Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS
TECHNICAL FIELD
The present invention relates generally to the use of one or more low-potency
sweetener(s) to improve one or more sweetness characteristics of one or more
high-
intensity sweetener(s). The present invention thus also relates to
compositions
comprising a mixture of at least one high-intensity sweetener and at least one
low-
potency sweetener. The present invention further relates to the use of a
combination of
at least one high-intensity sweetener and at least one low-potency sweetener
as a
sweetness modifier wh+en used in combination with at least one other sweetener
and/or as a sweetener. The present invention further relates to the use of one
or more
mogroside(s) as a sweetness enhancer in sweetened compositions and said
sweetened compositions. The present invention further relates to methods of
making
the sweeteners and compositions disclosed herein.
BACKGROUND
Sweetness in comestible products, that is products intended to be taken by
mouth
either for permanent ingestion or temporarily for expectoration, is often a
desirable
characteristic. Traditionally, sweetness has been provided by the addition of
one or
more sweeteners, particularly low-potency, nutritive sweeteners such as
sucrose
(table sugar), fructose, glucose, xylose, arabinose, rhamnose, sugar alcohols
such as
erythritol, xylitol, mannitol, sorbitol and inositol as well as sugar syrups
such as high
fructose corn syrup and starch syrup. These deliver considerable sweetness
without
any undesirable aftertaste. However, it is desirable to use a reduced amounts
of these
sweeteners to reduce the caloric value of the comestible product. It is
therefore
desirable to provide alternative sweeteners that can reduce the caloric value
of the
comestible product whilst maintaining the same or a similar sweetness taste.
High-intensity sweeteners (HIS) have been used for this purpose. High-
intensity
sweeteners may be natural or artificial and have a sweetness that can be
several
hundred times that of sucrose and thus can theoretically replace a much larger
quantity
of sugar in a composition. Examples of high-intensity sweeteners include
sucralose,
saccharin, aspartame, acesulfame potassium (AceK), neotame, advantame,
sterviol
glycosides, including stevioside, rebaudioside A, rebaudioside D or steviol
glycoside
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mixture preparations with rebaudioside A and/or stevioside as predominant
components. However, these substances generally have the drawback that they
may
impart undesirable off-tastes to comestible products, typically bitter,
metallic or liquorice
tastes, or an undesirable lingering sweetness.
It is therefore desirable to provide alternative and/or improved sweetness
modifying
composition and sweetened compositions to address one or more of these issues.
SUMMARY
In accordance with a first aspect of the present invention there is provided a
sweetness
modifying composition comprising:
one or more high-intensity sweetener(s) selected from the group consisting of
steviol glycosides and/or mogrosides; and
one or more low-intensity sweetener(s) selected from the group consisting of
cellobiose, psicose, cyclamate and/or 11-0-mogroside V;
wherein the sweetness modifying composition increases the sweetness of a
sweetened composition by more than the sweetness of the sweetness modifying
composition alone; and/or
wherein the ratio of the one or more high-intensity sweetener(s) to the one or
more low-potency sweetener(s) ranges from about 2:1 to about 12:1.
In accordance with a second aspect of the present invention there is provided
a
sweetened composition comprising:
at least one sweetener present in an amount having a sweetness equal to or
greater than about 1.5 % (w/v) sucrose equivalence; and
a sweetness modifying composition according to any aspect or embodiment of
the present invention.
In accordance with a third aspect of the present invention there is provided a
use of
one or more low-potency sweetener(s) selected from the group consisting of
cellobiose, psicose, cyclamate and/or 11-0-mogroside V to improve one or more
sweetness characteristic(s) of a sweetened composition comprising one or more
high-
intensity sweetener(s) selected from the group consisting of steviol
glycosides and/or
mogrosides, wherein the total concentration of the one or more low-potency
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sweetener(s) and the one or more high-potency sweetener(s) that is used has a
sweetness of less than 1.5 % (w/v) sucrose equivalence.
In accordance with a fourth aspect of the present invention there is provided
a method
of enhancing the sweetness of a sweetened composition, the method comprising
providing a base composition comprising at least one sweetener present in an
amount
at or above its sweetness recognition threshold and/or having a sweetness
equal to or
greater than about 1.5 % (w/v) sucrose equivalence, and adding one or more
high-
intensity sweetener(s) selected from the group consisting of steviol
glycosides and/or
mogrosides and one or more low-potency sweetener(s) selected from the group
consisting of cellobiose, psicose, cyclamate and/or 11-0-mogroside V, wherein
the
ratio of the one or more high-intensity sweetener(s) to the one or more low-
potency
sweetener(s) is from about 2:1 to about 12:1; and/or wherein the one or more
high-
intensity sweetener(s) are added in a total amount equal to or greater than
about 15
ppm and optionally equal to or less than about 50 ppm, and the one or more low-
potency sweetener(s) are added in a total amount equal to or greater than
about 2 ppm
and optionally equal to or less than about 12 ppm; and/or wherein the total
concentration of the one or more high-intensity sweetener(s) and the one or
more low-
potency sweetener(s) that is added has a sweetness less than 1.5 % (w/v)
sucrose
equivalence.
In accordance with a fifth aspect of the present invention there is provided a
method of
making a sweetness modifying composition according to any aspect or embodiment
of
the present invention, the method comprising combining one or more high-
intensity
sweetener(s) and one or more low-potency sweetener(s).
In accordance with a sixth aspect of the present invention there is provided a
method of
making a sweetened composition according to any aspect or embodiment of the
present invention, the method comprising combining the base composition, one
or
more high-intensity sweetener(s), one or more low-intensity sweetener(s) and
at least
one other sweetener.
In accordance with a seventh aspect of the present invention there is provided
a
sweetened composition comprising at least one sweetener present in an amount
having a sweetness equal to or greater than 1.5 % (w/v) sucrose equivalence;
and one
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or more sweetness enhancer(s) selected from mogroside IV, siamenoside and
neomogroside.
In accordance with an eighth aspect of the present invention there is provided
a use of
one or more of mogroside IV, siamenoside and neomogroside to enhance the
sweetness of a sweetened composition. Thus, in a further aspect there is
provided a
method for enhancing the sweetness of a sweetened composition, the method
comprising providing a base composition and adding at least one sweetener and
one
or more sweetness enhancer(s) selected from mogroside IV, siamenoside and
neomogroside.
In accordance with a ninth aspect of the present invention there is provided a
method
of making a sweetened composition according to any aspect or embodiment of the
present invention, the method comprising combining the base composition, one
or
more sweetness enhancer(s) selected from mogroside IV, siamenoside and
neomogroside and at least one other sweetener.
In accordance with a tenth aspect of the present invention there is provided a
sweetened composition comprising one or more mogroside(s). The one or more
mogroside(s) may, for example, be present as a sweetness enhancer and thus be
present in an amount having a sweetness of less than 1.5 % (w/v) sucrose
equivalence. The sweetened composition will then further comprise at least one
sweetener present in an amount having a sweetness equal to or greater than 1.5
%
(w/v) sucrose equivalence.
In accordance with an eleventh aspect of the present invention there is
provided a use
of one or more mogroside(s) to enhance the sweetness of a sweetened
composition.
Thus, there is provided a method for enhancing the sweetness of a sweetened
composition, the method comprising providing a base composition and adding at
least
one sweetener and one or more mogroside(s).
In accordance with a twelfth aspect of the present invention there is provided
a method
of making a sweetened composition according to any aspect or embodiment of the
present invention, the method comprising combining the base composition, one
or
more mogroside(s) and at least one other sweetener.
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In certain embodiments of any aspect of the present invention the one or more
high-intensity sweetener may include or be mogroside V and/or the one or more
low-potency sweetener may include or be 11-0-mogroside V.
5 In certain embodiments of any aspect of the present invention the ratio
of the one or
more high-intensity sweetener(s) to the one or more low-potency sweetener(s)
is from
about 2:1 to about 12:1. In certain embodiments of any aspect of the present
invention
the ratio of the one or more high-intensity sweetener(s) to the one or more
low-potency
sweetener(s) is from about 5:1 to about 12:1. In certain embodiments of any
aspect of
the present invention the ratio of the one or more high-intensity sweetener(s)
to the one
or more low-potency sweetener(s) may be from about 6:1 to about 10:1.
In certain embodiments of any aspect of the present invention the one or more
high-intensity sweetener(s) may be present in a total amount ranging from
about 15
ppm to about 30 ppm and/or the one or more low-potency sweetener(s) may be
present in a total amount ranging from about 2 ppm to about 10 ppm. In certain
embodiments of any aspect of the present invention the one or more high-
intensity
sweetener(s) may be present in a total amount ranging from about 22 ppm to
about 28
ppm and/or the one or more low-potency sweetener(s) may be present in a total
amount ranging from about 2 ppm to about 5 ppm.
In certain embodiments of the seventh to twelfth aspect of the present
invention, the
one or more mogroside(s) or one or more sweetness enhancer(s) may be present
in an
amount ranging from about 15 ppm to about 50 ppm. In certain embodiments, the
one
or more mogroside(s) or one or more sweetness enhancer(s) may be present in an
amount ranging from about 15 ppm to about 35 ppm.
In certain embodiments of any aspect of the present invention the combination
of the
one or more high-intensity sweetener(s) and the one or more low-potency
sweetener(s)
alone may have a sweetness less than about 1.5 % (w/v) sucrose equivalence. In
particular, the concentration of the one or more high-intensity sweetener(s)
and the one
or more low-intensity sweetener(s) in a sweetened composition may have a
sweetness
less than about 1.5 % (w/v) sucrose equivalence.
In certain embodiments of the seventh to twelfth aspect of the present
invention, the
one or more mogroside(s) or one or more sweetness enhancer(s) may have a total
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sweetness less than about 1.5 % (w/v) sucrose equivalence. In certain
embodiments,
the one or more sweetness enhancer(s) increase the sweetness of a sweetened
composition by more than the total sweetness of the one or more sweetness
enhancer(s) alone.
In certain embodiments of any aspect of the present invention the combination
of the
one or more high-intensity sweetener(s) and the one or more low-potency
sweetener(s)
may increase the sweetness of a sweetened composition by more than the
sweetness
of the combination alone. In certain embodiments of any aspect of the present
invention the combination of the one or more high-intensity sweetener(s) and
the one
or more low-potency sweetener(s) may increase the sweetness of a composition
by
equal to or greater than about 1.25 % (w/v) sucrose equivalence.
In certain embodiments of any aspect of the present invention the one or more
low-potency sweetener(s) weaken the lingering sweet taste of a sweetened
composition comprising the one or more high-intensity sweetener(s) compared to
the
lingering sweet taste of the sweetened composition in the complete absence of
the one
or more low-potency sweetener(s).
In certain embodiments of any aspect of the present invention the one or more
low-potency sweetener(s) weakens the bitter and/or astringent taste of a
sweetened
composition comprising the one or more high-intensity sweetener(s) compared to
the
bitter and/or astringent taste of the sweetened composition in the complete
absence of
the one or more low-potency sweetener(s).
One or more (e.g. all) of the sweeteners used may be natural or synthetic
(artificial).
One or more of the sweeteners may, for example, be made by a biological
process or
by an enzymatic process or by a synthetic process.
Certain embodiments of any aspect of the present invention may provide one or
more
of the following advantages:
= increased sweetness in a composition;
= enhanced sweetness in a composition including at least one sweetener;
= decrease in the amount of caloric sweetener required to obtain desired
sweetness;
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= improvement of one or more sweetness characteristics to make sweet taste
more
similar to sugar (sucrose);
= weakening of lingering sweetness (e.g. decreasing the length of time the
sweet
taste remains and/or decreasing the intensity of the sweet taste more
rapidly);
= weakening of bitter taste and/or astringent taste and/or liquorice taste
and/or
metallic taste;
= improvement in sweetness impact (e.g. increasing the maximum intensity of
the
sweet taste and/or decreases the length of time for the sweet taste to be
detected) (e.g. decreasing the lingering sweetness).
The details, examples and preferences provided in relation to any particulate
one or
more of the stated aspects of the present invention will be further described
herein and
apply equally to all aspects of the present invention. Any combination of the
embodiments, examples and preferences described herein in all possible
variations
thereof is encompassed by the present invention unless otherwise indicated
herein, or
otherwise clearly contradicted by context.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a chromatogram of a Luo Han Guo extract (extract 2 of Table 1
below).
Figure 2 shows the chemical structures of mogrosides 1-6;
Figure 3 shows the LC-MS analysis of commercial Luo Han Guo extracts;
Figure 4 shows the Heteronuclear Single Quantum Coherence-Total Correlation
Spectroscopy (HSQC-TOCSY) (hsqcgpmlph) of iso-mogroside VI with different
mixing
time (d9). A: 10 ms mixing time. B: 30 ms mixing time. C: 60 ms mixing time.
D: 100 ms
mixing time. Because of the overlap of H-1 of Glc ll and H-6a of Glc III, HSQC-
COSY
correlation intensity of Glc II was not analyzed here;
Figure 5 shows HSQC-TOCSY (hsqcgpmlph) peak intensity quantification of iso-
mogroside VI glucopyranosyls with different mixing time. (* C-3 and C-5
signals on
HSQC-TOCSY appeared overlap for 100 ms mixing time. The total integration of C-
3
and C-5 was therefore used in the bar chart);
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Figure 6 shows the strategy to elucidate mogroside sugar chain (*Numbers of 0-
2 to C-
6 appeared under certain mixing time might slightly change if adjusting peak
intensity
of HSQC-TOCSY. By observing the increasing intensity of 0-2 to 0-6 in
different
mixing time experiments connection sequence can be still determined. **There
is no
natural glycosylation on 0-3 of mogroside glucopyranosyl so far. 0-3
glycosylation on
glucopyranosyl would cause the downshift from 676 to 681 and can be easily
determined by HSQC-TOCSY experiments). The sequence of steps in Figure 6 can
be
outlined as follows:
In Step 1, Heteronuclear multiple bond correlation spectroscopy (HMBC) was
used to
determine anormeric C-1 and H-1 of the sugar. Start from the sugar link to
aglycone.
In Step 2, HSQC-TOCSY was used with 100ms mixing time to determine the whole
group of 0-2 to 0-6. HSQC-COSY or HSQC-TOCSY (d9=10 ms) to assign 0-2. HSQC-
TOCSY (d9=30 ms) to assign 0-3. HSQC-TOCSY (d9=60 ms) to assign 0-4. HSQC-
TOCSY (d9=100 ms) to assign 0-5 and 0-6. In Step 3, if a 0-2 downshift from -
675 to
-681, 0-4 downshift from -671 to -681 or 0-6 downshift from -662 to -69 is
observed,
check HMBC for glycosylation at these positions.**
If a 0-2 downshift from -675 to -681, 0-4 downshift from -671 to -681 or 0-6
downshift from -662 to -69, check HMBC for glycosylation at these positions.**
Figure 7 shows the chemical structure for iso-mogroside VI which has the
chemical
formula 066H112034 and an Exact Mass of 1448.70. This chemical structure is
designated Formula I; and
Figure 8 shows the chemical structure for 11-epi-mogrosideV which has the
chemical
formula 0601-1102029 and an Exact Mass of 1286.65. This chemical structure is
designated Formula II.
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DETAILED DESCRIPTION
The present invention is based on the surprising finding that a combination of
one or
more high-intensity sweetener(s) (e.g. mogroside V) and one or more low-
potency
sweetener(s) (e.g. 11-0-mogroside V) can act synergistically with at least one
other
sweetener (e.g. sucrose) to obtain a composition having a sweetness that is
greater
than the sum of the sweetness of the individual sweeteners. The present
invention is
further based on the surprising finding that one or more low-potency
sweetener(s) may
offset one or more negative sweetness characteristics of one or more high-
potency
sweetener(s). For example, the combination of one or more high-intensity
sweetener(s)
(e.g. mogroside V) and one or more low-potency sweetener(s) (e.g. 11-0-
mogroside V)
may provide improved sweetness characteristics in a sweetened composition
(i.e. a
composition comprising at least one other sweetener such as sucrose in an
amount
above its sweetness recognition threshold and/or an amount equal to or greater
than
about 1.5 % (w/v) sucrose equivalence) compared to using the one or more
high-intensity sweetener(s) alone. The sweetness characteristics may thus, for
example, be closer to the sweetness characteristics of sucrose.
Thus, there is provided herein various compositions comprising one or more
high-intensity sweetener(s) and one or more low-potency sweetener(s) as
disclosed
herein, particularly sweetened compositions comprising at least one sweetener
in an
amount above its sweetness recognition threshold and/or an amount equal to or
greater than about 1.5 % (w/v) sucrose equivalence and one or more high-
intensity
sweetener and one or more low-potency sweetener(s). The sweetened compositions
may also be referred to as comestible compositions. There is also provided
herein
various uses of one or more high-intensity sweetener(s) and one or more low-
potency
sweetener(s) as disclosed herein and methods of making the various
compositions
disclosed herein.
The present invention is further based on the surprising finding that
mogrosides such
as mogroside IV, siamenoside and neomogroside can act as sweetness enhancers
(i.e. can increase the sweetness of a sweetened composition by more than the
sweetness of the sweetness enhancer alone).
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Thus, there is provided herein various compositions, in particular sweetened
compositions, comprising one or more of mogroside IV, siamenoside and
neomogroside.
