Monatin and Reb A
Sample 1 2 3 4 5 6 7 8 9
Monatin
4.2 4.2 5.2 5.2 5.9 5.9 6.6 6.6 7.4
(% SEV)
.
Reb A
0 3.0 0 2.2 0 1.4 0 0.7 0
(% SEV)
.
.
Total
4.2 7.2 5.2 7.4 5.9 7.3 6.6 7.3 ' 7.4
(% SEV)
_
aMonatin
13.1 13.1 17.6 17.6 21.2 21.2 25.4 25.4
31.6
(PPm)
_. . .
bReb A .
0 73.2 0 50.6 0 29.7 0 14.6 0
(PPln)
Initial monatin concentration.
bInitial Reb A concentration.
{0060] Total indole concentration for each sample was measured at various
points using the
methodology described above under Example I and is shown in Table 9 below.
(The values
in parentheses represent the percentage of total indole remaining relative to
the initial
concentration.) For each comparison, after exposure to sunlight for ten weeks,
the sample
containing monatin and Reb A had a higher remaining indole concentration as
compared to
the sample containing monatin alone. Table 10 shows the stability of the Reb A
in the blend
over time. (The values in parentheses represent the percentage of Reb A
remaining relative
to the initial concentration.)
21
CA 02821306 2013 06 11
WO 2012/083251
PCT/US2011/065643
Table 9. Total Indole Concentration (ppm)
Sample
Time
1 2 3 4 5 6 7 8 9
(Weeks)
0 13.1 13.1 17.6 17.6 21.2 21.2 25.4 25.4
31.6
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0) (100.0)
2 11.3 12.5 15.3 15.6 18.5 19.0 22.6 22.2
26.4
(86.3) (95.4) (86.9) (88.6) (87.3) (89.6) (89.0) (87.4) (83.5)
4 8.8 11.5 14.0 13.3 18.4 18.7 19.8 21.5
17.6
(67.2) (87.8) (79.5) (75.6) (86.8) (88.2) (78.0) (84.6) (55,7)
6 9.2 10.5 10.5 13.2 17.9 19.6 14.9 20.1
20.3
(70.2) (80.2) (59.7) (75.0) (84.4) (92.5) (58.7) (79.1) (64.2)
8 6.2 8.6 6.2 11.3 17.5 20.3 13.1 19.8 5.2
(47.3) (65.6) (35.2) (64.2) (82.5) (95.8) (51.6)
(78.0) (16.5)
7.6 8.8 4.7 11.5 15.9 20.3 11.7 17.7 10.7
(58.0) (67.2) (26.7) (65.3) (75.0) (95.8) (46.1)
(69.7) (33.9)
22
CA 02821306 2013 06 11
WO 2012/083251 PCT/US2011/065643
Table 10. Reb A Concentration (ppm)
Sample
Time
1 2 3 4 5 6 7 8 9
(Weeks)
0 N/A 73.2 N/A 50.6 N/A 29.7 N/A 14.6 N/A
(100.0) (100.0) (100.0) (100.0)
2 N/A 71.7 N/A 50.2 N/A 27.2 N/A 15.7 N/A
(97.3) (99.2) (91.6) (107.5)
4 N/A 72.4 N/A 49.7 N/A 29.6 N/A 15.6 N/A
(98.9) (98.2) (99.7) (106.8)
6 N/A 71.9 N/A 49.8 N/A 27.2 N/A 13.9
N/A
(98.2) (98.4) (91.6) (95.2)
8 N/A 72.3 N/A 50.8 N/A 28.4 N/A 13.8 N/A
(98.8) (100.0) (95.6) (94.5)
N/A 72.2 N/A 50.1 N/A 26.9 N/A 13.8 N/A
(98.6) (99.0) 90.6) (94.5)
[0061] The color of each sample was also measured using a HunterLab ColorQuest
XE
colorimeter (Hunter Associates Laboratory, Inc., Reston, VA). As shown in
Table 11 below,
as the level of monatin in the solution increased, the value of color
intensity similarly
increased, with the exception of sample 5. The color intensity data also shows
that the yellow
discoloration was reduced by adding Reb A to the monatin-containing solution.
23
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 11. Yellow Color (I)*) Intensity Measured by Colorimeter
Sample
Time
1 2 3 4 5 6 7 8 9
(Weeks)
0 0.02 - 0.01 0.01 0.01 0.02 0.02 - 0.02 0,02
0.03
2 0.24 0.19 0.37 0.29 0.44 0.27 ' 1.14 0.45
1.17
4 1.11 0.42 0.60 0.48 0.54 0.22 1.50 0.60
3.64
6 1.23 0.82 1.65 0.73 0.71 0.18 2.88 0.72
3.34
8 1.96 1.56 3.53 1.36 0.82 0.21 3.54 0.79
7.66
1.53 1.42 " 3.20 1.46 1.08 0.99 3.99 1.03 5.23
-
[0062] The solutions were also evaluated for the presence of any off flavors.
As shown
below, for the majority of the samples containing monatin without Reb A, the
solutions
developed a musty off flavor over time, as compared to the solutions blended
with monatin
and Reb A, which did not exhibit a musty off flavor after 6 weeks. An off
flavor was
observed in all of the samples after 8 weeks. However, the intensity of the
off flavor was less
for the samples containing monatin and Reb A, as compared to the samples
containing
monatin only.
Table 12. Musty Off-Flavor Development (Odor Intensity Score)
Sample
-
Time 1 2 3 4 5 6 7 8 9
0 0 0 0 0 0 0 0 0
2 0 0 0 0 ' 0 0 1 0 1
_....
4 ' 0 0 0 0 0 0 1 0 - I
6 1 0 1 0 0 0 2 0 2
8 4 2 2 2 2 2 3 3 3
- ..
10 4 3 3 2 2 1 - 3 2 3
The intensity of musty off-flavor was rated on a scale from 0 to 9 where 0 -
none, 1 - trace, 2 = faint,
3 = slight, 4 = mild, 5 = moderate, 6 = definite, 7 = strong, 8 = very strong,
and 9 = extreme.
24
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Example 4
[0063] Lemon-lime flavored carbonated soft drinks were made using the formulas
shown in
the table below.
Table 13. Blends of Monatin and Reb A
Concentration (% by Weight)
Ingredients Sample 1 Sample 2 Sample 3 Sample 4
Water 99.601 99.599 99.595 99.575
Citric Acid 0.160 0.160 0.160 0.160
Trisodiuin Citrate 0.020 0.020 0.020 0.020
Sodium Benzoate 0.015 0.015 0.015 0.015
R,R-Monatin Salt 0.0040 0.0027 0.0017 0
Reb A 0.0000 0.0035 0.0081 0.0300
Lemon-lime Flavor 0.200 0.200 0.200 0.200
Total 100.000 100.000 100.000 100.000
[0064] The solutions were evaluated by a six-member sensory panel with
experience in
evaluating sweetness. A one-ounce sample of each solution, in random order,
was tasted cold
by each panelist and then rated for lingering sweetness intensity after ten
seconds. Some of
the panelists spit the sample out; others swallowed the sample ¨ each panelist
was consistent
with his or her methodology. The panelists arrived at a consensus score for
sweetness linger
for each sample.
Table 14. Sweetness Linger
Sample 1 Sample 2 Sample 3 Sample 4
Total % SEV 8.0 8.0 8.0 8.0
% SEV from Monatin 8.0 6.4 4.8 0
% SEV from Reb A 0 1.6 3.2 8.0
Sweetness Lingering 5 4 3 5
Seo rea
'Sweetness linger was rated on a scale from 1 to 9 where 1 = Not at all
lingering, 3 = Slightly
lingering sweetness, 5 = Moderately lingering sweetness, 7 = Very lingering
sweetness, and 9 ¨
Extremely lingering sweetness.
[0065] The linger scores in Table 14 show that the blends of monatin and Reb A
(Samples 2
and 3) had a reduced linger as compared to both the solution containing
monatin only
(Sample 1) and the solution containing Reb A only (Sample 4).
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Example 5
[0066] The relationship between sweetness intensity and time as it relates to
monatin, Reb A,
and blends of the two sweeteners was studied.
[0067] Samples were prepared to about 8% sucrose equivalent sweetness. The
blends were
formulated such that the ratio of sweetness from monatin to rebiana was about
100/0, 90/10,
80/20, 70/30, 60/40 and 0/100 (Table 15). The concentration of each sweetener
correlating to
the target sucrose equivalent value ("% SEV") was calculated from the Beidler
curve of
concentration response data for each of the individual sweeteners. The
standard form of the
Beidler equation is:
(a * C)
Sweetness = _____________________________
(b + C)
where C is the concentration of the sweetener (in mg/L), and a and h are
coefficients
calculated from the empirical measurements.
[0068] The Beidler equation for Reb A at room temperature in water is % SEV =
(11.26*C) /
(253.7+C) and for monatin at room temperature and pH 3 is % SEV = (16.32* C) /
(41.38 +
C), where C = concentration of sweetener in mg/L. All samples were prepared in
a citrate
buffer at pH 3.0 and room temperature.
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
Table 15. Formulas for Sample Solutions and Calculated (Theoretical) Sucrose
Equivalence Value
Monatin: Monatin Reb A Monatin Rel.) A
Total
Reb A (ppm) (ppm) (% SEV) (% SEV) (% SEV)
Ratio
100/0 43 0 8.3 0 8.3
90/10 36 19 7.5 0.8 8.3
80/20 29 42 6.8 1.6 8.4
70/30 24 69 6.0 2.4 8.4
60/40 19 101 5.2 3.2 8.4
0/100 0 623 0 8.0 8.0
,
Sucrose N/A N/A N/A N/A N/A
(reference)
[0069] Table 16 shows the formulation for each of the blends in Table 15, as
well as the 8%
sucrose reference sample.
Table 16. Formulations of the Monatin/Reb A Blends
Concentration (% by Weight)
Ingredients 100/0* 90/10* 80/20* 70/30* 60/40* 0/100* Sucrose
Water 99.816 99.815 , 99.813 99.810 99.808 99.758 91.82
Citric Acid 0.160 0.160 .... 0.160 0.160 0.160 0.160
0.16
Trisodiurn 0.020 0.020 0.020 0.020 0.020 0.020 0.02
Citrate
R,R- 0.0043 0.0036 0.0029 0.0024 0.0019 0 0
Monatin Salt
Reb A 0.0000 0.0019 0.0042 0.0069 0.0101 0.0623 0
Sucrose 0 0 0 0 0 0 8.00
Total 100.000 100.000 100.000 100.000 100.000 100.000 100.000
*Monatin to Reb A ratio
27
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
[0070] A panel of six trained and experienced panelists performed the time
intensity
evaluation testing. The panelists were instructed to put the entire one fluid
ounce sample in
his or her mouth and hold for ten seconds while continuously rating the
intensity of
sweetness, then swallow or spit the sample and continue rating sweetness
intensity for one
minute. Sweetness intensity was recorded using a computer mouse and Sensory
Information
Management System ("SIMS") 2000 software (Sensory Computer Systems,
Morristown, NJ).
The panelists moved the mouse to the right as the sweetness intensity
increased and to the left
as the intensity decreased. All samples were served at room temperature in a
balanced
random order, one at a time, and evaluated in duplicate. Panelists took a five-
minute break
between samples and cleansed their mouth with water and crackers. Results of
the time
intensity evaluation are shown in Figure 1.
[0071] The goal was for all of the samples in the study to have equivalent
sweetness. The
Reb A sample matched target sweetness of the sucrose reference very closely.
After
adjusting for R,R-monatin purity, the monatin sample and high-ratio monatin
blends were
sweeter than the sucrose reference (as outlined in Table 15). Surprisingly,
the sweetness of
monatin and rebiana blends did not fall somewhere between the 100% monatin and
100%
Reb A. Rather, the blends were all sweeter than either monatin or Reb A alone,
evidence of
an unexpected sweetness synergy between the two sweeteners.
Example 6
[0072] Sweetener samples were prepared to about 8 % SEV for quantitative
descriptive
analysis ("QDA") as described in Example 5.
[0073] A panel of nine trained and experienced panelists performed
quantitative descriptive
analysis ("QDA") on the samples using typical QDA methodology. The panelists
tasted a set
of control and test samples to develop the ballot with taste attributes, and
then were further
trained with reference solutions to practice scoring all of the samples on a
fifteen-point
intensity line scales developed for each attribute. Samples were served at
room temperature
in a balanced random order, one at a time, and evaluated in duplicate.
Panelists had a five-
minute break between evaluations and used water and crackers for rinsing.
Results of the
QDA testing are summarized in Table 17.
28
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 17: Quantitative Descriptive Analysis Results
Samples Time to Maximum Sour Bitter Metallic Drying
Reach Sweetness Astringent
Maximum
Sweetness
8% Sucrose 1.29 a 7.41 a 4.38 b 1.02 b 0.90 b 3.19 a
Monatin 1.57 a 7.27a 4.90 ab 1.22 b 0.85 b 3.47 a
Reb A 1.70a 6.34b 5.15 a 2.65a 1.46a 3.94a
90Monatin/ 1.48 a 7.38 a 4.69 ab 1.14 b 0.94 b 3.48 a
10Reb A
Blend
80Monatin/ 1.39 a 7.38 a 4.45 b 1.32 b 0.83 b 3.72 a
20Reb A
Blend
70Monatin/ 1.35 a 7.54 a 4.52 ab 1.56 b 0.92 b 4.03 a
30Reb A
Blend
60Monatin/ 1.61 a 7.65 a 4.77 ab 1.41 b 0.94 b 3.64 a
40Reb A
Blend
*Means followed by different letters are significantly different from each
other at p <0.05.
[0074] The data in Table 17 showed no difference between samples in time to
reach
maximum sweetness or in drying and astringent attributes. However, it was
surprising to
observe sweetness synergy between the monatin and Reb A blends, in that that
maximum
sweetness of the blends was greater than either monatin or Reb A alone. Blends
were also
less sour than either sweetener alone. Blending with monatin reduced the
bitter and metallic
attributes most closely attributed to Reb A.
Example 7
[0075] The sensory attributes of monatin, Reb A, and monatin/Reb A blends
using QDA
methodology were evaluated in water. A monatin solution and a Reb A solution
were first
formulated to match the sweetness of a 7% sucrose solution based on tasting
trials by a QDA
29
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
panel. Then the Beid ler equations for monatin and Reb A at room temperature,
neutral pH
were applied to formulate various blends of monatin and Reb A that had
equivalent
theoretical sweetness as the individual sweeteners (7% SEW). Formulations are
outlined in
Table 18.
Table 18. Test Sample Formulations
# Name Monatin Reb A Sucrose
Concentration Concentration Concentration
1 Sucrose 0% 0% 7%
2 Monatin 0.0036% 0% 0%
3 Reb A 0% 0.0417% 0%
4 80Monatin/20Reb A 0.0027% 0.0036% 0%
60Monatin/40Reb A 0.0019% 0.0084% 0%
6 40Monatin/60Reb A 0.0012% 0.0151% 0%
7 20Monatin/80Reb A 0.0006% 0.0251% 0%
[0076] Nine trained and experienced QDA panelists evaluated samples using
typical QDA
methodology. They trained with references, identified attributes to put on the
ballot, aligned
on tasting technique to be used, and practiced scoring the samples using the
ballot. Samples
were evaluated in individual sensory booths. All samples, except sucrose, were
evaluated in
a balanced sequential order one at a time. The sucrose sample was evaluated at
the end of
each session. Panelists were given 7% sucrose solution reference to calibrate
scoring before
the booth sessions. Samples size was 1.5 fluid ounces presented in a 2 ounce
soufflé cup and
samples were presented at room temperature. All samples were evaluated in
duplicate.
Panelists took a five-minute break between samples and were provided with
apples, carrots,
and sparking water and crackers to rinse and cleanse their palates during the
break. Results
of the QDA Analysis are summarized in Tables 19 and 20.