5 Compositions
There is provided herein various compositions comprising at least one high-
intensity
sweetener and at least one low-potency sweetener. There is also provided
herein
compositions comprising one or more mogroside(s), for example one or more of
10 mogroside IV, siamenoside and neomogroside. In certain embodiments, the
compositions are comestible compositions.
In certain embodiments, there is provided a sweetness modifying composition
comprising, consisting essentially of or consisting of at least one high-
intensity
sweetener selected from the group consisting of steviol glycosides and/or
mogrosides
and at least one low-potency sweetener selected from the group consisting of
cellobiose, psicose, cyclamate and/or 11-0-mogroside V. In certain
embodiments, the
sweetness modifying composition comprises, consists essentially of or consists
of one
high-intensity sweetener and one low-potency sweetener. The sweetness
modifying
composition may, for example, be a concentrate which may, for example, be
diluted in
a sweetened (e.g. comestible) composition to give the comestible composition a
desired sweetness. The term "sweetened composition" refers to a composition
comprising at least one sweetener present in an amount above its sweetness
recognition threshold and/or in an amount having a sweetness equal to or
greater than
about 1.5 % (w/v) sucrose equivalence.
In certain embodiments, there is provided a sweetened composition (e.g.
comestible
composition) comprising one or more mogroside(s), for example one or more of
mogroside IV, siamenoside and neomogroside. In certain embodiments, there is
provided a sweetened composition (e.g. comestible composition) comprising at
least
one high-intensity sweetener and at least one low-potency sweetener. The
combination
of the high-intensity sweetener(s) and low-potency sweetener(s) may be
referred to as
a sweetness modifying composition. The one or more mogroside(s), for example
the
one or more of mogroside IV, siamenoside and neomogroside may also be referred
to
herein as a sweetness modifying composition. Thus, in certain embodiments,
there is
provided a sweetened composition comprising at least one sweetener present in
an
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amount above its sweetness recognition threshold and/or in an amount having a
sweetness equal to or greater than about 1.5 % (w/v) sucrose equivalence and a
sweetness modifying composition comprising, consisting essentially of or
consisting of
at least one high-intensity sweetener and at least one low-potency sweetener.
The
sweetened composition may, for example, be a comestible composition.
The term "enhancing" when used in relation to a particular sweetness modifying
composition refers to a synergistic sweetening effect when the sweetness
modifying
composition is used in combination with at least one other sweetener. The
sweetness
modifying composition increases the sweetness of a sweetened composition by
more
than the sweetness of the sweetness modifying composition alone. In other
words, the
sweetness of a composition comprising at least one sweetener and at least one
sweetness modifying composition is greater than the sum of the sweetness of
all the
sweeteners in the composition. The sweetness modifying compositions described
herein are used in sweetened (e.g. comestible) compositions in amounts that
have no
detectable sweetness or no taste recognised as sweet (below its sweetness
recognition threshold). Typically, a sweetness modifying composition with a
sweetness
below 1.5 % (w/v) sucrose equivalence is accepted as being "not intrinsically
sweet" by
FEMA (Flavor & Extract Manufacturers Association). Sweetness modifiers may
also be
referred to as sweetness enhancers.
The sweetened composition comprising the sweetness modifying composition as
disclosed herein and at least one sweetener in an amount above its sweetness
recognition threshold and/or in an amount having a sweetness equal to or
greater than
about 1.5 % (w/v) sucrose equivalence may have a sweetness that is equal to or
more
than about 1.0 % (w/v) sucrose equivalence greater than the sweetness of the
sweetened composition in the absence of the sweetness modifying composition.
For
example, the sweetened composition may have a sweetness that is equal to or
more
than about 1.1 % (w/v) or equal to or more than about 1.15 % (w/v) or equal to
or more
than about 1.2 % (w/v) or equal to or more than about 1.25 % (w/v) sucrose
equivalence greater than the sweetness of the sweetened composition in the
absence
of the sweetness modifying composition. In other words, the sweetness
modifying
composition may increase the sweetness of a sweetened composition by equal to
or
more than about 1 % (w/v) or equal to or more than about 1.1 % (w/v) or equal
to or
more than about 1.15 % (w/v) or equal to or more than about 1.2 % (w/v) or
equal to or
more than about 1.25 % (w/v) sucrose equivalence. The comparative composition
is
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identical to the said composition except that it does not include the said
sweetness
modifying composition.
The term "sucrose equivalence" refers to the equivalence in sweetness of a
composition containing at least one non-sucrose sweetener to a reference
sucrose
solution. Typically, taste panellists are trained to detect sweetness of
reference sucrose
solutions containing between 1 % and 15 % sucrose (w/v). Other non-sucrose
sweeteners may then be tasted at a series of dilutions to determine the
concentration
of the non-sucrose sweetener that is as sweet (i.e. isosweet) to a given
sucrose
reference. The term "isosweet" refers to compositions that have equivalent
sweetness.
Typically, the sweetness of a given composition is measured with reference to
a
solution of sucrose. See "A Systematic Study of Concentration-Response
Relationships of Sweeteners," G.E. DuBois, D.E. Walters, S.S. Schiffman, Z.S.
Warwick, B.J. Booth, S.D. Pecore, K. Gibes, B.T. Carr, and L.M. Brands, in
Sweeteners: Discovery, Molecular Design and Chemoreception, D.E. Walters, F.T.
Orthoefer, and G.E. DuBois, Eds., American Chemical Society, Washington, DC
(1991), pp 261-276.
The combination of the one or more high-intensity sweetener(s) and the one or
more
low-potency sweetener(s) (e.g. the sweetness modifying composition) may, for
example, have a sweetness less than about 1.5 % (w/v) sucrose equivalence. For
example, the combination of the high-intensity sweetener(s) and the low-
potency
sweetener(s) (e.g. sweetness modifying composition) may have a sweetness equal
to
or less than about 1.45 % (w/v) sucrose equivalence or equal to or less than
about
1.4 % (w/v) sucrose equivalence or equal to or less than about 1.35 % (w/v)
sucrose
equivalence or equal to or less than about 1.3 % (w/v) sucrose equivalence.
For
example, the combination of the high-intensity sweetener(s) and the low-
potency
sweetener(s) (e.g. sweetness modifying composition) may have a sweetness equal
to
or greater than about 1 % (w/v) sucrose equivalence or equal to or greater
than about
1.1 % (w/v) sucrose equivalence or equal to or greater than about 1.15 % (w/v)
sucrose
equivalence or equal to or greater than about 1.2 % (w/v) sucrose equivalence
or equal
to or greater than about 1.25 % (w/v) sucrose equivalence or equal to or
greater than
about 1.3 % (w/v) sucrose equivalence.
The one or more mogroside(s), for example one or more sweetness enhancer(s)
selected from mogroside IV, siamenoside and neomogroside may, for example,
have a
sweetness less than about 1.5 % (w/v) sucrose equivalence. For example, the
one or
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more mogroside(s), for example one or more sweetness enhancer(s) selected from
mogroside IV, siamenoside and neomogroside may have a sweetness equal to or
less
than about 1.45 % (w/v) sucrose equivalence or equal to or less than about 1.4
% (w/v)
sucrose equivalence or equal to or less than about 1.35 % (w/v) sucrose
equivalence
or equal to or less than about 1.3 % (w/v) sucrose equivalence. For example,
the one
or more mogroside(s), for example one or more sweetness enhancer(s) selected
from
mogroside IV, siamenoside and neomogroside may have a sweetness equal to or
greater than about 1 % (w/v) sucrose equivalence or equal to or greater than
about
1.1 % (w/v) sucrose equivalence or equal to or greater than about 1.15 % (w/v)
sucrose
equivalence or equal to or greater than about 1.2 % (w/v) sucrose equivalence
or equal
to or greater than about 1.25 % (w/v) sucrose equivalence or equal to or
greater than
about 1.3 % (w/v) sucrose equivalence.
Each of the sweeteners and sweetness enhancers used in the compositions
disclosed
herein may be a natural or synthetic (artificial) sweetener. Examples of non-
naturally
occurring (i.e. synthetic) mogrosides are disclosed in WO 2017/075257, the
contents of
which are incorporated herein by reference. The term "natural sweetener"
refers to
sweeteners that are obtained from nature, including mixtures that may have
been
enzymatically treated (e.g. glycosylated) to form compounds not found in
nature (this
does not include purified compounds that have been enzymatically treated). For
example, a modified extract having a mogrol glycoside distribution that is
different (e.g.
enhanced) from the naturally occurring mogrol glycoside distribution may be
classed as
natural. For example, a mixture of glucosylated steviol glycosides and/or
glucosylated
mogrosides may be classed as natural. Each of the sweeteners used in the
compositions disclosed herein may be food-derived. A "food-derived" product
refers to
a product which is prepared under typical cooking conditions such as, for
example,
using temperatures similar to those used in cooking methods. In certain
embodiments,
the high-intensity sweetener and the low-potency sweetener used in the
compositions
disclosed herein (e.g. in the sweetness modifying composition disclosed
herein) are
both natural sweeteners. In certain embodiments, all of the sweeteners used in
the
compositions disclosed herein are natural.
The sweeteners disclosed herein may be used in pure or purified form and may
be
chemically synthesised, produced by biotechnological processes (e.g.
fermentation) or
isolated from a natural source (e.g. a botanical source including, without
limitation,
fruits, sugar cane, sugar beet).
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The one or more mogroside(s), for example the one or more of mogroside IV,
siamenoside and neomogroside may, for example, be at least 80 wt% pure. For
example, the one or more mogroside(s), for example one or more of mogroside
IV,
siamenoside and neomogroside may be at least about 85 wt% or at least about 90
wt%
or at least about 95 wt% or at least about 98 wt% or at least about 99 wt%
pure. For
example, the one or more mogroside(s), for example one or more of mogroside
IV,
siamenoside and neomogroside may be up to 100 wt% or up to 99 wt% pure.
The term "high-intensity sweetener" refers to compounds having a sweetness
that is at
least 100 times the sweetness of sucrose. In certain embodiments, the high-
intensity
sweetener has a sweetness that is at least about 120 or at least about 140 or
at least
about 150 or at least about 160 or at least about 180 or at least about 200 or
at least
about 220 or at least about 240 or at least about 250 or at least about 260 or
at least
about 280 or at least about 300 or at least about 320 or at least about 340 or
at least
about 350 or at least about 360 or at least about 380 or at least about 400 or
at least
about 420 or at least about 440 or at least about 450 times the sweetness of
sucrose.
The high-intensity sweetener may, for example, have a sweetness that is up to
1000
times the sweetness of sucrose. Although the high-intensity sweetener has a
sweetness that is at least 100 times the sweetness of sucrose, in the context
of its use
in a sweetness modifying composition as described herein, they will be used in
a
sweetened composition in an amount that does not have any detectable sweetness
or
be recognised as sweet (amounts providing a sweetness less than 1.5 % (w/v)
sucrose
equivalence, which is accepted as being "not intrinsically sweet" by FEMA.
The one or more high-intensity sweetener(s) may, for example, be one or more
steviol
glycosides and/or one or more mogrosides. For example, the one or more
high-intensity sweetener may be a mixture of steviol glycosides and
mogrosides. For
example, the one or more high-intensity sweeteners may be one or more steviol
glycosides. For example, the one or more high-intensity sweetener(s) may be
one or
more mogrosides. In certain embodiments, mogrosides may perform better than
steviol
glycosides in terms of sweetness enhancement and off-note reduction (e.g.
weakening
of lingering sweet after taste).
The high-intensity sweetener may, for example, be one or more steviol
glycoside(s).
Examples of steviol glycosides include, for example, stevioside (CAS: 57817-89-
7),
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rebaudioside A (CAS: 58543-16-1), rebaudioside B (CAS: 58543-17-2),
rebaudioside C
(CAS: 63550-99-2), rebaudioside D (CAS: 63279-13-0), rebaudioside E (CAS:
63279-
14-1), rebaudioside F (CAS: 438045-89-7), rebaudioside G (CAS: 127345-21-5),
rebaudioside H, rebaudioside I (CAS: 1220616-34-1), rebaudioside J,
rebaudioside K,
5 rebaudioside L, rebaudioside M (CAS: 1220616-44-3), rebaudioside N (CAS:
1220616-
46-5), rebaudioside 0 (CAS: 1220616-48-7), dulcoside A (CAS: 64432-06-0),
dulcoside
B (CAS: 63550-99-2), rubusoside (CAS: 64849-39-4) and Naringin Dihydrochalcone
(CAS: 18916-17-1).
10 The high-intensity sweetener may, for example, be one or more
mogroside(s).
In certain embodiments, the high-intensity sweetener may be one or more of the
mogrosides listed herein. In certain embodiments, the high-intensity sweetener
may be
one or more of mogroside IV, siamenoside, neomogroside and mogroside V
(including
all isomers thereof). For example, the high-intensity sweetener may be a
mixture of
15 mogroside IV, siamenoside and mogroside V (including all isomers
thereof). The one or
more mogroside(s) may, for example, be obtained or obtainable from Luo Han Guo
fruit
extracts.
The term "low-potency sweetener" refers to compounds having a sweetness that
is less
than 100 times the sweetness of sucrose. In certain embodiments, the low-
potency
sweetener has a sweetness that is up to about 95 times or up to about 90 times
or up
to about 85 times the sweetness of sucrose.
The one or more low-potency sweetener(s) are selected from one or more of
cellobiose, psicose, cyclamate and/or 11-0-mogroside V (CAS: 126105-11-1). For
example, the one or more low-intensity sweetener(s) may be one or more of
cellobiose,
psicose and 11-0-mogroside V.
In certain embodiments, the one or more high-intensity sweetener(s) includes
or is a
high-intensity mogroside. In certain embodiments, the one or more low-potency
sweetener(s) includes or is a low-potency mogroside. In certain embodiments,
the one
or more high-intensity sweetener includes or is a high-intensity mogroside and
the one
or more low-potency sweetener(s) includes or is a low-potency mogroside.
In certain embodiments, the one or more high-intensity sweetener includes or
is
mogroside V. In certain embodiments, the one or more low-potency sweetener
includes
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16
or is 11-0-mogroside V. In certain embodiments, the one or more high-intensity
sweetener includes or is mogroside V and the one or more low-potency sweetener
includes or is 11 -0-mogroside V.
Mogrosides are a group of triterpene glycosides and may be obtained from the
fruit Luo
Han Guo (Siraitia grosvenorii), also known as arhat fruit or longevity fruit
or swingle
fruit. Mogrosides make up approximately 1% of the flesh of the fresh fruit.
Through
extraction, an extract in the form of a powder containing up to 80% mogrosides
can be
obtained. Mogroside extract contains grosvenorine II, grosvenorine I, 11-0-
mogroside
II (I), 11-0-mogroside II (II), 11-0-mogroside II (III), mogroside II (I),
mogroside II (II),
mogroside II (III), 1 1-dehydroxy-mogroside III, 11-0-mogroside III, mogroside
III (I),
mogroside III (II), mogroside IV (I) (siamenoside), mogroside IV (II),
mogroside IV (III),
mogroside IV (IV), deoxymogroside V (I), deoxymogroside V (II), 11-0-mogroside
V (I),
mogroside V isomer, mogroside V, iso-mogroside V, 7-0-mogroside V,
11-0-mogroside VI, mogroside VI (I), mogroside VI (II), mogroside VI (III)
(neomogroside) and mogroside VI (IV). The precise amount of mogroside V may
vary
depending on the ripeness of the fruit and/or extraction process used.
Mogroside(s) include both mogroside(s) that occur in nature and mogrosides
that do
not occur in nature. Examples of mogrosides include, for example, grosvenorine
II,
grosvenorine I, 11-0-mogroside II (I), 11-0-mogroside II (II), 11-0-mogroside
II (III),
mogroside ll (I), mogroside ll (II), mogroside ll (III), 1 1-dehydroxy-
mogroside III,
11-0-mogroside III, mogroside III (I), mogroside III (II), mogroside Ille,
mogroside Illx,
mogroside IV (I) (siamenoside), mogroside IV (II), mogroside IV (III),
mogroside IV (IV),
deoxymogroside V (I), deoxymogroside V (II), 11-0-mogroside V (I), mogroside V
isomer, mogroside V, iso-mogroside V, 7-0-mogroside V, 11-0-mogroside VI,
mogroside VI (I), mogroside VI (II), mogroside VI (III) (neomogroside) and
mogroside
VI (IV). The mogroside(s) may, for example, be obtained or obtainable from Luo
Han
Guo extracts.
Mogroside V (CAS: 88901-36-4) is a glycoside of a cucurbitane derivative and
has the
chemical formula 0601-1102029 and the chemical structure shown below.
Mogroside V
can be found in certain plant extracts such as extracts from the fruit Luo Han
Guo
(Siraitia grosvenorii). Pure mogroside V has been found to have a sweetness of
at least
400 times the sweetness of sucrose.
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17
0
/
r =
8 6 =:õ._ I 0
6
- 0
:116
r
. 0
r
.t.
Siamenoside (CAS: 126105-12-2) is a cucurbitane found in the fruit of Siraitia
grosvenorii and has the following chemical structure.
Mogroside IV (CAS: 89590-95-4) is a triterpenic heteroside found in the fruit
of Siraitia
grosvenorii and has the following chemical structure.
HO*.
)H
OH
Neomogroside (CAS: 189307-15-1) is a cucurbitane glycoside also found in the
fruit of
Siraitia grosvenorii and has the following chemical structure.