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
Table 19. QDA Panel Results - Mean Intensity Scores for Flavor Attributes
Time Maximum Herbal Bitterness* Non- Sweet
Sucrose
Maximum Sweetness* Flavor* Aftertaste
Flavors*
Sweetness* Immediately**
Sucrose 1.1 c 6.1 a 0.5 c 0.3 d 0,7d 5.1 ab
Monatin 1.4 c 6.1 ab 0.9 bc 0.9 c 2.0 c 4.5 c
_ _____________________________________________________________________
Reb A ' 2.0 a 6.3 ab 1.8 a 2.1 a - 3.3 a 4.8 bc
80Monatin/ 1.4 be 7.0 c 0.8 be 0.8 cd 1.9 c 5.3 ab
20Reb A
60Monatin/ 1.3 c 6.9 c 0.8 bc 1.1 bc 2.4 be 5.5 a
40Reb A
40Monatin/ 1.8 a 6.7 be 1.3 ab ' 1.2 bc 2.6 b 5.1 ab
60Reb A
20Monatin/ 1.7 ab 7.1 c 1.3 ab 1.6 ab 2.5 b 5.4 a
80Reb A
*Means followed by different letters are significantly different from each
other at p < 0.05.
Table 20. Mean Intensity Scores for Aftertaste Attributes
(after 30 and 60 Seconds of Swallowing)
Samples Sweet Herbal Bitter Non-Sucrose Sweet Drying/
Aftertaste Aftertaste Aftertaste Flavors Aftertaste Astringent
L,,
30sec* 30sec 30sec* Aftertaste 60sec Afterfeel
30sec* 60sec*
Sucrose 2.2 b 0.3 a 0.2 b 0.5 d 0.8 a 2.2 c
Monatin 2.7 ab 0.4 a 0.8 b 1.7 be 1.6 a 2.9 b
Reb A 2.7 ab 0.9 a 1.4 a 2.5 a 1.3 a 3.0 ab
80Monatin/ 2.9 a 0.3 a 0.5 b 1.4 c 1.4 a 2.9 b
20Reb A
60Monatin/ 3.2 a 0.5 a 0.7 b 1.8 be 1.4 a 2.9 b
40Reb A
_ _
40Monatin/ 3.0 a 0.6 a 0.7 b 2.0 ab 1.4 a 3.0 ab
60Reb A
20Monatin/ 3.0 a 0.7 a 1.3 a 2.0 ab 1.5 a 3.3 a
80Reb A
*Means followed by different letters are significantly different from each
other at p <0.05.
31
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
[0077] The data show that there was no significant difference in maximum
sweetness
between the sucrose, monatin and Reb A samples, which all scored a sweetness
intensity
score of about 6. All of the monatin/Reb A blends were formulated to be
equally sweet to the
single sweeteners using data from the individual sweeteners' Beidler
equations, but
surprisingly, all of the blends were actually significantly sweeter than
predicted,
demonstrating sweetness synergy between the two sweeteners.
[0078] In summary, Reb A sample scored significantly higher in: time to reach
max
sweetness (slower onset); herbal flavor; bitter taste/aftertaste; and non-
sucrose
flavors/aftertaste compared to sucrose, monatin, 20Monatin/20Reb A, and
60Monatin/40Reb
A samples. The 80Monatin/20Reb A blend scored the closest to sucrose in
herbal, bitter, and
non-sucrose flavors attributes followed by monatin and 60Monatin/40Reb A
blend. The
80Monatin/20Reb A scored closer to sucrose in herbal, bitter and non-sucrose
flavor
attributes followed by monatin, and 60Monatin/40Reb A blend. Rd) A scored the
most
different in its flavor/aftertaste profile except for sweetness compared to
sucrose.
Example 8
[0079] Samples of blends were made and tested to further evaluate an effect of
Reb A on the
linger of monatin.
Table 21. Blends of Monatin and Reb A
Sample ID Monatin Reb A(ppin) Monatin Reb A Total
(PPm) Calculated
% SEV % SEV
% SEV
Sucrose 8.0
Monatin 40 0 8.0 0.0 8.0
90Mon/10Reb 33 19 7.2 0.8 8.0
80Mon/20Reb 27 42 6.4 1.6 8.0
70Mon/30Reb 22 69 5.6 2.4 8.0
60Mon/40Reb 17 101 4.8 3.2 8.0
32
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 22. Formulations of the Monatin/Reb A Blends of Table 20
Concentration (% by Weight)
=
Ingredients 100/0 90/10 80/20 70/30 60/40
Water 99.819 99.818 99.812 99.812 99.810
Citric Acid 0.160 0.160 0.160 0.160 0.160
Trisodium 0.020 0.020 0.020 0.020 0.020
Citrate
R,R-Monatin 0.0040 0.0033 0.0027 0.0022 0.0017
Salt
Reb A 0.0000 0.0019 0.0042 0.0069 0.0101
Total 100.000 100.000 100.000 100.000 100.000
[0080] Samples of the solutions were served at ambient temperature
(approximately 70 to
72 F). Six panelists were instructed to put the entire sample (about 1 ounce)
in their mouth,
and rate the initial intensity of sweetness using a 9-point intensity scale
(0=none and
9,--extrernely sweet) while holding the sample for 10 seconds and moving their
tongue around
in their mouth. Each of the panelists then swallowed or spit the sample, and
continued rating
sweetness intensity of each sample every 10 seconds. Al! samples were served
blind coded in
a balanced random order to the panelists. The panelists rated each sample in
duplicate. Table
23 shows the mean sweetness intensity of the six panelists at four specific
points in time.
Table 23. Mean Sweetness Intensity Results
Sample Description Initial lOsec 20sec 30sec
Sucrose 7.30 7.05 5.05 3.42
Monatin 7.63 7.35 5.70 3.90
90Mon/10Reb 7.28 6.48 4.97 2.88
80Mon/20Reb 7.76 7.50 5.45 3.69
70Mon/30Reb 7.68 7.26 5.42 3.63
60Mon/40Reb 7.45 7.04 5.39 3.65
[0081] Each panelist recorded the sweetness intensity at 10 second intervals
up to 80
seconds. Each panelist's rating for the duplicate samples was then averaged
and plotted. The
area under the curve (AUC) was calculated for each sample for each panelist. A
linger index
was calculated by dividing the AUC for each test sample by the AUC for sucrose
for that
panelist. This compares each panelist's linger response of the blends relative
to that
panelist's response to sucrose. The linger index for each panelist, as well as
the mean value
and standard deviation across panelists, is shown below.
33
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
Table 24. Linger Index Values
Panelist Panelist Panelist Panelist Panelist Panelist
Standard
1 2 3 4 5 6
Mean Deviation
Sucrose 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00
Monatin 1.13 0.86 1.44 1.20 1.22 1.17 1.17 0.18
90Mon/10Reb 0.83 0.84 1.28 1.16 1.09 0.75 0.99 0.21
80Mon/20Reb 1.32 0.97 1.32 1.18 1.12 0.89 1.13 0.18
70Mon/30Reb 1.06 0.84 1.30 1.37 1.15 0.96 1.11 0.20
60Mon/40Reb 0.97 1.00 1.27 1.00 0.89 1.11 1.04 0.13
Monatin without Reb A had a mean linger index value of 1.17.
Example 9
[0082] Solutions containing blends of monatin with other steviol glycosides
(i.e., stevia leaf
extract, rebaudioside-B, rebaudioside-D, stevioside) were exposed to light and
underwent
similar evaluations as described above for monatin and Reb A blends. Lemon-
lime flavored
carbonated beverages were exposed to fluorescent light (about 5900 Lux)
continuously in a
light box at 25 C for nine weeks. Solutions of about 9 inM citric
acid/trisodium citrate (pH
of approximately 3.0) in high purity water were sweetened with monatin (about
6.4 % SEV)
and equirnolar steviol glycoside concentrations (about 1.6 % SEV), flavored
with lemon-lime
flavor, and preserved with approximately 150 ppm sodium benzoate. The
solutions were
carbonated in four liter batch sizes using an Armfield carbonator with
approximately 3.5
liters of carbon dioxide (0'CO2") per liter of solution and then transferred
to 12 ounce clear,
plastic bottles. Caps were placed on the bottles after filling. Multiple
bottles of each sample
were created since a bottle was sacrificed when the measurements below were
taken.