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18
_
= ,
- I I
- 1
11-0-Mogroside V (CAS: 126105-11-1) is derived from mogroside V and has the
following chemical structure. It is also found in plant extracts such as
extracts from the
fruit Luo Han Guo (Siraitia grosvenorii). 11-0-mogroside V has been found to
have a
sweetness that is about 84 times the sweetness of sucrose.
OH OH
HO' " OH
OH OH
0,, õOH
I
OH
Ho'
o,HOOH
I I I:I OH
- -
11100
OH
The ratio of the one or more high-intensity sweetener(s) to the one or more
low-potency sweetener(s) is equal to or greater than about 2:1. For example,
the ratio
of the one or more high-intensity sweetener(s) to the one or more low-potency
sweetener(s) may be equal to or greater than about 2.5:1 or equal to or
greater than
about 3:1 or equal to or greater than about 3.5:1 or equal to or greater than
about 4:1
or equal to or greater than about 4.5:1 or equal to or greater than about 5:1
or equal to
or greater than about 5.5:1 or equal to or greater than about 6:1 or equal to
or greater
than about 6.5:1 or equal to or greater than about 7:1 or equal to or greater
than about
7.5:1 or equal to or greater than about 8:1. The ratio of the high-intensity
sweetener to
the low-potency sweetener is equal to or less than about 12:1. For example,
the ratio of
the one or more high-intensity sweetener(s) to the one or more low-potency
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19
sweetener(s) may be equal to or less than about 11.5:1 or equal to or less
than about
11:1 or equal to or less than about 10.5:1 or equal to or less than about 10:1
or equal
to or less than about 9.5:1 or equal to or less than about 9:1 or equal to or
less than
about 8.5:1. For example, the ratio of the one or more high-intensity
sweetener(s) to
the one or more low-potency sweetener(s) may range from about 5:1 to about
11:1 or
from about 6:1 to about 10:1 or from about 6.5:1 to about 9.5:1 or from about
7:1 to
about 9:1 or from about 7.5:1 to about 8.5:1.
In certain embodiments, the ratio of the one or more high-intensity
sweetener(s) to the
one or more low-potency sweetener(s) is from about 2:1 to about 12:1 or from
about
4:1 to about 12:1 or from about 5:1 to about 12:1 or from about 6:1 to about
10:1 or
from about 7:1 to about 9:1. The ratio may be weight or volume ratio. The
ratio only
applies to the high-intensity sweetener(s) and low-potency sweeteners in the
sweetness modifying composition (high-intensity and low-potency sweeteners
that are
used in a sweetened composition in an amount below the sweetness recognition
threshold or having less than 1.5 % (w/v) sucrose equivalence).
The one or more high-intensity sweetener(s) may be present in a composition in
a total
amount equal to or greater than about 15 ppm. For example, the one or more
high-intensity sweetener(s) may be present in a composition in a total amount
equal to
or greater than about 16 ppm or equal to or greater than about 17 ppm or equal
to or
greater than about 18 ppm or equal to or greater than about 19 ppm or equal to
or
greater than about 20 ppm or equal to or greater than about 21 ppm or equal to
or
greater than about 22 ppm or equal to or greater than about 23 ppm or equal to
or
greater than about 24 ppm or equal to or greater than about 25 ppm. For
example, the
one or more high-intensity sweetener(s) may be present in a composition in a
total
amount equal to or less than about 50 ppm or equal to or less than about 48
ppm or
equal to or less than about 46 ppm or equal to or less than about 45 ppm or
equal to or
less than about 44 ppm or equal to or less than about 42 ppm or equal to or
less than
about 40 ppm or equal to or less than about 38 ppm or equal to or less than
about 36
ppm or equal to or less than about 35 ppm or equal to or less than about 34
ppm or
equal to or less than about 32 ppm or equal to or less than about 30 ppm. For
example,
the one or more high-intensity sweetener(s) may be present in a composition in
a total
amount ranging from about 15 ppm to about 50 ppm or from about 15 ppm to about
45
ppm or from about 15 ppm to about 40 ppm or from about 15 ppm to about 35 ppm
or
from about 15 ppm to about 30 ppm. For example, the one or more high-intensity
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sweetener(s) may be present in a composition in a total amount ranging from
about 15
ppm to about 30 ppm or from about 20 ppm to about 30 ppm or from about 22 ppm
to
about 28 ppm or from about 23 ppm to about 27 ppm or from about 24 ppm to
about 26
ppm. For example, the one or more high-intensity sweetener(s) may be present
in a
5 composition in a total amount of about 20 ppm or about 25 ppm. The
composition may,
for example, be a sweetened composition comprising at least one sweetener in
an
amount having a sweetness above its sweetness recognition threshold and/or
equal to
or greater than about 1.5 % (w/v) sucrose equivalence.
10 The one or more low-potency sweetener(s) may be present in a composition
in a total
amount equal to or greater than about 2 ppm. For example, the one or more
low-potency sweetener(s) may be present in a composition in a total amount
equal to
or greater than about 3 ppm. For example, the one or more low-potency
sweetener(s)
may be present in a composition in a total amount equal to or less than about
12 ppm
15 or equal to or less than about 11 ppm or equal to or less than about 10
ppm or equal to
or less than about 9 ppm or equal to or less than about 8 ppm or equal to or
less than
about 7 ppm or equal to or less than about 6 ppm or equal to or less than
about 5 ppm.
For example, the one or more low-potency sweetener(s) may be present in a
composition in a total amount ranging from about 2 ppm to about 12 ppm or from
about
20 2 ppm to about 10 ppm or from about 2 ppm to about 5 ppm, for example in
a total
amount of about 3 ppm. The composition may, for example, comprise at least one
sweetener other than the combination of the high-intensity sweetener and the
low-potency sweetener (e.g. sweetness modifying composition) as disclosed
herein.
The concentration ranges may, for example, be particularly suitable for liquid
compositions such as beverages or compositions that do not comprise any
proteins or
fats. In compositions having a base such as milk and yogurt or other
compositions that
do comprise proteins and fats, higher concentrations of the one or more high-
intensity
sweetener(s) and one or more low-potency sweetener(s) may be used. For
example,
concentrations that are about 1.5 times higher than the concentrations used
for liquid
.. compositions or compositions that do not comprise any proteins or fats may
be used.
For example, concentrations that are from about 1.5 times to about 3 times
higher than
the concentrations used for liquid compositions or compositions that do not
comprise
any proteins or fats may be used.
Therefore, for example, the one or more high-intensity sweetener(s) may be
present in
a composition (e.g. a composition having a base such as milk and yoghurt or
other
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21
compositions that comprise proteins and/or fats) in a total amount ranging
from about
20 ppm to about 75 ppm, for example from about 22 ppm to about 74 ppm or from
about 24 ppm to about 72 ppm or from about 25 ppm to about 70 ppm or from
about
26 ppm to about 68 ppm or from about 28 ppm to about 66 ppm or from about 30
ppm
to about 65 ppm or from about 30 ppm to about 60 ppm or from about 30 ppm to
about
55 ppm or from about 30 ppm to about 50 ppm or from about 30 ppm to about 45
ppm.
Therefore, for example, the one or more low-potency sweetener(s) may be
present in a
composition (e.g. a composition having a base such as milk and yoghurt or
other
compositions that comprise proteins and/or fats) in a total amount ranging
from about
3 ppm to about 20 ppm or from about 4 ppm to about 18 ppm or from about 4 ppm
to
about 16 ppm or from about 5 ppm to about 15 ppm or from about 6 ppm to about
15
ppm.
In certain embodiments, a sweetened composition comprises at least one
sweetener in
an amount having a sweetness above its sweetness recognition threshold and/or
equal
to or greater than about 1.5 % (w/v) sucrose equivalence and a sweetness
modifying
composition consisting of 15 ppm to about 50 ppm of one or more high-intensity
sweetener(s) as described herein and 2 ppm to 12 ppm of one or more low-
potency
sweetener(s) as described herein. In certain embodiments, a comestible
composition
comprises at least one sweetener and a sweetness modifying composition
consisting
of 15 ppm to about 30 ppm of one or more high-intensity sweetener(s) as
described
herein and 2 ppm to 10 ppm of one or more low-potency sweetener(s) as
described
herein. In certain embodiments, a comestible composition comprises at least
one
sweetener and a sweetness modifying composition consisting of 20 ppm to about
ppm of one or more high-intensity sweetener(s) as described herein and 2 ppm
to
10 ppm of one or more low-potency sweetener(s) as described herein. In certain
embodiments, a comestible composition comprises at least one sweetener and a
sweetness modifying composition consisting of 22 ppm to about 28 ppm of one or
more
30 high-intensity sweetener(s) as described herein and 2 ppm to 5 ppm of
one or more
low-potency sweetener(s) as described herein. In certain embodiments, the high-
intensity sweetener is mogroside V. In certain embodiments, the low-potency
sweetener is 11-0-mogroside V.
The one or more mogroside(s), for example the one or more of mogroside IV,
siamenoside and neomogroside may, for example, be present in a sweetened
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22
composition in a total amount equal to or greater than about 15 ppm. For
example, the
one or more mogroside(s), for example the one or more of mogroside IV,
siameonside
and neomogroside may, for example, be present in a sweetened composition in a
total
amount equal to or greater than about 16 ppm or equal to or greater than about
17 ppm
or equal to or greater than about 18 ppm or equal to or greater than about 19
ppm or
equal to or greater than about 20 ppm or equal to or greater than about 21 ppm
or
equal to or greater than about 22 ppm or equal to or greater than about 23 ppm
or
equal to or greater than about 24 ppm or equal to or greater than about 25
ppm. For
example, the one or more mogroside(s), for example the one or more of
mogroside IV,
siamenoside and neomogroside may, for example, be present in a sweetened
composition in a total amount equal to or less than about 50 ppm, for example
equal to
or less than about 45 ppm, for example equal to or less than about 40 ppm, for
example equal to or less than about 35 ppm. For example, the one or more
mogroside(s), for example the one or more of mogroside IV, siamenoside and
neomogroside may be present in a sweetened composition in a total amount
ranging
from about 15 ppm to about 50 ppm or from about 15 ppm to about 45 ppm or from
about 15 ppm to about 40 ppm or from about 15 ppm to about 35 ppm or from
about
ppm to about 35 ppm or from about 20 ppm to about 30 ppm.
20 The term "ppm" refers to part(s) per million by weight, for example the
weight of a
compound, such as Mogroside V (in milligrams) per kilogram of the product
containing
such compound (i.e. mg/Kg) or the weight of a compound (e.g. orally
consumable/comestible product of the present disclosure), such as Mogroside V
(in milligrams) per litre of the product containing such compound (i.e. mg/L)
or by
volume, for example, the volume of a compound, such as Mogroside V (in
millilitres)
per litre of the product containing such compound (i.e. ml/L).
The sweetness modifying compositions described herein may, for example,
comprise
higher concentrations of the high-intensity and low-intensity sweeteners and
are then
diluted in a sweetened composition to obtain the concentrations recited
herein.
A sweetened composition comprises at least one sweetener in an amount equal to
or
greater than its sweetness recognition threshold and/or an amount having a
sweetness
equal to or greater than about 1.5 % (w/v) sucrose equivalence. The term
"sweetness
recognition threshold" refers to the lowest known concentration of a compound
that is
perceivable as sweet by the human sense of taste. A sweetness equal to or
greater
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23
than about 1.5 % (w/v) sucrose equivalence is accepted as being "intrinsically
sweet"
by FEMA.
The at least one sweetener may be nutritive or non-nutritive. Nutritive
sweeteners add
caloric value to the foods that contain them while non-nutritive sweeteners
are very low
in calories or contain no calories at all. Aspartame, the only approved
nutritive high-
intensity sweetener contains more than 2 % of the calories in an equivalent
amount of
sugar as opposed to non-nutritive sweeteners that contain less than 2 % of the
calories
in an equivalent amount of sugar.
The at least one sweetener may, for example, be selected from one or more of
sucrose, fructose, glucose, xylose, arabinose, rhamnose, tagatose, allulose,
trehalose,
isomaltulose, acesulfame potassium (AceK), aspartame, steviol glycoside(s),
sucralose, high-fructose corn syrup, starch syrup, saccharin, sucralose,
neotame,
advantame, Luo Han Guo extract, neohespiridin, dihydrochalcone, naringin
dihydrochalcone, neohesperidin dihydrochalcone, rubusoside, rebaudioside A,
stevioside, stevia, trilobtain and sugar alcohols such as erythritol, xylitol,
mannitol,
sorbitol and inositol. Examples of sweeteners that may be used in the
sweetened
compositions are disclosed, for example, in WO 2016/038617, the contents of
which
are incorporated herein by reference.
The at least one sweetener may, for example, be selected from one or more of
sucrose, high-fructose corn syrup, acesulfame potassium (AceK), aspartame,
steviol
glycoside(s) and/or sucralose.
How to sweeten consumables using sweeteners in a sufficient amount is well-
known in
the art. Depending on the consumable, the amount of sweetener can be reduced
by
addition of a sweetness modifying composition as disclosed herein. For
example, a
reduction of about 10 to about 40 Brix or more may be achieved.
The at least one other sweetener present in an amount equal to or greater than
its
sweetness recognition threshold and/or an amount having a sweetness equal to
or
greater than about 1.5 % (w/v) sucrose equivalence may, for example, be used
in a
sweetened composition in an amount equal to or greater than about 0.01 %
(w/v). For
example, the at least one other sweetener may be used in a sweetened
composition in
an amount equal to or greater than about 0.1 % (w/v) or equal to or greater
than about
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24
0.5 % (w/v) or equal to or greater than about 1 % (w/v) or equal to or greater
than
about 2 % (w/v). For example, the at least one other sweetener may be used in
a
comestible composition in an amount equal to or less than about 20 % (w/v) or
equal to
or less than about 15% (w/v) or equal to or less than about 10 % (w/v) or
equal to or
less than about 8 % (w/v) or equal to or less than about 6 % (w/v) or equal to
or less
than about 5 % (w/v).
The at least one other sweetener present in an amount equal to or greater than
its
sweetness recognition threshold and/or an amount having a sweetness equal to
or
greater than about 1.5 % (w/v) sucrose equivalence may be used in the
sweetened
compositions disclosed herein (e.g. comestible composition) in amounts
isosweet to
about 2% (w/v) to about 15% (w/v) sucrose.
In certain embodiments, there is provided herein a sweetness modifying
composition
consisting of mogroside V and 11-0-mogroside V in a ratio ranging from about
2:1 to
about 12:1, for example from about 6:1 to about 10:1. This sweetness modifying
composition may, for example, be used as sweetness enhancer or modifier in a
comestible composition. The comestible composition may, for example, comprise
at
least one other sweetener such as sucrose. The mogroside V may, for example,
be
used in the comestible composition in an amount ranging from about 15 ppm to
about
ppm or from about 20 ppm to about 30 ppm (e.g. about 20 ppm or about 25 ppm).
The 11-0-mogroside V may be used in the comestible composition in an amount
ranging from about 2 ppm to about 12 ppm or from about 2 ppm to about 10 ppm
(e.g.
about 8.5 ppm or about 3 ppm). The at least one other sweetener may, for
example, be
25 present in the comestible composition in an amount isosweet to about 2 %
(w/v) to
about 15 % (w/v) sucrose.
The compositions may be in any suitable form, for example solid (e.g. powder,
granules, tablets) or in solution (e.g. aqueous solution) or in an emulsion or
in a
30 suspension. For example, the compositions may further comprise a diluent
or bulking
agent such as dietary fibre.
Comestible compositions as disclosed herein include, for example, the
following.
- Wet/liquid soups regardless of concentration or container, including frozen
soups. For
the purpose of this definition soup(s) means a food prepared from meat,
poultry, fish,
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vegetables, grains, fruit and other ingredients, cooked in a liquid which may
include
visible pieces of some or all of these ingredients. It may be clear (as a
broth) or thick
(as a chowder), smooth, pureed or chunky, ready-to-serve, semi-condensed or
condensed and may be served hot or cold, as a first course or as the main
course of a
5 meal or as a between meal snack (sipped like a beverage). Soup may be
used as an
ingredient for preparing other meal components and may range from broths
(consommé) to sauces (cream or cheese-based soups).
- Dehydrated and culinary foods, including cooking aid products such as:
powders,
10 granules, pastes, concentrated liquid products, including concentrated
bouillon,
bouillon and bouillon like products in pressed cubes, tablets or powder or
granulated
form, which are sold separately as a finished product or as an ingredient
within a
product, sauces and recipe mixes (regardless of technology).
15 - Meal solutions products such as: dehydrated and freeze dried soups,
including
dehydrated soup mixes, dehydrated instant soups, dehydrated ready-to-cook
soups,
dehydrated or ambient preparations of ready-made dishes, meals and single
serve
entrees including pasta, potato and rice dishes.
- Meal embellishment products such as: condiments, marinades, salad dressings,
20 salad toppings, dips, breading, batter mixes, shelf stable spreads,
barbecue sauces,
liquid recipe mixes, concentrates, sauces or sauce mixes, including recipe
mixes for
salad, sold as a finished product or as an ingredient within a product,
whether
dehydrated, liquid or frozen.
25 - Beverages, including beverage mixes and concentrates, including but
not limited to,
alcoholic and non-alcoholic ready to drink and dry powdered beverages,
carbonated
and non-carbonated beverages, e.g., sodas, fruit or vegetable juices,
alcoholic and
non-alcoholic beverages.