[0083] Total indolc concentrations (monatin and its intramolecular
rearrangement products,
monatin lactone and monatin lactam) were quantified by chromatography using an
Agilent
1290 Infinity UHPLC at 45 C using with a Waters Acquity UPLC HSS T3 2.1x1
00mm, 1.8
p.m C18 column (Waters Corp., Milford, MA) and UV detection at 280 nm. The
compounds
were eluted by a gradient mobile phase consisting of (A) 0.1% formic acid in
10 mM
ammonium formate solution and (B) 100% acetonitrile. The integrated peak areas
for the
three (3) compounds were expressed in terms of monatin concentration (ing/L)
based on a
standard curve prepared for monatin. Monatin lactone and monatin lactam were
quantified
using the monatin standard curve and empirically determined response factors.
34
CA 02821306 2013 06 11
WO 2012/083251
PCT/US2011/065643
[0084] Total indole concentrations are shown in Table 25 for each of the
blends. The percent
of the original indole concentration remaining is shown in parentheses below
each value. The
addition of RA80, rebaudioside-B ("Reb-B"), rebaudioside-D (`Reb-D"), and
stevioside did
not reduce the proportion of indole that degraded relative to the solution
containing monatin
alone (untreated).
CA 02821306 2013 06 11
WO 2012/083251
PCT/US2011/065643
Table 25. Total Indole Concentrations for Various Blends of Monatin and
Steviol
Glycosides Exposed to Light
Steviol Glycoside Blended with Monatin
Untreated Reb A RA80 Reb B Reb D Stevioside
Steviol
Glycoside 0 35 35 32 44 31
(ppm)
Steviol
Glycoside 0 0.0362 0.0374 0.0398 0.0390 0.0385
(mM)
Total Indole (ppm)
24.6 24.6 24.6 24.5 24.6 24.7
Week 0
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0)
23.8 24.0 24.4 23.3 23.8 19.2
Week 1
(96.6) (97.5) (99.1) (94.8) (96.6) (77.9)
23.7 24.1 25.0 23.4 23.6 23.8
Week 2
(96.3) (97.7) (101.5) (95.3) (96.0) (96.5)
24.7 22.6 23.8 22.0 22.6 23.5
Week 3
(100.3) (91.6) (96.8) (89.4) (91.7) (95.1)
23.2 21.7 23.0 20.9 22.8 23.6
Week 4
(94.3) (87.9) (93.6) (85.3) (92.7) (95.6)
23.7 23.0 22.5 22.4 23.6 23.4
Week 5
(96.6) (93.3) (91.6) (91.2) (96.0) (95.0)
21.7 19.8 21.3 18.8 21.1 20.8
Week 7
(88.3) (80.5) (86.6) (76.5) (85.7) (84.5)
19.4 19.7 20.6 16.6 18.6 19.5
Week 8
(78.7) (79.8) (83.8) (67.8) (75.4) (79.1)
15.2 16.9 15.2 15.4 13.8 15.0 '
Week 9
(61.8) (68.7) (61.8) (62.9) (56.1) (60.7)
36
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
[0085] The yellow color intensity (b*) of each sample was also measured using
a HunterLab
ColorQuest XE colorimeter (Hunter Associates Laboratory, Inc., Reston, VA). As
shown in
Table 26, the addition of RA80, Reb B, Reb D, and stevioside did not reduce
yellow
discoloration relative to the solution containing monatin alone (untreated).
Table 26. Yellow Color Development by Various Blends of Monatin and Steviol
Glycosides Exposed to Light
Steviol Glycoside Blended with Monatin
Untreated Reb A RA80 Reb B Reb D Stevioside
Steviol .
Glycoside 0 35 35 32 44 31
(PM)
Steviol
Glycoside 0 0.0362 0.0374 0.0398 0.0390 0.0385
(m M)
Yellow Color Intensity (b*) _
Week 0 0.12 0.12 0.14 0.13 0.11 0.12
Week 1 0.18 0.28 0.28 0.25 0.33 0.18
Week 2 0.31 ' 0.27 0.40 0.40 0.44 0.33
Week 3 0.26 0.45 0.37 0.58 - 0.50 0.35
Week 4 0.43 0.60 0.52 0.76 0.69 0.48
Week 5 ' 0.50 0.59 0.72 0.62 0.64 0.48
_____
Week 7 0.65 0.83 0.76 1.03 0.84 0.81
Week 8 1.25 ' 1.00 - 1.07 1.66 1.37 1.26
Week 9 1.52 0.94 1.63 1.36 1.84 1.60
[0086] The same samples sacrificed for indole and yellow color measurements
above were
tasted to evaluate the presence or absence of musty off-flavor (Table 27). The
addition of
RA80, Reb 13, Reb D. and stevioside did not delay the onset of musty off-
flavor (all samples
were musty after 2 weeks) but the intensity of the musty off-flavored tended
to be lower in
the presence of steviol glycosides compared to monatin alone (untreated).
37
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 27. Musty Off-Flavor Development by Various Blends of Monatin and
Steviol
Glycosides Exposed to Light
Steviol Glycoside Blended with Monatin
Untreated Reb A RA80 Reb 13 Reb D Stevioside
Steviol
Glycoside 0 35 35 32 44 31
(PPm)
Steviol
Glycoside 0 0.0362 0.0374 0.0398 0.0390 0.0385
(mM)
"Musty Off-Flavor Intensity Rating
Week 0 0 0 0 0 0 0
Week! 0 0 0 0 0 0
Week 2 3 2 2 3 2 3
Week 3 4 3 3 3 2 3
Week 4 4 4 3 3 2 3
Week 5 5 4 4 4 3 4
'The intensity of musty off-flavor was rated on a scale from 0 to 9 where 0 =
none, 1 = trace,
2 = faint, 3 = slight, 4 = mild, 5 = moderate, 6 = definite, 7 = strong, 8 =
very strong, and 9 ¨
extreme.
Example 10
[0087] Unflavored still beverages (non-carbonated) were exposed to fluorescent
light (-5900
Lux) continuously in a light box at 25 C for .10 days. Solutions of about 9 mM
citric
acid/trisodium citrate (pH of approximately 3.0) in high purity water were
sweetened with
monatin (about 6.4% SEV) and equirnolar concentrations of steviol glycoside
(about 1.6%
SEV) and preserved with approximately 150 ppm sodium benzoate. The solutions
were
transferred to 60 mL clear, plastic bottles. Caps were placed on the bottles
after filling.
Multiple bottles of each sample were created since duplicate bottles of each
treatment were
sacrificed when the measurements below were taken.
[0088] Total indole concentrations (monatin and its intramolecular
rearrangement products,
monatin lactone and monatin lactam) were quantified by reversed phase
chromatography
using an Agilent 1290 Infinity UHPLC at 45 C with a Waters Acquity UPLC HSS T3
38
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
2.1x100mm, 1.8 pm C18 column (Waters Corp., Milford, MA) and UV detection at
280 11111.
The compounds were eluted by a gradient mobile phase consisting of (A) 0.1%
formic acid in
mM ammonium formate solution and (B) 100% acetonitrile. The integrated peak
areas for
the three (3) compounds were expressed in terms of monatin concentration
(mg/L) based on a
standard curve prepared for monatin. Monatin lactone and monatin lactam were
quantified
using the monatin standard curve and empirically determined response factors.
[0089] Total indole concentrations are shown in Table 28 for each of the
blends. The percent
of the original indole concentration remaining is shown in parentheses below
each value. The
addition of Reb A reduced the proportion of indole that degraded relative to
the solution
containing monatin alone (untreated). However, addition of RA80, Reb B, Reb D,
and
stevioside did not reduce the proportion of indole that degraded. Thus, the
observed effects
for Reb A on indole recovery were unique compared to the other steviol
glycosides.