- Confectionery products, e.g., cakes, cookies, pies, candies, chewing gums,
gelatins,
ice creams, sorbets, puddings, jams, jellies, salad dressings, and other
condiments,
cereal, and other breakfast foods, canned fruits and fruit sauces and the
like.
- Dairy products such as milk, cheese, yoghurt.
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26
- Pharmaceutical compositions which may, for example, be in the form of a
syrup, an
emulsion, a suspension, a solution or other liquid form.
- Dental compositions including, for example, mouth freshening agents,
gargling
agents, mouth rinsing agents, toothpaste, tooth polish, dentifrices, mouth
sprays and
dental floss.
- Edible gel compositions
The compositions disclosed herein may further comprise a base composition. For
example, the comestible compositions disclosed herein may further comprise a
comestible base composition. This refers to all the ingredients necessary for
the
composition except the combination of the high-intensity sweetener and low-
potency
sweetener (e.g. sweetness modifying composition). The base composition may,
for
example, be a sweetened base composition comprising at least one other
sweetener
present in an amount equal to or greater than its sweetness recognition
threshold
and/or an amount having a sweetness equal to or greater than about 1.5 % (w/v)
sucrose equivalence. These will naturally vary in both nature and proportion,
depending on the nature and use of the composition, but they are well known in
the art
and may be used in art-recognised proportions. The formulation of such a base
composition for every conceivable purpose is therefore within the ordinary
skill in the
art.
The ingredients in a base composition may include, but are not limited to,
anti-caking
agents, anti-foaming agents, anti-oxidants, binders, colourants, diluents,
disintegrants,
emulsifiers, encapsulating agents or formulations, enzymes, fats, flavour-
enhancers,
flavouring agents, gums, lubricants, polysaccharides, preservatives, proteins,
solubilisers, solvents, stabilisers, sugar-derivatives, surfactants,
sweetening agents,
vitamins, waxes, and the like. Solvents which may be used are known to those
skilled
in the art and include e.g. ethanol, ethylene glycol, propylene glycol,
glycerine and
triacetin. Encapsulants and gums include maltodextrin, gum arabic, alginates,
gelatine,
modified starch, and polysaccharides.
Examples of additives, excipients, carriers, diluents or solvents for flavour
or fragrance
compounds may be found e.g. in "Perfume and Flavour Materials of Natural
Origin", S.
Arctander, Ed., Elizabeth, N.J., 1960; in "Perfume and Flavour Chemicals", S.
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27
Arctander, Ed., Vol. I & II, Allured Publishing Corporation, Carol Stream,
USA, 1994; in
"Flavourings", E. Ziegler and H. Ziegler (ed.), Wiley-VCH Weinheim, 1998, and
"CTFA
Cosmetic Ingredient Handbook", J.M. Nikitakis (ed.), 1st ed., The Cosmetic,
Toiletry
and Fragrance Association, Inc., Washington, 1988.
The proportion of the combination of the one or more high-intensity
sweetener(s) and
one or more low-potency sweetener(s) (e.g. sweetness modifying composition) or
the
one or more sweetness enhancer(s) selected from mogroside IV, siamenoside and
neomogroside will depend on the nature of the composition and the degree and
characteristics of the sweetness desired. The skilled person can readily
ascertain the
appropriate proportion in every case with only simple, non-inventive
experimentation.
The amounts and proportions disclosed herein are exemplary only and the
flavourist
may seek particular effects by working outside this range, and it should be
regarded as
an indication only.
The pH of the composition disclosed herein may be any pH that does not
adversely
affect the taste of the sweetener blend. For example, the pH may range from
about
1.8 to about 8 or from about 2 to about 5. A person skilled in the art would
be able to
identify a suitable concentration of each sweetener to use depending on the pH
of the
composition.
The use of the one or more low-potency sweetener(s) with the one or more high-
intensity sweetener(s) may, for example, improve one or more sweetness
characteristics in a sweetened composition in comparison to the use of the one
or more
high-intensity sweetener(s) alone. Thus, the sweetened compositions disclosed
herein
may, for example, have one or more improved sweetness characteristics compared
to
the sweetened compositions in the absence of the one or more low-potency
sweetener(s). The use of one or more sweetness enhancer(s) selected from
mogroside
IV, siamenoside and neomogroside may, for example, improve one or more
sweetness
characteristics of a sweetened composition in comparison to the use of a
different
sweetness enhancer such as Luo Han Guo extract in place of the one or more of
mogroside IV, siamenoside and neomogroside.
The sweetened compositions disclosed herein may, for example, have one or more
sweetness characteristics that are more similar to sucrose compared to the
sweetened
compositions in the absence of the one or more low-potency sweetener(s) or
compared
to the sweetened compositions comprising a different sweetness enhancer.
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28
The sweetened compositions disclosed herein may, for example, have a weakened
lingering sweet taste in compared to the sweetened compositions in the absence
of the
one or more low-potency sweetener(s) or compared to the sweetened compositions
comprising a different sweetness enhancer.
The sweetened compositions disclosed herein may, for example, have a weakened
bitter taste and/or astringent taste and/or metallic taste and/or liquorice
taste compared
to the sweetened compositions in the absence of the one or more low-potency
sweetener(s) or compared to the sweetened compositions comprising a different
sweetness enhancer.
The sweetened compositions disclosed herein may, for example, have a
strengthened
sweetness impact compared to the sweetened compositions in the absence of the
one
or more low-potency sweetener(s) or compared to the sweetened compositions
comprising a different sweetness enhancer.
The comparative sweetened composition is identical except that it does not
include any
of the one or more low-potency sweetener(s) or identical except that it
comprises a
different sweetness enhancer in place of the one or more mogroside(s), for
example
one or more of mogroside IV, siamenoside and neomogroside.
Uses
There is provided herein the use of a combination of one or more high-
intensity
sweetener(s) and one or more low-potency sweetener(s) to enhance the sweetness
of
a composition comprising at least one other sweetener present an amount equal
to or
greater than its sweetness recognition threshold and/or an amount having a
sweetness
equal to or greater than about 1.5 % (w/v) sucrose equivalence. The
combination of the
one or more high-intensity sweetener(s) and one or more low-intensity
sweetener(s)
has a sweetness less than 1.5 % (w/v) sucrose equivalence. The one or more
high-intensity sweetener(s), one or more low-potency sweetener(s) and at least
one
other sweetener may be in accordance with any embodiment disclosed herein.
There is provided herein the use of one or more mogroside(s), for example one
or
more of mogroside IV, siamenoside and neomogroside to enhance the sweetness of
a
composition comprising at least one other sweetener present an amount equal to
or
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29
greater than its sweetness recognition threshold and/or an amount having a
sweetness
equal to or greater than about 1.5 % (w/v) sucrose equivalence.
Thus, there is provided a method for enhancing the sweetness of a sweetened
composition, the method comprising providing a base composition comprising at
least
one sweetener in an amount having a sweetness above its sweetness recognition
threshold and/or an amount having a sweetness equal to or greater than about
1.5 %
(w/v) sucrose equivalence, and adding at least one low-potency sweetener, at
least
one high-intensity sweetener; or adding one or more mogroside(s), for example
one or
more of mogroside IV, siamenoside and neomogroside. Each component of the
final
composition may be added in any order to obtain the desired final composition.
The
method may, for example, comprise mixing the components.
The one or more high-intensity sweetener and/or the combination of the one or
more
high-intensity sweetener and the one or more low-potency sweetener (e.g. the
sweetness modifying composition) and/or the one or more mogroside(s), for
example
one or more of mogroside IV, siamenoside and neomogroside may, for example,
increase the sweetness of a sweetened composition by equal to or more than
about
1.0 % (w/v) sucrose equivalence. For example, the high-intensity sweetener(s)
and/or
.. the combination of the high-intensity sweetener(s) and the low-potency
sweetener(s)
and/or the one or more mogroside(s), for example one or more of mogroside IV,
siamenoside and neomogroside may increase the sweetness of a sweetened
composition by equal to or greater than about 1.1 % (w/v) sucrose equivalence
or
equal to or greater than about 1.15 % (w/v) sucrose equivalence or equal to or
greater
than about 1.2 % (w/v) sucrose equivalence or equal to or greater than about
1.25 %
(w/v) sucrose equivalence. The composition may, for example, be a composition
comprising at least one other sweetener.
There is also provided herein the use of one or more low-potency sweetener(s)
to
improve one or more sweetness characteristics of a sweetened composition
comprising one or more high-intensity sweetener(s). The one or more high-
intensity
sweetener(s) and one or more low-potency sweetener(s) are used in amounts
having a
sweetness of less than about 1.5 % (w/v) sucrose equivalence.
Thus, there is provided a method for improving one or more sweetness
characteristics
of a sweetened composition comprising one or more high-intensity sweetener(s)
in an
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amount having a sweetness less than 1.5 % (w/v) sucrose equivalence, the
method
comprising providing a composition comprising one or more high-intensity
sweetener(s)
and adding one or more low-potency sweetener(s). Each component of the final
composition may be added in any order to obtain the desired final composition.
The
5 method may, for example, comprise mixing the components.
The improvement of one or more sweetness characteristics of a sweetened
composition comprising a high-intensity sweetener may, for example, provide
sweetness characteristics that are more similar to the sweetness
characteristics of
10 sucrose.
The sweetness characteristics may refer to the flavour profile (taste
profile), which
refers to the intensity of the flavour and perceptual attributes of a given
compound.
Exemplary flavour attributes of sweetness are sweetness intensity, bitterness,
black
15 liquorice etc.
The sweetness characteristics may refer to the temporal profile, which refers
to the
changes in perception of sweetness over time. Every sweetener exhibits a
characteristic appearance time (AT) and extinction time (ET). Most high-
potency
20 sweeteners, in contrast to carbohydrate sweeteners, display prolonged ET
(lingering).
Generally, the detected sucrose equivalence spikes to a maximal response
level, then
tapers off over time. The longer the taper, the greater the detected sweetness
linger of
a compound.
25 In certain embodiments, the one or more low-potency sweetener(s) may be
used to
weaken the lingering sweet taste of the sweetened composition comprising one
or
more high-intensity sweetener(s). In other words, the low-potency sweetener
may be
used to decrease the extinction time (ET) of the sweetened composition
comprising
one or more high-intensity sweetener(s). This relates to the undesirable
lingering of the
30 sweetness taste in the mouth after the composition is initially ingested
or expectorated.
The lingering sweet taste may, for example, refer to the length of time that
the
sweetness taste remains after it is initially detected, how rapidly the
intensity of the
sweetness taste decreases or fades after it is initially detected and the
intensity of the
sweetness taste after it is initially detected. The one or more low-potency
sweetener(s)
may, for example, decrease the length of time that the sweetness taste remains
after it
is initially detected and/or increase the speed at which the sweetness taste
decreases
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31
after it is initially detected and/or decrease the intensity of the sweetness
taste after it is
initially detected.
In certain embodiments, the one or more low-potency sweetener(s) may be used
to
weaken the bitter taste and/or astringent taste and/or metallic taste and/or
liquorice
taste of the sweetened composition comprising the one or more high-intensity
sweetener(s). The term "liquorice" refers to a sweet taste of a compound.
In certain embodiments, the one or more low-potency sweetener(s) may be used
to
strengthen the sweetness impact of the sweetened composition comprising the
one or
more high-intensity sweetener(s). The sweetness impact relates to the length
of time it
takes before the sweetness is initially detected and the intensity at which
the
sweetness is initially detected. The one or more low-potency sweetener(s) may,
for
example, decrease the amount of time before the sweetness is initially
detected and/or
increase the intensity at which the sweetness is initially detected.
The degree of sweetness and other sweetness characteristics described herein
may be
evaluated by a tasting panel of trained experts, for example as described in
the
examples below.
Manufacturing Methods
There is further provided herein methods of making the compositions disclosed
herein.
The compositions may be in accordance with any embodiment disclosed herein.
The methods may comprise combining each component of a desired composition in
the desired proportions and optionally mixing the components together. The
components may be combined and mixed in any suitable order.
A person skilled in the art would identify a suitable method to make the
composition
(e.g. suitable order in which to combine or mix the components) depending on
the
nature of the composition and the degree and characteristics of the sweetness
desired.
The methods may, for example, comprise providing a desired base composition
and
adding the sweeteners thereto.
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Each of the sweeteners disclosed herein may be made by a synthetic process or
by a
biological (e.g. enzymatic) process or a fermentation process or may be
isolated from a
natural source such as a plant or fruit.
The process may, for example, comprise contacting at least one mogrol
precursor
substrate with a mogroside pathway enzyme. The enzyme may, for example, be
present in a cell lysate or may be present in a host cell (e.g. a recombinant
host cell).
The enzyme may, for example, be a UGT enzyme (UDP-glucuronosyltransferase).
For example, a mogroside compound may be made by the biosynthetic pathway
disclosed in WO 2013/076577 or WO 2014/086842, the contents of which are
incorporated herein by reference.
For example, mogroside V may be made by the biosynthetic pathway disclosed in
ltkin et al., "The biosynthetic pathway of the nonsugar, high-intensity
sweetener
mogroside V from Siraitia grosvenorir, PNAS, 7 November 2016, E7619 ¨ E7628
and
WO 2016/038617, the contents of which are incorporated herein by reference.
For example, a mogroside compound may be made by modifying (e.g.
redistributing
glycoside content) another mogroside compound. For example, a mogroside
compound may be made by redistributing glycoside content of another mogroside
compound using acid or enzymes as disclosed in WO 2014/150127, the contents of
which are incorporated herein by reference.
The process may, for example, comprise extracting one or more sweetener
compounds from a natural source such as a plant or fruit. This may, for
example, be
followed by a purification step to yield a high-intensity sweetener, low-
intensity
sweetener or mixture of sweeteners (e.g. mixture of high-intensity sweeteners
such as
a mixture of mogrosides). The extract may, for example, have a relatively high
content
of mogroside V and/or 11 ¨0-mogroside V (e.g. at least about 30 wt% or at
least about
40 wt%). This may, for example, involve fractioning, for example by flash
chromatography. One or more mogroside compounds (e.g. mogroside V) may be
obtained from Luo Han Guo (Siraitia grosvenorii) fruit.
When a fermentation process is used to make the target product (e.g. target
mogroside
product), the target can be extracted from the aqueous fermentation reaction
medium
using an appropriate solvent (e.g., heptane) followed by fractional
distillation. The
chemical composition of each fraction can be measured quantitatively by GC/MS
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33
(gas chromatography mass spectrometry). Fractions can be blended to generate
the
desired mogroside compounds (e.g. mogroside V and 11-0-mogroside V) for use in
flavour or other applications.
Verification of acceptability of the final blended product can be carried out
by direct
comparison to a reference mogroside flavouring product (for example, an
existing
natural flavouring commercial product obtained from a commercial supplier).
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34
EXAMPLES
Example 1
Methods
Luo Han Guo fruit extracts obtained from Blue California (Tomas, Rancho Santa
Margarita, California) (extract 4), Azile LCC (Rolling Hills Est, California)
(extracts 1
and 2) and Chr. Olesen Group (Gentofte, Denmark) (extract 3) were analysed to
identify the compounds present in the extract.
Sample solutions of the extracts were prepared by dissolving 16.52 jig of the
sample in
25.0 mL solvent (acetonitrile/water 20/80 v/v). From this solution 100 iL were
transferred into a HPLC vial and 900 iL solvent was added (66.1 ppm solution).
From
the sample solution 10 iL was transferred to a HPLC vial and 990 iL solvent
was
added (6.61 ppm solution). Both the 66.1 and 6.61 ppm solutions were injected
twice
on the LC-MS.
Calibration (reference) solutions of mogroside V were made by dissolving 9.22
mg
mogroside V (98.5 % mogroside V obtained from AAPIN chemicals Ltd.,
Oxfordshire,
UK) in 10.0 mL solvent (acetonitrile/water 20/80 v/v). The stock solution was
stored in
the freezer and used to prepare solutions of mogroside V at various
concentrations
(0.11 ppm, 0.34 ppm, 1.02 ppm, 3.07 ppm and 9.22 ppm). These solutions were
also
injected twice on the LC-MS.
2 iL of each solution was injected on an Acquity C18 BEH 1.7 pm 150 x 2.1 mm
column (Waters, Milford, Massachusetts, United States) at 40 C. Compounds were
eluted using a mixture of acetonitrile and 0.1% formic acid in water starting
at
20% acetonitrile going up to 50% acetonitrile in 14 minutes. The gradient was
back on
the starting values in 1 minute and stabilized for 5 minutes. The flow was set
on 400 iL
during the whole run.
Eluted compounds were detected using liquid chromatography mass spectrometry
(LC/MS). The mass spectrometer was operating in ESI negative mode measuring
150 to 2000 Amu with a resolution of 70000. Gas flow rates were sheath 60, aux
20
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and sweep 3. Capillary temperature and aux gas heater temperature were set on
380 C and 400 C respectively.
The % of each component in the extract was calculated using the following
equation
5 and calibrated against a curve of the various concentration calibration
mogroside V
(reference) solutions described above.
a re. c. r cf
rc.r = __________ . 100
511"
10 .. ratio = % component
area = component area in sample (average area from 2 injections)
V = sample solvent volume in litres
d = sample dilution (from sample solution to vial)
slope = slope from mogroside V calibration curve with b (intercept) = 0
15 SW = sample weight in mg
Results
Figure 1 shows a chromatogram of a Luo Han Guo extract (extract 2 of Table 1
below).
Table 1 shows the composition of four different Luo Han Guo extracts.
Mogroside V is
the mogroside having the highest concentration in all four extracts (about 45
wt% in
extract 1).
0
Table 1.
w
=
Retention Time (Rt) Concentration in Concentration in
Concentration in Concentration in oe
Name
w
(minutes) Extract 1 (wt%) Extract 2 (wt%)
Extract 3 (wt%) Extract 4 (wt%) =
=
(...)
1.99 Grosvenorine II 0.64 0.44
0.00 0.45
1.52 Grosvenorine 1 1.38 0.84
0.00 0.62
7.44 11-0-mogroside 11 (1) 0.01 0.02
0.03 0.03
P
11-0-mogroside II
8.54 0.01 0.01
0.01 0.01 .
,õ
0
11-0-mogroside II
,
,
9.51 0.03 0.01
0.04 0.02 ,
,
(III)
0
7.21 Mogroside 11 (1) 0.04 0.08
0.11 0.14
8.43 Mogroside 11 (11) 0.06 0.06
0.05 0.05
oo
9.43 Mogroside 11 (111) 0.51 0.20
0.53 0.27 n
1-i
m
oo
w
11-dehydroxy-
=
9.24 0.04 0.02
0.03 0.01
oe
mogroside III
O-
o,
4.
(...)
w
4.
C
w
5.97 11-0-mogroside III 0.22 0.11
0.19 0.09 =
,-.
oe
i-J
w
=
,-.
6.20 Mogroside III (I) 1.61 0.92
1.42 0.71 =
(...,
7.88 Mogroside III (II) 0.29 0.24
0.27 0.28
5.13 Siamenoside 1.84 1.81
2.95 2.20
P
5.30 Mogroside IV (II) 0.42 0.50
0.51 0.88 0
0
5.67 Mogroside IV (III) 2.18 1.91
3.19 2.31
0
,
,
,
,
,
6.05 Mogroside IV (IV) 0.09 0.08
0.14 0.10 0"
Deoxymogroside V
6.58 1.30 1.42
1.54 1.36
(I)
Deoxymogroside V
7.43 0.38 0.38
0.39 0.32
(II) oo
n
1-i
11-0-mogroside V
m
4.13 4.99 4.70
4.89 4.97 oo
(I)
w
=
,-.
oe
'a
3.87 Mogroside V isomer 0.54 0.58
0.60 0.56 c,
4.
(...,
w
4.
C
w
4.54 Mogroside V 45.42 43.88
43.94 41.67 =
,-.
oe
i-J
w
=
,-.
4.89 Iso-Mogroside V 2.20 2.10
2.05 1.74 =
(...,
2.08 7-0-mogroside V 0.19 0.15
0.15 0.18
3.23 11-0-mogroside VI 0.33 0.27
0.26 0.25
P
3.72 Mogroside VI (I) 0.80 0.66
0.83 0.64 0
0
oe
.
3.93 Mogroside VI (II) 0.66 0.52
0.40 0.43
0
,
,
,
Mogroside VI (III)
4.22 1.19 0.96 0.88 0.74
(Neomogroside)
4.67 Mogroside VI (IV) 0.19 0.16
0.14 0.45
Total 67.58 63.04
65.57 61.50
oo
n
1-i
m
oo
w
=
,-.
oe
'a
c,
4.
(...,
w
4.
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Example 2
Methods
A Luo Han Guo fruit extract obtained from Azile LCC (Rolling Hills Est,
California)
(extract 1 of Example 1 above) containing about 68 wt% mogrosides was
fractionated
by reverse phase (C-18) flash chromatography.
Compounds were eluted using a mixture of methanol (Me0H) in water starting at
30%
Me0H followed by a linear gradient of 30-80% Me0H then finally the column was
flushed with 80% Me0H. The solvents were introduced at the flow rate of
30m1/min
throughout the separation procedure. Eluted compounds were visualized with a
UV
detector set at 210 nm and a coronal light scattering detector. The % of each
component in the extract was calculated using the equation described in
Example 1
above.
Collected fractions were pooled according to Table 2 below, and then freeze
dried to
powders. The powder corresponding to various pooled fractions as given in
Table 2
below was dissolved in various concentrations on top of 5% sucrose. The taste
of
these samples was compared by three expert panellists (trained flavourists) to
controls
of 5% sucrose. Thus the sweetness enhancement effect of each fraction or pool
of
fractions exhibited in 5% sucrose was determined.
Results
The results are indicated in the table below. The whole extract was collected
into 22
fractions. Fractions 1-10 contain no mogroside V.
Table 2.
Fraction # Mogroside Dose level
(combined) V content of Fraction Taste evaluation (on top of 5%
(0/0) (PPrn) sucrose)
1( tube 1-7) 0 15
2(tube 8-12) 0 15 Some in this set have sugary notes,
most
3(tube 13-16) 0 15 also have fermented off notes
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4 (tube 17-20) 0 15
5 (tube 21-24) 0 15 Some astringency in this part
6 (tube 25-28) 0 15
Sweetness suppressed
7 (tube 29-32) 0 15
Bitter, metallic, fermented off notes
8 (tube 33-34) 0 15
No enhancement
9 (tube 35-36) 0 15
10 (tube 37) 0 15 Fermented, typical white dog notes,
astringent, cooked delayed sweet
11 (tube 38) 1.15 45 Sweeter, some mouthfeel, some upfront
12 (tube 39) 15.9 45 Slightly higher in enhancement, less
off
notes
13 (tube 40) 64.5 30 Sweeter, higher licorice, less dirty
fermented, sl higher astringency
14 (tube 41- 100 25 Very lingering, very licorice, dirty
sweet,
42) numbing, sharp sweetness, mouth drying
15 (tube 43- 97 25 Strong fermented dirty note, higher
44) sweet, numbing delayed sharp, strong
licorice, linger, metallic
16 (tube 45- 70.3 25
46)
17 (tube 47) 18.7 45 Astringent, some enhancement, mostly
18 (tube 48) 4.6 45 licorice lingering
19 (tube 49) 1.95 45
20 (tube 50- 1.56 5
51) Negative notes, typical fermented
21 (tube 52- 0 5 lingering
54)
22 (tube 55- 0
end)
Fraction 1-10 and 19-22 have an off-taste, which is the character of Luo Han
Guo fruit,
no sweet enhancement impact.
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The better sweetness enhancement effect was observed within fractions 12-17,
which
contains mainly mogrosides. When the fractions almost have pure mogroside V,
the
lingering, dirty fermented note is more noticeable, such as fractions 14 and
15. Thus,
pure mogroside V has inherent lingering off-taste.
Fraction 12 was the cleanest sweet, but less upfront due to small percentage
mogroside V. Fraction 13 has better sweet quality, but slightly higher
astringency. 11-
0-mogroside V and mogroside V are the two major mogrosides in those two
fractions,
but with different ratio (F12 mogroside V:11-0-mogroside V is 4:9 and F13
mogroside
V:11-0-mogroside V is 13:3).
Mogroside V was very sweet, judged to be 425 times sweeter than sucrose, while
11-
0-mogroside V is rated as 84 times sweeter than sucrose.
Example 3
Methods
A Luo Han Guo fruit extract obtained from Azile LCC (Rolling Hills Est,
California)
(extract 1 of Example 1 above) containing about 68 wt% mogrosides was
fractionated
and the composition of each fraction determined by the chromatography method
described above in relation to Example 1.
Each fraction was combined with a solution of 5 % sucrose and the taste of
these
samples was compared by three expert panelists (trained flavourists) to
controls of 5%
sucrose.
Results
Table 3 shows the chemical composition of fractions 11 to 20 of the extract.
C
t..)
Table 3.
=
,-,
cio
Retention
t..)
o
Time (Rt) Name F11 F12 F13 F14 F15
F16 F17 F18 F19 F20
o
(...)
(minutes)
1.99 Grosvenorine II 0.02 0.01 0.00 0.00 0.00 0.00
0.01 0.00 0.00 0.00
1.52 Grosvenorine I 0.01 0.00 0.00 0.00 0.00 0.01
0.01 0.01 0.00 0.01
7.44 11-0-mogroside II (I) 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
P
8.54 11-0-mogroside II (II) 0.00 0.00 0.00 0.00 0.00
0.00 0.01 0.02 0.02 0.01
c,
4=,
N
0.
Iv
9.51 11-0-mogroside II (111) 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 0.00
,
,
,
,
,
7.21 Mogroside 11 (I) 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00 " 8.43 Mogroside II (11) 0.00 0.00
0.00 0.00 0.00 0.00 0.02 0.06 0.10 0.06
9.43 Mogroside II (111) 0.00 0.00 0.00 0.00 0.00
0.01 0.03 0.03 0.03 0.04
11-dehydroxy-mogroside
9.24 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.00
III
1-d
n
5.97 11-0-mogroside III 0.00 0.00 0.00 0.00 0.00 0.02
0.46 4.87 8.66 5.65
m
1-d
6.20 Mogroside III (I) 0.00 0.00 0.00 0.00 0.00 0.01
0.08 1.52 14.8 82.6 t..)
o
,-,
cio
7.88 Mogroside III (II) 0.04 0.06 0.04 0.02 0.02
0.03 0.05 0.05 0.04 0.12 O-
o,
.6.
(...)
t..)
.6.
C
t..)
5.13 Mogroside IV (I) 0.01 0.01 0.05 0.63
4.27 17.5 21.2 5.99 1.03 0.25
,-,
cio
5.30 Mogroside IV (II) 0.01 0.01 0.01 0.01
0.08 1.86 12.0 12.7 5.87 1.08
t..)
o
,-,
o
5.67 Mogroside IV (III) 0.01 0.01 0.01 0.01
0.03 0.00 19.2 64.3 86.7 36.6 (44
6.05 Mogroside IV (IV) 0.00 0.00 0.00 0.00
0.00 0.01 0.01 1.32 2.54 2.39
6.58 Deoxymogroside V (I) 0.00 0.00 0.00 0.00 0.00 0.00
0.00 0.03 0.05 3.75
P
7.43 Deoxymogroside V (II) 0.00 0.00 0.00 0.00 0.00
0.00 0.00 0.00 0.01 0.01 0
0
4=,
Iv
4.13 11-0-mogroside V (I) 22.1 35.5 14.2 1.97 0.22 0.17
0.19 0.13 0.11 0.10 ,
,
,
,
,
"
0
3.87 Mogroside V isomer 5.54 4.68 1.14 0.11
0.02 0.02 0.02 0.01 0.01 0.01
4.54 Mogroside V 1.14 15.9 64.5 104 97.5 70.8
18.7 4.59 1.95 1.56
4.89 Iso-Mogroside V 0.03 0.18 1.24 3.88
6.86 9.19 3.38 0.70 0.14 0.11
1-d
2.08 7-0-mogroside V 0.02 0.00 0.04 0.04
0.06 0.05 0.02 0.01 0.00 0.00 n
1-i
m
1-d
3.23 11-0-mogroside VI 0.88 0.12 0.01 0.00
0.00 0.00 0.00 0.00 0.00 0.00 t..)
o
,-,
cio
3.72 Mogroside VI (I) 9.58 4.90 0.56 0.09
0.01 0.00 0.01 0.00 0.00 0.00 O-
o
.6.
(44
t..)
.6.
C
t..)
3.93 Mogroside VI (II) 0.65 2.45 2.37 0.00 0.17 0.05
0.03 0.02 0.01 0.01 =
,-,
cio
i-J
t..)
Mogroside VI (III)
=
4.22 0.80 3.96 4.77 2.01 0.00 0.15
0.07 0.04 0.03 0.03
o
(...)
(Neomogroside)
4.67 Mogroside VI (IV) 0.00 0.01 0.07 0.25 0.55 1.03
0.56 0.12 0.02 0.01
Total 40.8 67.9 89.0 112.6 109.8 100.9
76.0 96.6 122.2 134.5
P
.
,õ
.
4=,
,';'
4=,
0.
Iv
o
r
u,
1
r
r
1
Iv
o
.0
n
1-i
m
1-d
t..)
o
,-,
cio
O-
o
.6.
(...)
t..)
.6.
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Table 4 shows the tasting results for fractions 12 to 15. The results are
similar to the
results obtained in Example 2.
Table 4.
Iso-sweet Comments for
Fraction Level Iso-sweet
Comments for 5% Sucrose Tasting
(PPrn) tasting
Slightly higher in enhancement, less off-
notes, this was the cleanest sweet of all
the samples, less upfront sweet than Luo
Han Guo extract, good quality of sweet
except for lower upfront sweet
impression.
Closest to Luo
Han Guo extract,
F12 45 The
other fractions have different off-
less overall off-
notes compared to Luo Han Guo extract,
notes
but are comparable in terms of quality of
sweet.
Basically, numbing/irritating effects are
stronger than "wet dog" fermented
effects, typical in Luo Han Guo extract
Very close in
Sweeter, higher licorice, less dirty
F13 30 sweetness,
fermented, sl higher astringency
lingering
Very lingering, very licorice, dirty sweet,
Slightly sweeter, lower
overall quality of sweet compared
very likely would to Luo
Han Guo extract, numbing, sharp
match iso-sweet
sweetness, mouth drying, irritating,
F14 25 of Luo Han Guo lingers
extract, less dirty
finish than Luo Fractions 14 and 15 have a sharp,
Han Guo extract
numbing, burn, which negatively effects
potency and quality of sweet
F15 25 Very close in Strong
fermented dirty note, higher
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profile, less off-
sweet, numbing, delayed sharp, strong
notes licorice, lingers, metallic
Example 4
Mogroside V and 11-0-mogroside V were isolated from extracts of Luo Han Guo
using
Agilent 1100 preparative HPLC system with a Phenomenex Luna 018 (2) column (5
m, 210 X 21.4 mm), and combined to form solutions of various concentrations.
These solutions were combined with a solution containing 5 % (w/v) sucrose and
0.03
% (w/v) citric acid to give test samples and evaluated by a sweet sensitive
taste panel
of five experts (trained flavourists).
The results are shown in Table 5 below.
20
30
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Table 5.
Mogroside Content Mogroside V 11-0-Mogroside V Taste evaluation
Concentration Concentration
(Dom) (Dom)
Mogroside V 20 0 Less sweet, less
upfront, musty, similar
like base
Mogroside V 25 0 Good impact, strong
lingering, metallic,
woody.
Mogroside V 30 0 Sweet lingering,
astringent, sl licorice,
upfront
11-0-mogroside V 0 45 Linger, metallic,
clean
finish, not as sweet as
mogroside V
Mogroside V + 11-0- 20 3 sweet, cinnamic,
mogroside V
lingering, woody, fruity
sweet
Mogroside V + 11-0- 20 5 Slight lingering, low
mogroside V
impact sweet, astringent
Mogroside V + 11-0- 20 8.5 lingering, slight
fruity,
mogroside V mouthfeel, full,
fruity
sweet
Mogroside V + 11-0- 25 3 Preferred, sugar like
mogroside V impact, increase
sweetness
Mogroside V + 11-0- 25 5
Moderate impact, bitter
mogroside V finish
Mogroside V + 11-0- 25 8.5 Sweeter, slightly
dry,
mogroside V slightly lingering,
sl
fruity, cleanest,
mouthf eel
Luo Han Guo extract 45 (total dose level of Luo Han Guo Clean impact, some
extract) lingering, low sweet
impact
It was surprisingly found that blending 11-0-mogroside V with mogroside V
improves
the sweet quality compared to mogroside V alone. 11-0-mogroside V on top of
mogroside V helped to reduce sweetness lingering (weaken later sweetness
taste) and
reduce astringent and bitter aftertastes compared to mogroside V alone. Thus,
the
11-0-mogroside V made the sweetness taste more similar to sugar than mogroside
V
alone (i.e. assists in providing a temporal profile that is closer to sugar).
This enables
the use of higher concentrations of mogroside V to obtain higher sweetness
whilst
eliminating the disadvantages associated with using higher concentrations of
this
sweetener (e.g. lingering, bitter and astringent aftertastes). This was
surprising given
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that mogroside V is the most potent mogroside sweetener and 11-0-mogroside V
is of
much lower potency.
Example 5
A sweet sensitive taste panel ranked the sweetness of solutions of a mixture
of
sweeteners ("Mixture 1") in relation to sucrose solutions at a range of
concentrations to
determine sucrose equivalence. Mixture 1 was a combination of fractions 11 to
18 of
Example 2 and contained 8.16 wt% 11-0-mogroside V and 61.6 wt% mogroside V.
The results are shown in Table 6 below.
Table 6.
35 ppm 25 ppm 30 ppm 1 % 1.5
''/o
Panelist
Mixture 1 Mixture 1 Mixture 1
Sucrose Sucrose
1 3 2 1 4 5
2 1 2 5 3 4
3 1 2 3 4 5
4 2 3 1 4 5
5 1 3 4 2 5
6 2 5 1 3 4
7 3 2 5 1 4
8 3 2 1 4 5
9 3 1 2 4 5
10 3 2 1 4 5
Total 22 24 24 33 47
The data demonstrates that mixture 1 has a sweetness below 1 % sucrose
equivalence
(as determined by seven panellists), which is accepted as "not intrinsically
sweet" by
FEMA. Therefore, mixture 1 is suitable for use as sweetness modifiers or
blends at the
indicated concentrations because it does not have any detectable sweetness at
these
levels.