39
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 28. Total Ind le Concentrations for Various Blends of Monatin and
Steviol
Glycosides Exposed to Light
Steviol Glycoside Blended with Monatin
Untreated Reb A RA80 Reb B Reb D Stevioside
Steviol
Glycoside 0 35 35 32 44 31
(PPm)
Steviol
Glycoside 0 0.0362 0.0374 0.0398 0.0390 0.0385
(mM)
Total Indole (ppm)
26.1 25.8 25.8 25.7 25.9 26.2
Day 0
(100.0) (100.0) (100.0) (100.0) (100.0) (100.0)
26.2 25.4 25.5 25.1 26.2 25.4
Day 2
(100.4) (98.4) (98.8) (97.7) (101.2) (96.9)
23.7 24.0 22.7 22.6 23.3 22.1
Day 4
(90.8) (93.0) (88.0) (87.9) (90.0) (84.4)
19.4 20.0 18.6 18.7 19.4 19.0
Day 6
(74.3) (77.5) (72.1) (72.8) (74.9) (72.5)
15.8 16.8 13.5 12.5 13.9 13.0
Day 8
(60.5) (65.1) (52.3) (48.6) (53.7) (49.6)
15.7 16.2 14.1 13.3 14.5 13.4
Day 10
(60.2) (62.8) (54.7) (51.8) (56.0) (51.1)
[0090] The color of each sample was also measured using a HunterLab ColorQuest
XE
colorimeter (Hunter Associates Laboratory, Inc., Reston, VA). As shown in
Table 29, the
addition of RA80, Reb B, Reb D, and stevioside did not reduce yellow
discoloration relative
to the solution containing monatin alone (untreated). However, addition of Reb
A reduced
yellow discoloration and so its ability to reduce discoloration by monatin
solutions exposed
to light was unique compared to the other steviol glycosides.
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 29. Yellow Color Development by Various Blends of Monatin and Steviol
Glycosides Exposed to Light
Steviol Glycoside Blended with Monatin
Untreated Reb A RA80 Reb B Reb D
Stevioside
Steviol
Glycoside 0 35 35 32 44 31
(PPm)
Steviol
Glycoside 0 0.0362 0.0374 0.0398 0.0390 0.0385
(mM)
Yellow Color Intensity (b*)
Day 0 0.02 0.04 0.02 0.03 0.03 0.06
Day 2 0.12 0.11 0.15 0.22 0.16 0.19
Day 4 0.40 0.34 0.64 0.60 0.52 0.71
Day 6 0.53 0.42 0.70 0.68 0.59 0.81
Day 8 1.44 1.23 1.99 2.24 1.90 2.18
Day 10 1.51 1.33 1.83 2.05 1.77 2.06
Example 11
[0091] Whether mixtures of monatin, stevia, and /o han guo, exhibit
quantitative sweetness
synergy in binary blends with each other was investigated.
[0092] Quantitative synergy was identified by evaluating the sweetness
response for each of
the sweeteners alone, creating concentration-response ("C/R") curves for each
sweetener, and
then evaluating the sweetness of the various blends against predicted results
extrapolated
from the C/R curves.
[0093] Monatin (2R, 4R-monatin monopotassium salt) and stevia (95%
rebaudioside A) were
obtained from Cargill. Lo han guo (PureLo 46% mogroside V) was obtained from
Biovittoria. Al] sweetener samples and sucrose references were prepared in a
citric/citrate
buffer consisting of citric acid monohydrate (0.158%, w/v) and trisodium
citrate dihydrate
(0.022%, w/v).
41
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
[0094] Sweetness evaluations were made by matching the sweetness of the test
solutions to
that of a known sucrose standard. A panel of 8-10 trained, experienced judges
was presented
with a range (2-12%; w/v) of sucrose reference solutions and a single test
sample. Each test
solution was monatin, Reb A, lo hart guo, or a blend. Panelists were
instructed to taste the
test sample and then to identify the sucrose reference sample that matched the
perceived
sweetness of the test sample. If the sweetness of the test sample was judged
to fall between
that of two references, panelists were instructed to estimate sweetness to one
decimal point.
A rigorous rinsing procedure was adopted following each sip of test or
reference solution to
minimize the potential for sweetness adaptation and carry-over. Panelists
evaluated each
sample in duplicate. Results for each test sample were averaged.
[0095] To create the C/R curves, each sweetener was evaluated at 4 or 5 evenly
spaced points
across a range of sucrose equivalence values ("% SEV"). Monatin was evaluated
at 10-40
ppm, stevia at 120-600 ppm, and lo han guo at 200-800 ppm. The sweetener
concentrations
tested and measured % SEV values are summarized in Table 30. The Beidler
equation was
applied to these data to create an individual C/R curve for each sweetener.
Table 30. Concentration-Response Determinations of Monatin, Reb A, and
La han gun
Sweetener Concentration (ppm) % SEV (% sucrose)
Monatin 10 3.9
20 6.2
30 7.8
40 9.1
Stevia 120 3.8
240 5.3
360 5.8
480 6.1
600 7.1
Lo han guo 200 3.9
350 4.6
500 6.0
650 6.9
800 7.0
[0096] Binary blends of the sweeteners were then prepared for the synergy
evaluations. The
% SEV target for the blends was 7-8% SEV, with varying ratios of sweetness
contribution
from each sweetener. The predicted % SEV for each blend, extrapolated from the
Beidler
42
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
C/R curves prepared from the data set gathered in this specific study from
this specific panel,
and the actual % SEV measured by sensory panel are reported in Table 31.
Table 31. Sucrose Equivalence of Theoretical 8% Sucrose Blends
Sweetener Calculated % Sweetener Predicted % Actual % %
SEV
Blend SEV Ratio Blend SEV SEV Difference
(% sucrose Concentration (% sucrose (% sucrose (% sucrose
equivalence) (ppm) , equivalence) equivalence) _equivalence)
Stevia/ 1.6/5.8 65/475 7.4 7.4 0
Lo han guo
2.6/5.1 120/355 7.6 7.1 -0.5
3.3/4.3 175/265 7.6 7.2 -0.4
3.9/3.4 240/185 7.3 6.7 -0.6
4.7/2.4 340/115 7.1 6.3 -0.8
Monatin/Stevia 2.2/4.7 5/340 6.9 8.3 1.4
3.2/3.9 8/240 7.1 8.9 1.8
4.1/3.3 11/175 7.4 , 8.9 1.5
5.1/2.6 15/120 7.7 8.5 0.8
6.0/1.6 19/65 7.6 8.4 0.8
Monatin/ 2.2/5.8 5/475 8.0 8.1 0.1
Lo han guo _
3.2/5.1 8/355 8.3 8.0 -0.3
4.1/4.3 11/265 8.4 8.3 -0.1
5.1/3.4 15/185 8.5 . 8.7 0.2
6.0/2.4 19/115 8.4 7.9 -0.5
[0097] The results demonstrate that stevia/To han guo blends are almost
additive, as are
monatin/ lo han guo blends. However, monatin/stevia blends demonstrated
quantitative
synergy in that the measured sweetness was much higher than predicted. The
percent
increased sweetness of the inonatin/stevia blends is summarized in Table 32.
Table 32. Synergy of Monatin/Stevia Blends
Sweetener Concentrations Predicted Measured Sweetness
Blend (PPm) % SEV % SEV Synergy
(%)
Monatin/Stevia 5/340 6.9 8.3 20.3
8/240 7.1 8.9 25.3
11/175 7.4 8.9 20.3
15/120 7.7 8.5 10.4
19/65 7.6 8.4 10.5
43
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Example 12
[0098] The attributes of monatin and Reb A blends in a tabletop sweetener
application were
investigated. Ingredients and the lot numbers of ingredients used to make
tabletop sweetener
formulations are identified in Table 33.
Table 33. Ingredients Used
Ingredients Lot Number
Dextrose* 03186883
R, R, Monatin* 1392
Reb A* 1040
Sugar Retail
*Available from Cargill Inc., Minneapolis, MN
[0099] Tabletop sweeteners were formulated to be equisweet to two teaspoons of
sucrose
(8.5 g). The amount of monatin and Reb A used in each formulation was
calculated from the
Beidler equations for each sweetener, determined previously in Example 5. The
Beidler
equation relating sucrose equivalence value ("% SEV") to anhydrous monatin
concentration
(mg/L) in water was derived as: % SEV =26.7* [monatin] / (69.6 + [monatin]).