A concentration of mixture 1 having an iso-sweet threshold close to 1 % (35
ppm) was
selected and added to 5 % (w/v) sucrose solutions. This solution was then
ranked
against 5, 6, 6.5 and 7 % (w/v) sucrose solutions. This was repeated for 45
ppm Luo
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Han Guo extract. The average score of each solution was determined. The
results are
shown in Table 7 below.
Table 7.
Sweetener Average Score
Mixture 1 (35 ppm) 6.5
Luo Han Guo extract (45 ppm) 6.4
It was surprisingly found that mixture 1 and Luo Han Guo extract act as
sweetness
enhancers since the increase in sweetness of the 5 % (w/v) sucrose solution to
which
they were added was greater than the sweetness of the sweetener alone.
The taste of various concentrations of mixture 1 was tested by an expert panel
of three
people (trained flavourists) in solutions containing 5 % sucrose and 0.03 %
citric acid.
The taste was compared to the Luo Han Guo extract used in Example 2 (obtained
from
Azile LCC (Rolling Hills Est, California) (extract 1 of Example 1 above) and
containing
about 68 wt% mogrosides) (combined with the same 5 % sucrose and 0.03 % citric
acid solution). The results are shown in Table 8.
Table 8.
Sample Panelist 1 Panelist 2 Panelist 3
Luo Han Guo Sweeter, round, sl Good impact, low Most rounded
extract (45 ppm) linger baggage sweet profile
Vitamin note,
Mixture 1 (35 ppm) Linger, sweet Sweetest, lingering oxidized,
most
sweet overall
Lower sweet, least
Mixture 1 (30 ppm) Sweeter Clean sweet
baggage
SI lower in Lingering, clean,
Mixture 1 (25 ppm) Clean sweet
sweetness least sweet
Overall, mixture 1 provides a better sweet quality (less baggage, sweeter)
than the Luo
Han Guo extract.
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Example 6
A sweet sensitive taste panel ranked the sweetness of solutions of various
sweeteners
(mogroside V, mogroside IV, siamenoside, neomogroside, 11-0-mogroside V) in
5 relation to sucrose solutions at a range of concentrations to determine
sucrose
equivalence. The sweeteners were obtained using an Agilent 1100 preparative
HPLC
system with a Phenomenex Luna 018 (2) column (5 pm, 210 X 21.4 mm). The
results
are shown in Tables 9 to 13.
10 Table 9.
20 ppm 25 ppm
0.5% 1% 1.5%
Panelist Mogroside Mogroside
Sucrose Sucrose
Sucrose
V V
1 3 1 4 2 5
2 1 3 2 4 5
3 1 2 4 3 5
4 1 2 4 3 5
5 1 2 3 4 5
6 1 3 2 5 4
Total 8 13 19 21 29
Table 10.
25 ppm 30 ppm
Sucrose 0.5 Sucrose 1 Sucrose 1.5
Panelist Mogroside Mogroside
0/0 0/0 0/0
IV IV
1 3 1 4 2 5
2 2 3 1 4 5
3 1 2 3 4 5
4 2 4 3 1 5
5 2 1 3 4 5
6 1 2 3 4 5
7 2 1 3 4 5
Total 13 14 20 23 35
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Table 11.
Sucrose Siamenoside Siamenoside Sucrose 1 Sucrose
Panelist
0.5 ''/o 20 ppm 25 ppm % 1.5
''/o
1 1 4 3 2 5
2 1 3 2 4 5
3 2 1 3 4 5
4 1 4 2 3 5
1 2 4 3 5
6 1 3 2 4 5
7 1 2 3 4 5
Total 8 19 19 24 35
Table 12.
Mogroside
V (25 ppm)
Sucrose 0.5 Sucrose 1 Sucrose 1.5
Panelist + 11-0-
% % %
Mogroside
V (3 ppm)
1 1 2 3 4
2 3 2 1 4
3 1 3 2 4
4 1 2 4 3
5 1 2 3 4
6 1 3 2 4
7 1 3 2 4
Total 9 17 17 27
5
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Table 13.
25 ppm 30 ppm
0.5% 1% 1.5%
Panelist Neomogros Neomogros
Sucrose Sucrose
Sucrose
ide ide
1 1 4 2 3 5
2 3 1 2 4 5
3 2 1 3 4 5
4 1 2 4 3 5
3 1 5 2 4
6 1 2 4 3 5
7 2 1 3 4 5
8 1 2 3 4 5
9 1 2 3 4 5
Total 15 16 29 31 44
The data demonstrates that mogroside V (25 ppm), mogroside IV (30 ppm),
siamenoside (25 ppm), mogroside V (25 ppm) in combination with 11-0-mogroside
V
5 (3 ppm) and neomogroside (30 ppm) all have a sweetness below 1.5 %
sucrose
equivalence (as determined by seven panellists), which is accepted as "not
intrinsically
sweet" by FEMA. Therefore, these compounds and mixtures are suitable for use
as
sweetness modifiers at the indicated concentrations because they do not have
any
detectable sweetness at these levels.
Concentrations of the tested sweeteners were selected with an iso-sweet
threshold
close to 1 % and added to 5 % (w/v) sucrose solutions. These solutions were
then
ranked against 5, 6, 6.5 and 7 % (w/v) sucrose solutions. The average score of
each
solution was determined. The results are shown in Table14 below.
Table 14.
Sweetener Average Score
Mogroside V (25 ppm) 6.2
Mogroside IV (30 ppm) 6.2
Siamenoside (25 ppm) 6.4
Neomogroside (30 ppm) 6.35
It was surprisingly found that mogroside V, siamenoside, neomogroside and
mogroside
V act as sweetness enhancers since the increase in sweetness of the 5 % (w/v)
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sucrose solution to which they were added was greater than the sweetness of
the
sweetener alone.
The taste of these sweeteners was tested by an expert panel of three people
(trained
flavourists) in solutions containing 5 % sucrose and 0.03 % citric acid. The
taste was
compared to the Luo Han Guo extract used in Example 2 (obtained from Azile LCC
(Rolling Hills Est, California) (extract 1 of Example 1 above) and containing
about 68
wt% mogrosides). Mogroside IV, siamenoside and neomogroside are all better
than
Luo Han Guo extract in terms of sweet quality when added to 5 % sucrose and
0.03 %
citric acid. These 3 compounds provide a sugar like taste with less lingering
sweet
taste. Siamenoside was described as having "more sweet body, sweeter, rounder
with
a little more upfront and more round lasting sweet". Mogroside IV was
described as
having "good and similar sweetness as mogroside V". Neomogroside was described
as
having "sweetness, but slightly bitter aftertaste". The results for mogroside
V are shown
in Table 15.
Table 15.
Sample Description
Astringent, fuller sweet, less sharp, mid
Luo Han Guo extract (45 ppm)
sweet, lingering off note, sweeter, fruity
More astringent, more acidic, more back
80 % Mogroside V (20 ppm)
end, missing upfront fullness, flat, closest
to Largo, slight acidic
Most sweet, acidic, slightly stronger than
80 % Mogroside V (25 ppm) #2,
sweeter overall, sweeter than largo,
linger
In general, the tasters agreed that 80% mogroside V does not have the same
full round
sweet profile as the Luo Han Guo extract. The 80% mogroside V is more acidic
tasting
when applied to a sugar/acid/water solution.
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Example 7
Methods
Mogroside V, siamenoside, mogroside IV and neomogroside were obtained using an
Agilent 1100 preparative HPLC system with a Phenomenex Luna 018 (2) column
(Slim, 210 X 21.4 mm)..
The mogroside V, siamenoside, mogroside IV and neomogroside were each added to
a solution containing 5 % sucrose and 0.03 % citric acid in a concentration of
25 ppm (mogroside V), 25 ppm (siamenoside), 30 ppm (mogroside IV) and
30 ppm (neomogroside) respectively.
These test solutions were tasted by an expert panel of seven people. For
various
aspects of sweet taste (upfront sweet, overall sweet, lingering sweet,
astringent,
volatile off-taste), each panellist scored the test solutions in comparison to
the base
solution (solution of 5 % sucrose and 0.03 % citric acid).
A score of 0 indicated that the taste aspect was the same, 1 indicates
slightly higher, 2
indicates higher, 3 indicates much higher, -1 indicates slightly lower, -2
indicates lower
and -3 indicates much lower. The average score for each test solution for each
taste
aspect was calculated. The results are shown in Table 16 below.
Table 16.
Mogroside V Siamenoside Mogroside IV
Neomogroside
Overall Sweet 1 1.8 0.4 1.4
Upfront Sweet 0.8 1.4 0.4 1
Lingering
0.4 0.6 0 0.4
Sweet
Astringent 0 0.4 0 0
Volatile Off-
0 0.2 0 0
Note
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Example 8
As shown above, siamenoside, neomogroside and mogroside IV all have similar or
better sweet taste quality on top of 5 % sucrose and 0.03 % citric acid
compared to
5 mogroside V. Therefore, the taste of 11-0-mogroside V with each of these
mogrosides
is evaluated as shown in Table 17.
Table 17.
11-0
Siamenoside
Mogroside mogroside V
concentration
content concentration
(PPm) (PPm)
Siamenoside 25 0
Siamenoside + 11-
25 3
0 mogroside V
Siamenoside + 11-
25 5
0-mogroside V
Siamenoside + 11-
25 8.5
0-mogroside V
11-0
Mogroside IV
mogroside V
concentration
concentration
(PPm) (PPm)
Mogroside IV 30 0
Mogroside IV+ 11-
30 3
0-mogroside V
Mogroside IV + 11-
30 5
0-mogroside V
Mogroside IV +11-
30 8.5
0-mogroside V
11-0-
Neomogroside
concentration mogroside V
concentration
(PPm) (PPm)
Neomogroside 30 0
Neomogroside+11-
30 3
0 mogroside V
Neomogroside
+11-0-mogroside 30 5
V
Neomogroside
+11-0-mogroside 30 8.5
V
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Example 9
The taste of mogroside V with and without 11-0-mogroside V in various milk or
yoghurt
bases is evaluated as shown in Table 18. The iso-sweet threshold for mogroside
V in
milk and yoghurt is also evaluated.
A milk base (2% fat) includes 2% fat milk and 5 % sucrose. A non-fat yoghurt
base
includes plain non-fat yoghurt and 5 % sucrose. A full fat yoghurt base
includes plain
full fat yoghurt and 5 % sucrose. A higher dose level is used for milk and
yoghurt
compositions due to the fat, protein and other ingredients. Luo Han Guo
extract is used
at 75 ppm for these applications.
Table 18.
Mogroside Mogroside 11-0 mogroside
concentration V concentration
content
(PPm) (PPm)
Mogroside V 40 0
Mogroside V +
11-0 40 5
mogroside V
Mogroside V +
11-0- 40 10
mogroside V
Mogroside V +
11-0- 40 15
mogroside V
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Example 10
The identification of new minor cucurbitane glycosides from Siraitia
grosvenorii
Introduction
Siraitia grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang is a herbaceous
perennial
vine of Cucurbitaceae family endemic to southern China and northern Thailand.
The
fruit of S. grosvenorii, commonly known as luo han guo' has been used for
traditional
medicine in China for centuries as a treatment of respiratory infection,
bronchitis,
gastritis, constipation etc. Modern pharmacological research have confirmed
that S.
grosvenorii extracts or its components possess variety of bioactivities, such
as
antibacterial, anti-inflammation, anti-diabetic, anti-cancer, and
immunostimulatory [1].
Luo Han Guo has been used as a sweetener in food and beverages in China. It is
now
one of the best known natural high intensity sweeteners throughout the world.
Since
cucurbitane glycoside mogroside V has been discovered as the sweet principle
of S.
grosvenorii, more than 40 cucurbitane triterpenoids have been reported from S.
grosvenorii so far [1-4]. Food and flavor industry researchers have been
actively
discovering and adding more new compounds into the mogroside pool in order to
find
new mogrosides with better sweet performance [5-7]. New molecules under known
natural sweetener categories with better sweet performance have been a sought-
after
for food and flavor industries. The commercialization of rebaubioside M (also
known as
rebaubioside X) is a good example. Even it is a minor natural product from
Stevia (less
than 0.1%) discovered in 2010,
rebaubioside M quickly progressed into
commercialization stage thanks to cost reduction by technology development in
plant
biology, molecular biology and enzyme engineering [8, 9]. Rebaudioside M
received
Letter of No Objection concerning its Generally Recognized as Safe (GRAS)
status
from US FDA in 2013, 2014 and 2017 (GRN No. 473, 512 and 667) [10-12].
We have been conducting investigations to seek the best performance mogrosides
or
their combinations by using commercial Luo Han Guo extracts [13]. Herein, we
report
two new minor cucurbitane glycosides from S. grosvenorii and emphasize our new
oligosaccharide elucidation strategy based on HSQC-TOCSY experiments with
different mixing times.
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Materials and Methods
General experimental procedures
Optical rotations were measure with a Rudolph Autopol IV polarimeter. The NMR
spectra were recorded on Bruker DRX Avance 300 or 500 spectrometers. Chemical
shifts are given in 5 (ppm) referring to the residual solvent peak. Low
pressure
chromatography was performed on Biotage Flash System SP1. Preparative HPLC was
performed on an Agilent 1100 preparative HPLC system with a Phenomenex Lunar
C18(2) column (5 pm, 210 x 21.4 mm) or a TSKgel Amide-80 (5 pm, 300 x 21.5 mm)
(Tosoh Bioscience LLC). Analytical HPLC was performed on an Agilent 1100
analytical
HPLC system equipped with ESA Corona CAD detector. LC-MS was performed using
Waters Q-Tof micro mass spectrometer coupled with Waters 2795 separation
module.
Plant material
The Luo Han Guo extract (commercial name Swingle, -60% mogrosides) was
purchased from Blue California Co., Ltd.
Instrumentation
Chromatographic conditions: The chromatography was performed on a Waters
Acquity
H UPLC. Separation was carried out at 25 C using a 1.0 x100 mm, Acquity UPLC
HSS
T3 column (Waters), with a particle size of 1.8 mm, equipped with a 0.2 mm
prefilter.
Solvent A was water and solvent B was acetonitrile, both solvents contained
0.1%
formic acid. Injection volume was set to 10 pl. The chromatography flow rate
was 200
I/min. Sample was eluted from the LC column using the following linear
gradient
(curve number 6): 0-40min: 90% A-30% A; 40-45 min: 30-10% A; 45-50 min: 10% A;
50-51 min 10%-90% A, 51-55 min 90% A for re-equilibration.
Mass spectrometry
The U-HPLC system was coupled to a hybrid quadrupole orthogonal time-of-flight
(TOF) mass spectrometer (SYNAPT G2 HDMS, Waters MS Technologies, Manchester,
UK). The mass spectrometer was operated in the positive electrospray
ionization mode
(ES1 ). The sample cone voltage 40, capillary voltage 0.7 kv, source
temperature 40 C,
desolvation temperature 450 C, desolvation gas flow 800 L/h, and cone gas
flow 50
Uh were optimized. Leucine enkephalin was used as the lock mass [M+H] at m/ z
556.2771. Sodium formate solution was used for external instrument
calibration.
Purification
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3 g Luo Han Guo extract was dissolved in 15 mL water and loaded onto a pre-
equilibrated 0-18 Snap cartridge (KP-018-HS, 120 g, 132 mL column volume). The
gradient system (A: water; B: methanol) used was: 30% 2 CV, 30%-80% 10 CV,
80%-100% 2 CV, 100% 2 CV. The flow rate was 30 mL/min. Fractions were
collected
for 27 mL per tube. Four loading of total 12 g Luo Han Guo was fractionated.
All the
fractions were analyzed by analytical HPLC to locate the fractions with the
target
mogrosides (isocratic mobile phase: 24% acetonitrile in water. Column: Luna
018 Slim
4.6x150mm). Fractions 36-38 with iso-mogroside VI and 11-epi-mogroside V were
combined to evaporate solvents. Further preparative HPLC purification of
fractions 36-
38 afforded iso-mogroside VI (1, 22 mg) and 11-epi-mogroside V (2, 17 mg) (24%
acetonitrile in water, 10 mUmin, retention time 13.1 min and 14.3 min,
respectively).
11-oxo-mogroside V (4) and neomogroside (3) were mainly in flash fractions 39-
40 with
11-oxo-mogroside V as the major component. On reverse phase 0-18 preparative
HPLC, neomogroside appeared as a tail shoulder of 11-oxo-mogroside (24%
acetonitrile in water, 10 mUmin, retention time 17.0 min and 18.0 min,
respectively).
Collection of the peak front gave 105 mg of the compound 11-oxo-mogroside V
(4).
Further purification of the shoulder neomogroside (3, 15 mg) was achieved by
preparative HPLC on TSKgel Amide-80 (65% acetonitrile in water, 20 mL/min, rt
15.5
min).
iso-mogroside VI (/).White amorphous powder; [a]20D -8.2 (c 0.12, Me0H); For
1H
NMR and 130 spectroscopic data, see Tables 1;-HRESIMS: m/z 1449.7075 [M+H] -
(calcd. for 0661-1113034, 1447.7113, .82.6 PPm).
epi-mogroside V (2).White amorphous powder; [a]20D +4.5 (c 0.13, Me0H); For 1H
NMR and 130 spectroscopic data, see Tables 1; HRESIMS: m/z 1287.6558 [M+H]-
(calcd. for 060E1103029, 1287.6585, ,82.1 PPm)=
Acid hydrolysis and determination of absolute configuration of sugars
Compounds 1(1.2 mg) or 2 (1.8 mg) were incubated in 1 mL 1 M HCI at 80 '0 for
3
hrs. After hydrolysis, the solution was extracted with Et0Ac (1 mL X3). The
remaining
aqueous solutions were evaporated by blowing nitrogen gas and freezing dried.
The
absolute configuration of the sugar in the residue was determined by GC-MS
analysis
of its 0-silylated derivative and comparing with the derivatives of D-glucose
and L-
glucose standards. Briefly, the sugar residues, D-glucose (2 mg) or L-glucose
(2 mg)
were dissolved in pyridine (0.5 mL). 0.1 M L-cysteine methyl ester
hydrochloride
(Aldrich, Milwaukee, WI) in pyridine (0.5 mL) was added into the solution. The
mixture
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was kept at 60 C for 2 h and dried by blowing nitr ogen gas. The residue was
added
with 1-trimethylsilylimidazole (Fluka, Buchs, Switzerland) (0.5 mL) and
incubated under
60 C for 1 h. The mixture was partitioned by addin g n-hexane and water (1.0
mL
each). The n-hexane extract was analyzed by GC-MS under the following
conditions:
5 capillary column HP-5M5 (30 m x 0.25 mm x 0.25 jim, Agilent); column
temperature,
180 to 230 C at a ramp of 5 C/min ; injection tem perature, 250 cC; carrier,
He gas;
split ratio, 20:1. The 0-silylated derivatives of D-glucose and L-glucose
showed
retention time at 16.02 and 16.39 min, respectively. By comparing the
retention time
and co-chromatography, the sugar residues after acid hydrolysis of 1 and 2
were
10 .. determined to be D-glucose.
Reduction of 11-oxo-mogroside V with NaBH4
25 mg of 11-oxo-mogroside V (4) was dissolved in 50% dioxane and added with 20
mg NaBH4 and heated at 50 C for 3 days. The reaction mixture was periodically
15 .. analyzed by HPLC to monitor the reaction progress. After the reaction,
the mixture was
acidified by acetate acid and concentrated to dryness by blowing nitrogen gas.
The
residue was re-dissolved in water and passed through a pre-equilibrated 0-18
SPE
column. The methanol eluents from SPE column were concentrated. The residue
was
then separated by semi-preparative HPLC. The two reduced products had same
20 retention time and molecular weight as the isolated mogroside V and 11-
epi-mogroside
V by LC-MS analysis and co-chromatography on analytical HPLC. The 1-D and 2-D
NMR data also confirmed that the structures of the two reduced products were
mogroside V and 11-epi-mogroside V.
Results and Discussion
Isolation and elucidation of iso-mogroside VI (1) and 11-epi-mogroside V (2)
During the course of investigating a commercial Luo Han Guo extracts with 60%
mogrosides by LC-MS, several mogrosides with six or five sugar moieties in the
extracts attracted our attention (Figure 2 and 3). Since there were little
report on the
sweet properties of mogroside V and VI isomers, we decided to purify and
identify
these isomers for our evaluation. The concentrations of 1, 2, 3 and 4 in the
extracts
were estimated to be 0.8%, 0.5%, 0.6%, 4.9%, respectively, according to the
universal
Corona detector. After fractionated on flash chromatography system and
followed by
preparative HPLC purification, the four targeted mogrosides 1-4 were purified
and
determined to be iso-mogroside VI (1), 11-epi-mogroside V (2), neomogroside
(3), 11-
oxo-mogroside V(4).
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The molecular formula of 1 was deduced as 066H112034 by its HR-ESI-MS spectral
data
([M-FI]- m/z, 1447.6957, calcd. for 0661-1111034, 1447.6957). The NMR spectral
data of 1
suggested the structure of a hexasaccharide triterpenoid saponin: 30 of the 66
carbons
were assigned to the triterpenoid aglycone, and 36 of 66 to six hexose
moieties. The
130 and 1H NMR spectra of 1 showed the signals of seven singlet tertiary
methyls, a
doublet secondary methyl, and an olefinic methine (Table 19), which suggested
a
typical (24R)-cucurbit-5-ene-313,11a,24,25-tetraol mogrol aglycone. The mogrol
aglycone of 1 was further confirmed by extensive analysis of its 1H, 130, and
2D
(COSY, TOCSY, HSQC and NOESY) NMR data, as well as comparison with NMR
data of mogroside V standard.
Table 19. 1H NMR and 130 NMR spectroscopic data for iso-mogroside VI and 11-
epi-
mogroside V (1H 300 MHz and 13075 MHz in pyridine-d5/D20 10:1)
iso-mogroside VI 11-epi-mogroside V
oc oc
1 1.93, 2.86 26.9 1.68, 1.93 24.8
2 2.20, 2.38 29.5 1.92, 2.42 29.6
3 3.65 (brs, 7.5) 87.6 3.68 (brs, 7.8) 87.5
4 - 42.4 - 42.1
5 - 144.5 - 143.2
6 5.45 (d, 4.8) 118.5 5.47 (d, 6.6) 119.4
7 1.65, 2.27 24.6 1.64, 2.22 26.1
8 1.59 43.6 1.99 40.9
9 - 40.2 - 40.3
10 2.80 (brd, 13) 36.7 2.09 40.1
11 4.15 77.8 4.05 72.5
12 2.12 40.8 1.99, 2.15 39.4
13 - 47.5 - 46.0
14 - 49.8 - 50.0
1.03, 1.10 34.7 1.16 35.5
16 1.40, 2.05 28.6 1.40, 2.10 28.6
17 1.72 51.0 1.65 51.7
18 0.86 s 17.1 1.29 s 18.3
19 1.28 s 26.3 1.25 s 23.2
1.46 36.5 1.56 36.8
21 1.04 (d, 6.2) 19.2 1.13 (d, 5.9) 19.3
22 1.67, 1.83 33.4 1.69, 1.95 33.6
23 1.53, 1.84 29.5 1.56, 1.85 29.5
24 3.71 (d, 8.2) 92.3 3.71 (d, 8.6) 92.6
- 72.9 - 73.0
26 1.29 s 26.9 1.30 s 27.2
27 1.41 s 24.6 1.44 s 24.9
28 1.14 s 27.8 1.03 s 28.4
29 1.45 s 26.2 1.46 s 26.5
0.88 s 19.5 0.81 s 18.4
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11 o-mogroside VI -epi-
mogroside
is v
Glc-I
1 4.73 (d, 7.9) 106.8 4.77 (d,
7.9) 106.6
2 3.86 75.1 3.89 75
3 4.11 77.8 4.18 77.6
4 3.99 71.4 3.93 71.1
4.01 77.2 4 76.9
6 4.26, 4.69 70.1 4.26, 4.73
69.7
Glc-II
1 5.10 (d, 7.8) 105.1 5.10 (d,
7.8) 104.8
2 3.99 75.0 3.99 74.7
3 4.22 77.8 4.15 77.7
4 3.96 71.5 3.9 71.4
5 4.11 78.0 4.16 78.0
6 4.26, 4.47 62.5 4.26, 4.47
62.3
Glc-II I
1 4.85 (d, 7.5) 103.6591
4.85 (d, 7.5) 103.5
2 4.12 81.6 4.22 80.9
3 4.21 78.3 4.23 78.3
4 3.93 71.4 3.99 70.9
5 4.01 76.4 4.02 76.1
6 3.92, 4.81 70.0 3.90, 4.80
69.7
Glc-IV
1 4.78 (d, 7.5) 104.6 4.78 (d,
7.5) 104.3
2 3.95 74.5 3.99 74.9
3 4.18 77.7 4.21 77.7
4 3.87 71.2 3.91 71
5 4.00 78.2 4.13 77.8
6 4.26, 4.47 62.4 4.26, 4.47
62.1
Glc-V
1 5.43 (d, 7.8) 104.7 5.50 (d,
7.8) 104.5
2 4.05 75.4 4 75.4
3 4.14 76.4 4.15 77.9
4 4.14 82.0 3.92 72.0
5 3.86 76.5 4.02 78.0
6 4.25, 4.42 62.6 4.25, 4.52
62.9
Glc-VI
1 5.03 (d, 7.7) 104.8
2 3.99 74.6
3 4.16 77.7
4 3.94 71.3
5 4.07 77.9
6 4.37, 4.44 62.3
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GC-MS analysis of water-soluble acid hydrolysis products of 1 showed that D-
glucose
was the only monosaccharide in the structure of 1. The HSQC spectra clearly
displayed the anomeric cross-peaks of six glucosyls: Glc-I ((5c 106.8 and (5H
4.73), Glc-
II (bc 105.1 and (5H 5.10), Glc-Ill (5c 103.7 and (5H 4.85), Glc-IV (5c 104.6
and 5H 4.78),
Glc-V (5c 104.7 and (5H 5.43), Glc-VI (5c 104.8 and 5H 5.03). The
stereochemistry of all
the six glucopyranosyls were determined to be 13 configuration from their
anomeric
proton coupling constants 34.411, H2. From HSQC-TOCSY experiment (hsqcgpmlph)
with
100 ms mixing time, the glucopranosyl carbon signals can be divided into six
groups
(Figure 4). The oligosaccharide elucidation was started from the
glucopyranosyl
connected at 0-3 of the cucurbitane aglycone. Glc-I was determined to link
with
aglycone 0-3 according to the HMBC correlation of its anomeric proton ((5H
4.73, d, J=
7.9 Hz) with aglycone 0-3 ((5c 87.6) and the NOESY correlation of Glc-I H-1
and
aglycone H-3. The 130 signals of Glc-I ((5c 75.1, 77.8, 71.4, 77.2, 70.1) as
determined
by HSQC-TOCSY missed a typical 0-6 carbon signal at around (5c 62. The
downfield
shift of Glc-I 0-6 ((5c 70.1) indicated glycosylation at this position. By
comparing HSQC-
TOCSY spectra (hsqcgpmlph) with increased mixing time from 10, 30, 60, and 100
ms,
the magnetization transfer relay can be observed gradually extending from 0-2
to 0-6
(Figure 4). As shown in Figure 4, HSQC-TOCSY under 10 ms mixing time displayed
the correlation of glucopyranosyl H-1 and 0-2. Under 30 ms mixing time, the
correlation
of H-1 and 0-3 appeared in addition to H-1 and 0-2 correlation. Under 60 ms,
the
carbon chain as indicated by the HSQC-TOCSY correlation extend to 0-4. The
full
HSQC-TOCSY correlation of H-1 with 0-2 to 0-6 can be observed under 100 ms.
Therefore, signals of 0-2 to 0-6 can be unambiguously assigned. The linkage of
Glc-II
to Glc-I was established by the HMBC correlation of anomeric Glc-II H-1 ((5H
5.10, d,
J=7.8 Hz) to Glc-I 0-6 ((5c 70.1). The 130 signals of Glc-II ((5c 75.0, 77.8,
71.5, 78.0,
62.5) suggested no glycosylation on Glc-II. As a result, the sugar chain on
aglycone 0-
3 was furnished as 3-0-(13-D-glucopyranosyl(1¨>6)-13-D-glucopyranosyl.
HMBC correlation of anomeric proton ((5H 4.85, d, J=7.5 Hz) to aglycone carbon
signal
((5c 92.3) indicated the connection of Glc-Ill H-1 to aglycone 0-24. The 130
pattern of
Glc-Ill ((5c 81.6, 78.3, 71.4, 76.4, 70.0) suggested 0-2 and 0-6 glycosylation
shifts.
Analysis of HSQC-TOCSY with 10, 30, 60, and 100 ms mixing time resulted in the
sequential assignment and confirmation of 0-2 and 0-6 downfield shifts. Glc-IV
was
determined to connect to 0-6 of Glc-Ill as from its H-1 ((5H 4.78, d, J=7.5
Hz) HMBC
correlation with 0-6 of Glc-Ill ((5c 70.0). Glc-IV was a regular terminal
glucopyranosyl
without any substitution ((5c 74.5, 77.7, 71.2, 78.2, 62.4). The linkage of
Glc-V to 0-2 of
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Glc-Ill was established by HMBC correlation of anomeric Glc-V H-1 (5H 5.43, d,
J=7.8
Hz) to Glc-Ill 0-2 (5c 81.6). The relatively down-field shift of Glc-V H-1 (5H
5.43) was
consistent with previous reports with similar structure. The 130 chemical
shift of 0-4
normally at (5c 70-71 was missing in the Glc-V signal set (5c 104.7, 75.4,
76.4, 82.0,
76.5, 62.6), which suggested glycosylation at 0-4. By observing the 0-2 to 0-6
relay
from HSQC-TOCSY with 10, 30, 60 and 100 ms mixing time, 5c 82.0 was clearly
assigned to 0-4 of Glc-V (Figure 4). HMBC cross-peak between 0-4 of Glc-V (5c
82.0)
and H-1 of Glc-VI (5H 5.03, d, J=7.7 Hz) further confirmed that Glc-VI linked
to Glc-V at
this position. Glc-VI was a terminal glucopyranosyl without further sugar
branch. Based
upon the above evidences, the structure of iso-mogroside (1) was assigned as 3-
0-13-
D-glucopyranosyl(1¨>6)-13-D-glucopyranosyl-mogrol-24-0-13-D-glucopyranosyl-
(1¨>6-
[13-D-glucopyranosyl-(1-4)-13-D-glucopyranosyl-(1¨>2)]-13-D-glucopyranosyl.
Compound 2 was assigned a molecular formula of 0601-62029 from its HR-ESI-MS
data
(EM-H] - m/z, 1285.6429). The NMR data of the oligosaccharide portion of 2
were
superimposable with those of mogroside V. Detailed 2-D NMR experiments
including
HSQC, HMBC, NOESY, COSY and HSQC-TOCSY confirmed that 2 had the same
sugar moieties as mogroside V. Attentions were then turned onto the aglycone
NMR
data. The HMBC correlation between C-11 and H3-19 revealed significant upfield
shift
.. of C-11 (5c 72.5) as compared with mogroside V (5c 77.8). Further
assignment of
aglycone data by 2-D NMR experiments showed that major 130 chemical shift
changes
occurred on 0-8, C-10 and C-12 when comparing with the data of mogroside V
(Table
19). This suggested 13-0H instead of a-OH at C-11. The 13-0H stereo structure
of 2 was
further established by NOE correlations between H-8 and H3-18, 19; H-10 and H3-
28,
H3-30; H-11 and H3-30; H-17 and H3-30. There were one natural 11-13-0H
cucurbitane
and one semi-synthetic 11-13-0H cucurbitane reported before [14, 15]. The 130
NMR
data of compound 2 aglycone had a good match with the data of the semi-
synthetic 11-
f3-0H cucurbitane glycoside, which was recorded in pyridine-d6[14]. The 130
NMR data
of natural ii -13-OH cucurbitane by Matsuda et al was obtained in methanol-d4
and were
quite different in terms of chemical shifts at C-11, 0-8, C-10 and C-12 [15].
To further
confirm the 11-13-0H structure of 2, semi-synthesis of 2 was carried out by
chemical
reduction of 11-oxo-mogroside V (4) to the 11-13-0H and 11-a-OH isomers of
mogroside V. By LC-MS, HPLC co-chromatography and NMR data analysis, the semi-
synthetic 11-epi-mogroside V was determined to be identical to the isolated 11-
epi-
mogroside V. Therefore, the structure of 11-epi-mogroside V (2) was elucidated
as 3-
0-13-D-glucopyranosyl(1 ¨>6)-13-D-glucopyranosy1-11 13-OH-mogrol-24-0-13-D-
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glucopyranosyl-(1¨>2)-[13-D-glucopyranosyl-(1¨>6)]-13-D-glucopyranosyl. To our
best of
knowledge, this is the first report of natural mogroside with a 11-13 hydroxyl
group.
HSQC-TOCSY with different mixing time for oligosaccharide chain elucidation.
5 Gheysen et al investigated TOCSY experiments with different mixing time
and
concluded 100 ms as the optimal spin lock time to discriminate D-glucose, D-
galactose
and D-mannose [16]. Through their results, we noticed that the spin lock time
could
significantly affect the magnetization transfer efficiency of H-1 of D-
glucose. The
correlation between H-1 and H-2 through H-6 gradually extended to H-6 as the
spin
10 lock time increased. Inspired from their investigation, we hypothesized
that by
increasing spin lock time of HSQC-TOCSY, we should be able to see that the
correlations of glucose H-1 with 0-2 to 0-6 gradually extend from 0-2 to 0-6
as the
chain of the magnetization transfer extends. HSQC-TOCSY with increasing spin
lock
time should tell the carbon sequence information, which would be very useful
for
15 oligosaccharide elucidation and assignment. Figure 4 showed HSQC-TOCSY
(hsqcgpmlph) of iso-mogroside VI with 10, 30, 60 and 100 ms mixing time. The
cross-
peaks in Figure 4 were quantified by their integrals and compared in Figure 5.