For Reb A:
% SEV=11.26* [Reb A] / (253+ [Reb A]). Dextrose was considered to be 75% as
sweet as
sucrose in formulating. Packet weight for each sweetener was 1 gram using
dextrose for the
majority of the bulk.
[00100] Six sweetener blends with varying ratios of monatin and Reb A were
evaluated in
this study. The blends of monatin and Reb A were expressed on a sweetness
basis. Because
of monatin's greater sweetness potency it provided 90% of the sweetness in the
combination.
The other blends ratios were about 100/0, 75/25, 50/50, 25/75 and 0/100 of
monatin to Reb
A.
[00101] To prepare each sweetener sample for tasting, one packet (1 gram) of
each
sweetener blend was added to 170g of reverse osmosis-purified water and
stirred to dissolve.
Sensory attributes of these products were determined by an expert panel of
sixteen
applications and sensory scientists from the Cargill Research Center who were
all
experienced in working with and tasting monatin and Reb A. Samples were
evaluated in
individual sensory booths. Panelists were offered water and crackers to rinse
their palates
and then waited one minute between samples. Three samples were evaluated in
each tasting
44
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
session. All three tasting sessions were completed in one day. The blind coded
samples were
presented in a balanced rotation and served at room temperature in 2 fluid
ounce soufflé cups.
[00102] The panelists were asked to evaluate each sample using a nine-point
scale. A blind
coded sucrose sample was also included in the test and evaluated with the
other samples. Six
attributes were evaluated, including sweetness intensity and sweetness linger.
Mean scores
for each of the attributes are compiled in Table 34.
Table 34. Average Scores for Various Blends of Monatin and Reb A
Compared to Sucrose
Ratio of % Sweetness from Monatin/Reb A
Attribute 100%
90/10 75/25 50/50 25/75 100% Sucrose
Monatin Reb A Control
Sweetness 4.2 4.3 4.9 4.4 4.1 4.3 4.6
Onset*
Sweetness 5.1 4.9 6.1 5.4 5.9 5.3 4.8
Intensity**
Sweetness 3.8 3.9 4.1 4.1 3.2 3.3 4.6
Quality*** _
Sweetness 6.1 6.2 6.6 6.4 6.4 6.3 5.2
Linger**
Bitterness 5.7 5.8 5.9 5.6 6.6 6.5 - 5.3
Intensity**
*9-poin1 onset scale (1-"Extremely Slower"; 5-"Samc as Control"; 9=" Extremely
Faster")
**9-point linger scale (1-"Extremely Less"; 5-"Same as Control"; Extremely
More")
***9-point quality scale (1-"Extremely Worse"; 5="Same as Control"; 9="
Extremely Better")
[00103] The data show that the sweetness intensity of the 75/25 (6.1), 50/50
(5.4), and 25/75
(5.9) blends was greater than the sweetness intensity of either monatin (5.1)
or Reb A (5.3)
alone. This result is evidence of the sweetness synergy between the two
sweeteners.
[00104] The greater sweetness intensity may also have driven the higher
overall quality, and
sweetness onset scores for the 75/25 (4.1 and 4.9) and 50/50 (4.1 and 4.4)
blends relative to
either monatin (3.8 and 4.2) or Reb A (3.3 and 4.3) alone. The lower
difference from control
score for the blends indicated the quality was better and closer to that of
the sucrose control.
[00105] No substantial differences were observed between treatments with
respect to
sweetness linger. Though blends of monatin and Reb A typically reduce the
lingering
sweetness, it is likely that that the higher sweetness intensity of the blends
also increased the
sweetness linger such that no difference was observed between the blends and
either monatin
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
or Reb A alone. It was also observed that greater ratios of monatin in the
blends reduced the
bitterness of Reb A.
Example 13
[00106] The objective of this study was to demonstrate sweetness synergy
between monatin
and Reb A at 4% SEV (Table 35), and monatin and RA80 at 7% SEV (Table 36).
[00107] A group of 7 to 8 food scientists participated in this study. The
participants were all
experienced tasters and have been working with monatin and Reb A samples.
Sample
concentrations (Tables 35 and 36) at 4% and 7% SEV were determined using
Beidler
equations of each sweetener. Samples were mixed in filtered water, and tasted
at room
temperature. Each participant received 1.5oz of sample in 2oz souffle cups.
They were served
a set of two samples at a time, asked to taste the samples, and indicate which
sample was
sweeter than the other. In each set, one sample was the blend, and the other
was either
monatin or a Reb A sample.
Table 35. Samples at 4% SEV Calculated Using Beidler Curves for Each
Sweetener
Monatin Reb A Relative Sweetness
Contribution
(PP111) (PPI11) (Monatin/Reb A Blend)
12 0 100/0
0 140 0/100
9 19 80/20
Table 36. Samples at 7% SEV Calculated Using Beidler Curves for Each
Sweetener
Monatin RA80 (ppm) Relative Sweetness Contribution
(PPm) (Monatin/RA80 Blend)
25 0 100/0
0 376 0/100
18 38 80/20
46
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 37, Tasting Results for Monatin and Reb A Samples at 4% SEV
Sample Set Total # of # of Panelists Choosing
SEV
Panelists the Blend as Sweeter
4% Monatin vs. 7 6 (86%)
80Mon/20RebA Blend
4% Reb A vs. 8 4(50%)
80Mon/20RebA Blend
[00108] As the data in Table 37 show, the 80/20 blend of monatin and Reb A was
found to
be sweeter compared to monatin alone by 86% of the panelists and compared to
Reb A alone
by 50% of the panelists at 4% SEV.
Table 38. Tasting Results for Monatin and RA80 Samples at 7% SEV
Sample Set Total 4 of # of Panelists Choosing
SEV
Panelists the Blend as Sweeter
7% Monatin vs. 7 7 (100%)
80Mon/20RebA80
7% RA80 vs. 80Mon/20 7 7 (100%)
RebA80
[00109] As the data in Table 38 show, the 80/20 blend of monatin and RA80 was
found to
be sweeter compared to both monatin and RA80 alone by 100% of the panelists at
7% SEV.
[00110] These results demonstrate examples of sweetness synergy between
monatin and Reb
A samples at different % SEV levels. When the sweeteners blend together the
resulting
sweetness of the blend is higher than that of the individual sweeteners.
Example 14
[00111] The objective of this study was to assess sweetness linger of no-sugar
added
applesauce using monatin only compared to variations using blends of monatin
with Reb A
(95% rebaudioside-A). In this study, monatin (2R, 4R-monatin rnonopotassium
salt) and Reb
A, both available from Cargill, were combined with commercially-available
MOTT'S
47
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Natural no-sugar added applesauce. This natural applesauce contained 12 grams
of sugar
from apples in a 1/2 cup serving (123 grams). Additional sweetness from
monatin only or
monatin/Reb A blends in the natural applesauce was about 10% SEV, yielding a
similar
sweetness intensity as M011 S Original applesauce (25 grams of sugar per 1/4
serving).
Three different sweetened variations of the natural applesauce were prepared
(Table 39).
Table 39. Formulated Levels of Monatin and Reb A in Commercially Available
Natural, No-Sugar Added Applesauce
Monatin Reb A Relative Sweetness
Contribution
(ppm) (PPm)
(Monatin/Reb A)
42 0 100/0
30 55 77/23
20 140 56/44
[00112] Sensory evaluation of the sweetened applesauces was conducted by a
panel of 6
application and sensory scientists from the Cargill Research Center, who were
all
experienced in working with and tasting monatin and Reb A. Panelists were
provided water
and crackers to rinse their palates between tasting samples. Applesauce
samples were served
at refrigeration temperature (4 C) in coded 2 ounce souffle cups.
[00113] Two different paired sets of applesauce were evaluated by panelists in
a balanced
rotation in a single day: (a) 100% monatin sweetened and 77%/23% monatin/ Reb
A
sweetened blend and (b) 100% monatin sweetened and 56%/44% monatin/ Reb A
sweetened
blend. For each set, panelists were instructed to consume the entire sample,
cleanse their
palate, consume the entire second sample, then select the coded sample which
had a longer
lingering sweetness. Results are shown in Table 40.