The
peak intensity (as presented by the integrals) could be an indication of their
distance
from H-1 in some cases. For example, all the 0-3 peaks were significantly
weaker than
20 0-2 peaks under 30 ms mixing time experiments. However, under 60 ms 0-3
peaks
become bigger than 0-2 peaks. To ensure correct elucidation, the carbon
sequence
should be determined through the overview of all the HSQC-TOCSY spectra with
different mixing time, not just by the peak intensity under one mixing time.
25 Traditionally, NMR elucidation and assignment of sugar chain of saponins
start from
the sugar linked to aglycone. By HMBC or NOESY, the well-resolved anomeric H-1
and
0-1 signals can be identified. Then through COSY correlations and matching 3J
(H,H)
coupling constants, the proton signals of the monosaccharide can be assigned.
Since a
large coupling constant (>7 Hz) typically indicate two neighboring axial C¨H
bonds and
30 small coupling constant (<4 Hz) for an axial¨equatorial or
equatorial¨equatorial C¨H
bond, the type of monosaccharide can be determined. NOE correlations are
useful for
confirmation of the stereochemistry of axial-axial, axial-equatorial or
equatorial-
equatorial relations. The carbon signals of the saccharide (0-2 to 0-6) are
assigned
according to HSQC or HMQC. The chemical shifts of the carbon signals as
determined
35 by HSQC/HMQC are very important information to confirm the
monosaccharide type
since the pattern of 0-1 to 0-6 chemical shifts for different type of
monosaccharides is
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characteristic and consistent. Through the observation of carbon chemical
shifts
changes, the glycosylation position on the sugar chain can be identified and
further
confirmed by HMBC correlation. In summary, traditional way to elucidate
saponin sugar
is: HMBC ¨> C-1, H-1 ¨> COSY ¨> H-2 to H-6 ¨> HSQC/HMQC ¨> 0-2 to 0-6, then
assisted and confirmed by coupling constant analysis and NOESY experiment.
1I-1-1H TOCSY (Total Correlated Spectroscopy also known as HOHAHA ¨
Homonuclear Hartmann Hahn) experiment could be a big help to divide the
complicated sugar proton signals into groups. The transfer of magnetization
during the
TOCSY spin lock from the anomeric H-1 to the end of the furanose or pyranose
ring
will depend on the magnitude of the intervening 3J (H,H) scalar coupling
constants.
Neighboring axial-axial protons with large coupling constant (>7 Hz) allow a
fast
transfer of magnetization, whereas axial-equatorial or equatorial-equatorial
with small
coupling constant (<4 Hz) will considerably reduce transfer efficiency.
Therefore,
TOCSY experiment not only can be used to group proton signals into spin
systems, but
also provide the stereochemistry information of the saccharide. For example,
we
should be able to see the magnetization relay of glucose through H-1 to H-6
with the
right mixing time. For galactose, there is no magnetization relay over H-4
even with 200
ms mixing time.
However, for the case of mogrosides with five or six sets of glucopyranosyl
signals,
using COSY and TOCSY to connect H-1 to H-6 can be quite tricky. The proton
signals
of the mogroside glucopyranosyls have very similar chemical shifts and appear
crowded in a small range from 5H 3.8 to 4.5. It is hard to make clear COSY
connections
through such poorly-resolved proton signals. The glucopyranosyl carbon signals
are
also very close and the HSQC cross-peaks heavily overlap to each other , which
make
the elucidation and assignment even more difficult.
Previously, HSQC-TOCSY have been applied in the structure elucidation and
assignment of saponins by grouping carbon signals in each spin system together
[17,
18]. Through our investigation, we demonstrated for the first time that the
signal
sequence within the glucopyranosyl carbon group can be identified by applying
different mixing time in HSQC-TOCSY experiments.
Figure 6 summarized the new HSQC-TOCSY based strategy to elucidate the
glucopyranosyl oligosaccharide chain of mogrosides as follows: In Step 1,
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Heteronuclear multiple bond correlation spectroscopy (HMBC) was used to
determine
anormeric C-1 and H-1 of the sugar. Start from the sugar link to aglycone. In
Step 2,
HSQC-TOCSY was used with 100ms mixing time to determine the whole group of 0-2
to 0-6. HSQC-COSY or HSQC-TOCSY (d9=10 ms) to assign 0-2. HSQC-TOCSY
(d9=30 ms) to assign 0-3. HSQC-TOCSY (d9=60 ms) to assign 0-4. HSQC-TOCSY
(d9=100 ms) to assign 0-5 and 0-6. In Step 3, if a 0-2 downshift from -675 to -
681,
0-4 downshift from -671 to -681 or 0-6 downshift from -662 to -69 is observed,
check
HMBC for glycosylation at these positions.** If a 0-2 downshift from -675 to -
681, C-
4 downshift from -671 to -681 or 0-6 downshift from -662 to -69, check HMBC
for
glycosylation at these positions.**. 1-D NMR data such as 1H coupling
constants and
130 carbon signal pattern and 2-D NMR experiments such as NOESY, HMBC, TOCSY,
COSY and HSQC could assist the process and confirm the results. The new HSQC-
TOCSY based strategy may provide a simple, fast and unambiguous way for
elucidation and assignment of glucopyranosyl chains of any new or known
mogrosides.
The strategy can also be adapted for elucidation and assignment of other
monosaccharides and oligosaccharides.
Structures of neomogroside and mogroside VI
Compound 3 was determined to be neomogroside by extensive 1-D and 2-D NMR
analysis, as well as comparison with literature data [19]. For the
oligosaccharide chain
elucidation of 3, signals were assigned by HSQC-TOCSY and TOCSY experiments
with different mixing time at 10, 30, 60, 100 ms. The linkage of the six
saccharides
were made by their NOESY and HMBC correlations. The oligosaccharide chain on 0-
3
of aglycone can be clearly assigned as 13-D-glucopyranosyl-(1¨>2)-13-D-
glucopyranosyl-
(1¨>6)-13-D-glucopyranosyl. The glucopyranosyl on 0-24 of aglycone was
branched
with a 13-D-glucopyranosyl-(1¨>2) and a 13-D-glucopyranosyl-(1¨>6).
Neomogroside was firstly discovered from S. grosvenorii and described by Si et
al.
[19]. Searching neomogroside in Scifinder returned the CAS number 189307-15-1.
However, the incorrect structure of neomogroside was given in Scifinder even
though
the literature referred by Scifinder was the 1996 article by Si et al. The
incorrect
structure of 189307-15-1 was given as 3-0-B-D-glucopyranosyl-(1¨>2) -[13-D-
glucopyranosyl-(1¨>6)]-13-D-glucopyranosyl-mogrol-24-043-D-glucopyranosyl-
(1¨>6)-
[13-D-glucopyranosyl-(1¨>2)]-13-D-glucopyranosyl (structure of 6 in Figure 2)
in Scifinder.
The report of neomogroside by Si et al was written in Chinese and published in
a
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Chinese journal in 1996. The accessibility and misunderstanding of this
article might
lead to the incorrect structure in Scifinder.
In Scifinder, neomogroside and mogroside VI had the same CAS number 189307-15-
1
.. and same structure. Takemoto et al firstly reported mogroside VI from S.
grosvenorii
[2]. But it only referred a pure mogroside with a molecular formula of
066H112034 and no
structure was determined [2]. Prakash et al. reported the structure and NMR
data of
mogroside VI as a known compound in their article published in 2011 [6]. In
their
article, the structure of mogroside VI was assigned as the structure of 6 in
Figure 2.
Prakash mentioned that the structure elucidation of mogroside VI was made by
NMR
analysis and also by comparing with the literature values. However, no
citation was
given for the literature values.
For known compounds, comparison of NMR data with literature data could be
useful for
structure determination. However, the complexity of mogroside NMR data makes
it
difficult to determine the structure mainly by comparison of NMR data with
literature
data. 1H NMR data of known mogrosides in different reports showed variations
due to
different NMR solvents used (the ratio of pyridine and D20 could cause signal
shifts) or
simply incorrect assignments.
Even though 130 NMR data are quite consistent and have better resolution than
1H
NMR data, structure determination of oligosaccharide chain of known mogrosides
cannot be relied on directly comparing 130 NMR data with literature data.
Considering
the case of neomogroside, if the Glu-VI glucopyranosyl-(1¨>2) branched on Glu-
I, Glu-
II, Glu-Ill, Glu-IV, or GluV, the five isomers may have very similar 130 NMR
data.
Rather than comparing 130 NMR data with literature data, extensive 2-D NMR
analysis
should be carefully done before the oligosaccharide chain of mogrosides are
unambiguously determined.
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Example 11
Sweet intensity of iso-mogroside VI and 11-epi-mogroside V
Methods
10 mg iso-mogroside VI (Figure 7) was dissolved in 31 mL water to make 100 ppm
iso-
mogroside VI solution. The working solution of 11-epi-mogroside V (Figure 8)
was 374
ppm (9.34 mg 11-epi-mogroside V in 25 mL water). A series of standard sucrose
solutions were prepared (0.50, 0.75, 1.00, 1.25, 1.50%) as sweetness
references.
Results
Four sweet sensitive panelists evaluated 100 ppm iso-mogroside VI and 374 ppm
11-
epi-mogroside V and the sucrose standards, and were asked to give sweet
equivalence concentrations to sucrose. The mean sweet equivalence
concentrations of
each compound were used to calculate the iso-sweet potency. The iso-sweet
potency
values of iso-mogroside VI and 11-epi-mogroside V were determined as 91 and 35
times of sweetness of sucrose, respectively (100 ppm iso-mogroside VI sweet
equivalent to 0.91% sucrose; 374 ppm 11-epi-mogroside V sweet equivalent to
1.31%
sucrose).
Example 12
Methods
lso-Mogroside VI and 11-epi-mogroside V were obtained as described in Example
10.
The iso-Mogroside VI and 11-epi-mogroside V were each added to a solution
containing 5 % sucrose and 0.03 % citric acid in a concentration of 25 ppm.
These test
solutions were tasted by an expert panel of seven people. For various aspects
of sweet
taste (overall sweet, upfront sweet, lingering sweet, astringent, volatile off-
note), each
panellist scored the test solutions in comparison to the base solution
(solution of 5 %
sucrose and 0.03 % citric acid). A score of 0 indicated that the taste aspect
was the
same, 1 indicates slightly higher, 2 indicates higher, 3 indicates much
higher, -1
indicates slightly lower, -2 indicates lower and -3 indicates much lower.
Results
The average score for each test solution for each taste aspect was calculated.
The
results are shown in Table 20 below.
Table 20
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lso-Mogroside Epi-Mogroside
VI V
Overall Sweet 0.2 0.17
Upfront Sweet 0.2 0.33
Lingering
0.2 0
Sweet
Astringent 0 0
Volatile Off-
0 0
Note
Higher upfront
sweetness
offsets slightly
Comments Slightly acidic lower linger to
give overall
higher
sweetness
Two new minor cucurbitane glycosides along with known 11-oxo-mogroside and
neomogroside were purified from the commercial extracts of Luo Han Guo
(Siraitia
grosvenorii (Swingle) C. Jeffrey ex Lu et Z. Y. Zhang). By extensive NMR and
LC-MS
analyses and chemical synthesis, the structures of the two new compounds iso-
5 mogroside VI (1) and 11-epi-mogroside V (2) were elucidated as 3-0-13-D-
glucopyranosyl(1¨>6)-13-D-glucopyranosyl-mogrol-24-0-(13-D-glucopyranosyl-
(1¨>6)-[13-
D-glucopyranosyl-(1-4)-13-D-glucopyranosyl-(1¨>2)]-13-D-glucopyranosyl and 3-0-
13-D-
glucopyranosyl(1¨>6)-13-D-glucopyranosy1-1113-0H-mogrol-24-0-13-D-
glucopyranosyl-
(1¨>6)-[13-D-glucopyranosyl-(1¨>2)]-13-D-glucopyranosyl, respectively. The
sweet
10 potency of iso-mogroside VI and 11-epi-mogroside V were evaluated as 91
and 35
times of sweetness of sucrose, respectively (100 ppm iso-mogroside VI sweet
equivalent to 0.91% sucrose; 374 ppm 11-epi-mogroside V sweet equivalent to
1.31%
sucrose). Through our course of identifying the new and known mogrosides with
five or
six glucopyranosyls, a new strategy for glucopyranosyl sugar chain elucidation
and
15 assignment was developed. The new strategy based on HSQC-TOCSY experiments
with different mixing times featured a quick and unambiguous elucidation and
assignment of glucopyranosyl oligosaccharide chains. The previous confusion
regarding the structures of neomogroside and mogroside VI was reviewed and
clarified
after the confirmation of neomogroside structure by our extensive NMR spectral
20 analysis.
The foregoing broadly describes certain embodiments of the present invention
without limitation. Variations and modifications as will be readily apparent
to those
skilled in the art are intended to be within the scope of the present
invention as defined
in and by the appended claims.
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REFERENCES
1. Chun, L.; Li-Mei, L.; Feng, S.; Zhi-Min, W.; Hai-Ru, H.; Li, D.
JIANG, T.-L.
Chemistry and pharmacology of Siraitia grosvenorii: A review. Chinese journal
of natural medicines, 2014, 12,89-102.
2. Takemoto, T.; Arihara, S.; Nakajima, T. Okuhira, M. Studies on the
constituents
of Fructus Momordicae. I. On the sweet principle. Yakugaku Zasshi, 1983, 103,
1151-4.
3. Takemoto, T.; Arihara, S.; Nakajima, T. Okuhira, M. Studies on the
constituents
of Fructus Momordicae. III. Structure of mogrosides. Yakugaku zasshi: Journal
of the Pharmaceutical Society of Japan, 1983, 103, 1167-1173.
4. Takemoto, T.; Arihara, S.; Nakajima, T. Okuhira, M. Studies on the
constituents
of Fructus momordicae. II. Structure of sapogenin. Yakugaku zasshi: Journal of
the Pharmaceutical Society of Japan, 1983, 103, 1155.
5. Chaturvedula, V. S. P. Meneni, S. R. A New Cucurbitane Glycoside from
Siraitia grosvenorii. Nat Prod Commun, 2015, 10, 1521-3.
6. Chaturvedula, V. S. P. Prakash, I. Cucurbitane Glycosides from Siraitia
grosvenorii. J. Carbohydr. Chem., 2011, 30, 16-26.
7. Patron, A.; Manam, R.; Colquitt, J.; Servant, N.; Noriega, C. E.;
Hammaker, J.
R. Gonsalves, N. High intensity sweeteners. Patent W02017075257A2, May 4,
2017.
8. Prakash, I.; Markosyan, A. Bunders, C. Development of next generation
stevia
sweetener: rebaudioside M. Foods, 2014, 3, 162-175.
9. Ohta, M.; Sasa, S.; Inoue, A.; Tamai, T.; Fujita, I.; Morita, K.
Matsuura, F.
Characterization of novel steviol glycosides from leaves of Stevia rebaudiana
Morita. Journal of Applied Glycoscience (Japan), 2010.
10. U.S. Food and Drug Administration.
https://wvvw.accessdata.fda.qoviscribts/fdcc/?set=GRASNotices&id=51 2.
(accessed May 25, 2017).
11. U.S. Food and
Drug Administration.
https://www.accessdataida.aov/scribts/fdcc/?set=brasnotices&id=667.
(accessed May 25, 2017).
12. U.S. Food and Drug Administration.
https://www.accessdataida.qoviscribts/fdcc/?set=GRASNotices&id=473.
(accessed May 25, 2017).
13. Jia, Z. Yang, X., Novel Sweetener ISO-Mogroside V. 2008, Europe Patent
EP2188300 B1.
14. Kasai, R.; Matsumoto, K.; Nie, R.-L.; Morita, T.; Awazu, A.; Zhou,
J. Tanaka, 0.
Sweet and bitter cucurbitane glycosides from Hemsleya carnosiflora.
Phytochemistry, 1987, 26, 1371-1376.
15. Matsuda, H.; Nakashima, S.; Abdel-Halim, 0. B.; Morikawa, T. Yoshikawa,
M.
Cucurbitane-type triterpenes with anti-proliferative effects on U937 cells
from an
egyptian natural medicine, Bryonia cretica: structures of new triterpene
glycosides, bryoniaosides A and B. Chemical and pharmaceutical bulletin,
2010, 58, 747-751.
16. Gheysen, K.; Mihai, C.; Conrath, K. Martins, J. C. Rapid identification
of
common hexapyranose monosaccharide units by a simple TOCSY matching
approach. Chemistry¨A European Journal, 2008, 14, 8869-8878.
17. Tabatadze, N.; Elias, R.; Faure, R.; Gerkens, P.; De Pauw-Gillet, M.
C.;
Kemertelidze, E.; Chea, A. 011ivier, E. Cytotoxic triterpenoid saponins from
the
roots of Cephalaria gigantea. Chemical and pharmaceutical bulletin, 2007, 55,
102-105.
18. Mshvildadze, V.; Elias, R.; Faure, R.; Rondeau, D.; Debrauwer, L.;
Dekanosidze, G.; Kemertelidze, E. Balansard, G. Triterpenoid saponins from
CA 03064334 2019-11-20
WO 2018/220103 PCT/EP2018/064324
72
leaves of Hedera pastuchowii. Chemical and pharmaceutical bulletin, 2004, 52,
1411-1415.
19. Si, J.; Chen, D.; Chang, Q. Shen, L. Isolation and determination of
cucurbitane-
glycosides from fresh fruits of Siraitia grosvenorii. Zhiwu Xuebao, 1996, 38,
489-494.