48
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 40. Number (n ¨ 6) of Panelists Indicating Longer Lingering Sweetness of
Applesauces Sweetened with Monatin Only Compared to Those with Monatin/
Reb A Blends
77% 56%
100% Monatin/ 100% Monatin/
Monatin 23% Monatin 44%
Reb A Reb A
6 0 4 2
[00114] The monatin-sweetened applesauce was identified as having more
lingering
sweetness by all panelists in comparison to the applesauce sweetened with
either the
77%/23% or the 56%/44% blend of monatin to Reb A.
[00115] These results indicate that the presence of stevia-based sweeteners,
such as Reb A,
reduced the lingering sweetness profile of monatin in applesauce. It should
also be noted that
this effect was observed in the presence of inherent fruit sugars from the
apples.
Example 15
[00116] The objective of this study was to assess sweetness linger of
sweetener blends
shown in Table 41 compared to monatin, Reb A, RA80, and sucrose over time.
Trained
Quantitative Descriptive Analysis ("QDA") panelists evaluated these blends and
individual
sweeteners mentioned above for sweetness intensity and linger at different
sweetness blend
ratios of 80/20, 60/40, and 50/50, and at different % SEV levels of 4%, 7%,
and 12%.
Table 41. Sweetener Blends
Sweetener Blend % SEV
1. *80Monatin/20RebA 4%, 7%, and 12%
2. 60Monatin/40RebA 7%
3. 50Monatin/50RebA 7%
4. 80Monatin/20RA80 7%
*In this blend 80% of total sweetness comes from monatin and 20% comes from
Reb
A; other blends are composed in a similar fashion.
49
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Trained Panel Method
[00117] Training: QDA panelists, highly trained and experienced on-call taste
panelists,
had two to three sessions of training prior to testing. The panelists were
trained with
reference sucrose samples to practice rating sweetness intensity on the scale,
and to determine
best technique for rating sweetness and sweetness linger. They determined the
best technique
to be to take a sip from the sample, hold sample in the mouth for about 10
seconds to allow
the sweetener to reach its peak sweetness, and rate the initial sweetness
intensity at peak
within 10 seconds. Then they spit out the samples to rate remaining sweetness
(linger) in their
mouth at every 10 seconds. They waited 10 - 15 minutes between samples and
rinsed with
sparkling water to help cleanse their palate and prevent carryover from
previous samples. All
of the sweeteners including the blends were adjusted to match in sweetness
intensity initially
at 10 seconds evaluation. Sweetness matching was carried out using panelists'
feedback
during training.
[00118] Testing: Panelists were served 1.5 oz of each sample in 2 oz soufflé
cups at room
temperature (68-72 F) for the testing session. They followed the technique
determined during
training: They took one sip of the sample, held the sample in their mouths for
10 seconds, and
rated sweetness intensity at peak. They then spit the sample, and rated
remaining sweetness
intensity every 10 seconds. The sweetness intensity was rated up to 50 seconds
for 4% SEV,
up to 70 seconds for 8% SEV, and up to 80 seconds for 12% SEV samples. The
panelists
evaluated the samples with a 10 - 15 minute break in between samples. They
rinsed their
mouths with sparkling water after their evaluation was finished. All samples
were evaluated
in a balanced sequential order one at a time. The panelists were given
appropriate sucrose
sample (e.g., 4%, 7%, or 12%) to calibrate before the testing session.
[00119] Ballot: Samples were rated on a 15 point sweetness intensity scale.
none strong
0 1 2 3 4 5 6 7 8 9 10 11 12 13
14 15
[00120] Data Analysis: Data was collected in replicate, and analyzed by ANOVA
at 95%
confidence interval using SPSS software (Version 15.0 for Windows).
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
[00121} QDA Panel Sample and Panelists Information: Tables 42 to 47 show
concentrations (%w/v) of each sample used, and number of panelists attended
each test.
Samples were prepared in filtered water.
Table 42. Concentrations of Monatin, Reb A, 80Monatin/20RebA Blend, and
Sucrose
Formulated at 7% SEV
Name Monatin Reb A Sucrose
Concentration Concentration Concentration
Sucrose 0% 0% 7%
Monatin 0.0036% 0% 0%
Reb A 0% 0.0417% 0%
80Mon/20RebA 0.0020% 0.0035% 0%
Panelist Number: nine trained panelists
Table 43. Concentrations of Monatin, Reb A, 60Monatin/40RebA Blend, and
Sucrose
Formulated at 7% SEV
Name Monatin Reb A Sucrose
Concentration Concentration Concentration
Sucrose 0% 0% 7%
Monatin 0.0036% 0% 0%
Reb A 0% 0.0417% 0%
60Monatin/40RebA 0.0016% 0.0069% 0%
Panelist Number: eleven trained panelists
Table 44. Concentrations of Monatin, Reb A, 50Monatin/50RebA Blend, and
Sucrose
Formulated at 7% SEV
Name Monatin Reb A Sucrose
Concentration Concentration Concentration
Sucrose 0% 0% 7%
Monatin 0.0036% 0% 0%
Reb A 0% 0.0417% 0%
50Monatin/50RcbA 0.0013% 0.0092% 0%
Panelist Number: ten trained panelists
51
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
Table 45. Concentrations of Monatin, Reb A, 80Monatin/20RebA Blend, and
Sucrose
Formulated at 4% SEV
Name Monatin Reb A Sucrose
Concentration Concentration Concentration
Sucrose 0% 0% 4%
Monatin 0.0015% 0% 0%
Reb A 0% 0.014% 0%
80Monatin/20RebA 0.0003% 0.0084% 0%
Panelist Number: eight trained panelists
Table 46. Concentrations of Monatin, Reb A, 80Monatin/20RebA Blend, and
Sucrose
Formulated at 12% SEV
Name Monatin Reb A Sucrose
Concentration Concentration Concentration
Sucrose 0% 0% 12%
Monatin 0.0090% 0% 0%
80Mon/20RebA 0.0050% 0.0062% 0%
Panelist Number: nine trained panelists
Table 47. Concentrations of Monatin, RA80, 80Monatin/20RA80 Blend, and Sucrose
Formulated at 7% SEV
Name Monatin RA80 Sucrose
Concentration Concentration Concentration
Sucrose 0% 0% 7%
Monatin 0.0036% 0% 0%
RA80 0% 0.0417% 0%
80Monatin/20RA80 0.0025% 0.0033% 0%
Panelist Number: six trained panelists
[00122] Results: Sweetness intensity scores collected over time can be seen in
the following
tables for inonatin, Reb A, and various blends of monatin/RebA (80/20, 60/40,
and 50/50)
samples formulated at 7%, 4%, and 12% SEV.
52
CA 02821306 2013 06 11
WO 2012/083251 PCT/US2011/065643
Table 48. Sweetness Intensity Change Over Time for Monatin, Reb A,
80monatin/20RebA, and Sucrose Formulated at 7% SEV
Samples lOsec 20sec 30sec 40sec 50sec 60sec 70sec
Monatin 7.0 a* 6.3 a 4.5 a 2.9 a 1.6 a 0.6 a
0.1 a
Reb A 7.0 a 6.2 a 4.4 a 2.4 ab 1.2 ab 0.4 ab
0.1 a
80Monatin/20RebA 6.9 a 5.6 b 3.7 b 2.1 be 1.0 be 0.3 be
0.1 a
Sucrose 6.9a 5.1 b 3.0 b 1.7c 0.6c 0.1 c
0.0 a
p-values 0.460 0.011 0.008 0.003 0.001 0.002
0.658
* Means followed by different letters are significantly different from each
other at p < 0.05.
Table 49, Sweetness Intensity Change Over Time for Monatin, Reb A,
60Monatin/40RebA, and Sucrose Formulated at 7% SEV
Samples lOsec 20sec
30sec 40sec 50sec 60sec 70sec
Monatin 6.9 a 5.8 a 4.0 a 2.7 a 1.8 a 0.8 a 0.4
a
Re-b-A--- = 6.7 a--6.1 a 4.2 a 2.8 a 1.6 ab 0.8 a 0.2
a
60Monatin/40RebA 7.0 a 5.7a 3.6a 2.1 b 1.3 b 0.4 b 0.2a
Sucrose 7.0 a 4.6 b 2.7 b 1.7 b 0.6 c 0.1 b
0.1 a
p-values 0.201 0.001 0.003 0.025 0.008 0.006
0.181
Table 50. Sweetness Intensity Change Over Time for Monatin, Reb A,
50Monatin/50RebA, and Sucrose Formulated at 7% SEV
Samples lOsec
20sec 30sec 40sec 50sec 60sec 70sec
Monatin 6.9 a 6.1 a 4.4 a 2.9 a 1.8 a 0.9 a
0.3 a
Reb A 6.8 a 6.3 a 4.8 a 3.1 a 2.0 a 1.0 a
-0.4 a--
50Monatin/50RebA 6.9a - 6.0 a 3.8 b 2.3 b 1.1 1-) 0.4 b
0.0 a
Sucrose 6.9 a 4.6 b 2.6 c 1.3 c 0.5 c - 0.0 c 0.0 a
p-values 0.771 0.000 0.000 0.000 0.000 0.000
0.004
Table Si. Sweetness Intensity Change Over Time for Monatin, Reb A,
80Monatin/20RebA, and Sucrose Formulated at 4% SEV
Samples lOsec 20sec 30sec 40sec 50sec
Monatin 3.84 a 3.38 a 2.31 a 1.31 a 0.44 a
Reb A 3.91 a 3.34a 2.09 a 1.19 a 0.34 a
80Monatin/20RebA 4.00 a 3.31 a 2.06 a 1.06 ab 0.34 a
'-Sucrose-- 4.03 a 2.75 b 1.50 b 0.66 b 0.16 a
p-values 0.329 0.025 0.014 0.021 0.18
53
CA 02821306 2013-06-11
WO 2012/083251 PCT/US2011/065643
Table 52. Sweetness Intensity Change Over Time for Monatin, 80Monatin/20RebA,
and
Sucrose Formulated at 12% SEV
Samples lOsec 20sec 30sec 40sec 50sec 60sec 70sec 80sec
Monatin 11.9 a10.6 a 7.9 a 5.5 a 3.6 a 2.3 a 1.2 a
0.5 a
80Monatin/20RebA* 11.0 a - 10.4 a 7.7a -5.0 b 3.2a 1.9 b 0.7
b 0.2 b
Sucrose 12.0 a 9.2 b 5.8 b 3.3 c 1.7 b 0.5 c 0.0
c 0.0 c
p-values 0.382 0.000 0.000 0.000 0.000 0.000 0.000
0.006
*A single Reb A sample was not included in the testing. Based on sweetness
dose response characteristics of
Rch A. a 12% SEV level with Reb A as a single sweetener cannot be reached.
Therefore Reb A was not
included here as the sweetness of all samples initially needed to match at 10
seconds of tasting.
[00123] Tables 48 to 52 show sweetness intensity scores and the change in the
scores over
time. Panelists scored sweetness intensity of each sample initially within 10
seconds when
the sweetness reached its peak while they were holding the sample in their
mouth. Then they
spit the samples, and continue evaluating the remaining sweetness (sweetness
linger) in their
mouth every 10 seconds until panelist no longer received any sweetness. As
seen from the
tables above, monatin, Reb A, and monatin/Reb A blend (80/20, 60/40, and
50/50) samples
scored the same or not statistically significantly different from each other
for sweetness
intensity at initial 10 seconds of evaluation. In other words initially all of
the samples
matched in sweetness intensity to each other. Over time a decrease in
sweetness intensities
were observed for all of the samples. The decrease in sweetness was faster for
the monatin
and Reb A blend samples compared to either monatin or Reb A samples
individually. In
other words Monatin/Reb A blends had less sweetness linger compared to the
either
sweetener alone. For example, in the case of 80Monatin/20RebA blend sample at
7% SEV
(Table 48), the sweetness scores for this blend were statistically
significantly lower than the
sweetness scores for monatin and Reb A samples at 20 and 30 seconds of
tasting. Similarly,
60Monatin/40RebA blend (Table 49), and 50Monatin/50RebA blend (Table 50)
samples at
7% SEV scored significantly lower in sweetness than both monatin and Reb A
samples at 40,
50, and 60 seconds of tasting. The 80Monatin/20RebA blend samples at 4%, and
12% SEV
(Tables 51 and 52) also showed similar results that the sweetness scores of
blend samples
were lower than the either sweetener alone indicating less sweetness linger
over time for the
blend samples. These results indicate that different blends (80/20, 60/40, and
50/50) of
monatin and Reb A had significantly reduced sweetness linger compared to
sweetness linger
of monatin and Reb A alone at different % SEV (7%, 4%, and 12%) levels.
54
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 53. Sweetness Intensity Change Over Time for Monatin, RA80,
80Monatin/20RA80, and Sucrose Formulated at 7 % SEV
Samples lOsec 20sec 30see 40sec 50sec 60sec 70see
Monatin 7.0 a 6.3 a 4.7 a 3.3 a 2.0 a 0.9 a 0.2 a
RA80 7.0 a 6.4 a 4.7 a 3.3 a 1.6 b 0.5 b 0.2 a
80Monatin/20RA80 7.0 a 5.8 b 4.1 a 2.6b 1.0 c 0.1 c 0.0 a
Sucrose 6.8 a 4.8 c 2.8 b 1.3 c 0.3 d 0.0 c 0.0 a
p-values 0.295 0.003 0.007 0.001 0.001 0.009 0.458
[00124] Table 53 shows another example of blending rnonatin with RA80. As the
data in
Table 53 show, the 80Monatin/20RA80 blend was significantly lower in sweetness
at 20, 40,
50, and 60 seconds of tasting compared to both monatin and RA80 samples
indicating less
sweetness linger for the blend over time.
Example 16
[00125] Methods: A roundtable consensus panel was conducted with 6 expert
tasters who
are research employees experienced in tasting and evaluating moriatin, and Reb
D samples.
Samples were prepared in filtered water and evaluated for sweetness linger. A
category scale
of 1 to 9 (1-Not at all lingering, 5=Moderately lingering, 9=Extremely
lingering sweetness)
was used to measure the sweetness linger. Samples were matched for sweetness
at 7% SEV
before the evaluation started by the expert tasters. Sample concentrations as
well as the lot
numbers can be found in Table 54. Tasters were served 1.5 oz of sample in 2 oz
souffle cups
at room temperature (68-72 F). They were asked to have a sip of the sample,
swirl the sample
in their mouths and hold it for 10 seconds. After 10 seconds, the tasters
either swallowed or
expectorated the sample, and rated the sweetness linger at 30, 60 and 90
seconds. Tasters
were asked to cleanse well with water and crackers in between tasting the
samples.
CA 02821306 2013-06-11
WO 2012/083251
PCT/US2011/065643
Table 54. Concentrations of Monatin, Reb D(92%), 80Monatin/20ROD Blend, and
Sucrose Formulated at 7% SEV
Name Lot # Monatin Reb D (92%) Sucrose
Concentration Concentration Concentration
Sucrose M3 WTBO 0% 0% 7%
Monatin 25001392 0.0036% 0% 0%
Reb D 00018279-1931 0% 0.030% 0%
80Monatin/20RebD 25001392/ 0.0020% 0.0035% 0%
00018279-1931
Table 55. Sweetness Linger Scores
Samples 30sec 60sec 90sec
Monatin 6.8 5.0 3.7
Reb D 5.8 4.3 3.0
80Monatin/20Reb D 5.8 4.3 3.2
Sucrose 4.2 2.7 2.2
[00126] Results: As the data in Table 55 show, sweetness linger scores of both
80Monatin/20RebD blend and Reb D samples were lower than that of the monatin
at 30, 60,
and 90 seconds of evaluation. In consensus expert tasters indicated that
80Mon/20RebD
blend, and Reb D were lingering less in sweetness than monatin, and closer to
the sucrose in
overall linger. In agreement with the previous trained panel results, this
example also showed
that blending monatin and Reb D decreased sweetness linger of monatin over
time.
[00127] It will be apparent to one of ordinary skill in the art from the
teachings herein that
specific embodiments and examples of the present invention may be directed to
one or a
combination of the above-indicated aspects, as well as other aspects.
56
