Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HIGH-PURITY STE VIOL GLYCOSIDES
TECHNICAL FIELD
The present invention relates to compositions comprising steviol glycosides,
including highly purified steviol glycoside compositions, and processes for
making the
same.
BACKGROUND OF THE INVENTION
High intensity sweeteners possess a sweetness level that is many times greater
than
the sweetness level of sucrose. They are essentially non-caloric and are
commonly used in
diet and reduced-calorie products, including foods and beverages. High
intensity
sweeteners do not elicit a glycemic response, making them suitable for use in
products
targeted to diabetics and others interested in controlling for their intake of
carbohydrates.
Steviol glycosides are a class of compounds found in the leaves of Stevia
rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family
native to
certain regions of South America. They are characterized structurally by a
single base,
steviol, differing by the presence of carbohydrate residues at positions C13
and C19. They
accumulate in Stevia leaves, composing approximately 10% - 20% of the total
dry weight.
On a dry weight basis, the four major glycosides found in the leaves of Stevia
typically
include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%)
and
dulcoside A (0.3%). Other known steviol glycosides include rebaudioside B, C,
D, E, F
and M, steviolbioside and rubusoside.
Although methods are known for preparing steviol glycosides from Stevia
rebaudiana, many of these methods are unsuitable for use commercially.
Accordingly, there remains a need for simple, efficient, and economical
methods
for preparing compositions comprising steviol glycosides, including highly
purified steviol
glycoside compositions.
SUMMARY OF THE INVENTION
The present invention provides a process for preparing a composition
comprising a
target steviol glycoside by contacting a starting composition comprising an
organic
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substrate with a microbial cell and/or enzyme preparation, thereby producing a
composition comprising a target steviol glycoside.
The starting composition can be any organic compound comprising at least one
carbon atom. In one embodiment, the starting composition is selected from the
group
.. consisting of steviol glycosides, polyols or sugar alcohols, various
carbohydrates.
The target steviol glycoside can be any steviol glycoside. In one embodiment,
the
target steviol glycoside is steviolmonoside, steviolmonoside A,
steviolbioside,
steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A
(rebaudioside KA),
stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3,
rebaudioside
AM or a synthetic steviol glycoside.
In one embodiment, the target steviol glycoside is rebaudioside AM.
In some preferred embodiments enzyme preparation comprising one or more
enzymes, or a microbial cell comprising one or more enzymes, capable of
converting the
starting composition to target steviol glycosides are used. The enzyme can be
located on
the surface and/or inside the cell. The enzyme preparation can be provided in
the form of a
whole cell suspension, a crude lysate or as purified enzyme(s). The enzyme
preparation
can be in free form or immobilized to a solid support made from inorganic or
organic
materials.
In some embodiments, a microbial cell comprises the necessary enzymes and
genes encoding thereof for converting the starting composition to target
steviol glycosides.
Accordingly, the present invention also provides a process for preparing a
composition
comprising a target steviol glycoside by contacting a starting composition
comprising an
organic substrate with a microbial cell comprising at least one enzyme capable
of
converting the starting composition to target steviol glycosides, thereby
producing a
medium comprising at least one target steviol glycoside.
The enzymes necessary for converting the starting composition to target
steviol
glycosides include the steviol biosynthesis enzymes, UDP-glucosyltransferases
(UGTs)
and/or UDP-recycling enzyme.
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In one embodiment, the steviol biosynthesis enzymes include mevalonate (MVA)
pathway enzymes.
In another embodiment, the steviol biosynthesis enzymes include non-mevalonate
2-C-methyl-D-erythrito1-4-phosphate pathway (MEP/DOXP) enzymes.
In one embodiment the steviol biosynthesis enzymes are selected from the group
including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase,
kaurene
synthase, kaurene oxidase, kaurenoic acid 13¨hydroxylase (KAH), steviol
synthetase,
deoxyxylulose 5 -phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate
reductoisomerase (DXR), 4-diphosphocytidy1-2-C-methyl-D-erythritol synthase
(CMS), 4-
diphosphocytidy1-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidy1-2-C-
methyl-D-erythritol 2,4- cyclodiphosphate synthase (MCS), 1-hydroxy-2-methy1-
2(E)-
butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-
diphosphate
reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase,
mevalonate
kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase,
cytochrome
P450 reductase etc.
The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of
adding at least one glucose unit to steviol and/or a steviol glycoside
substrate to provide
the target steviol glycoside.
As used hereinafter, the term "SuSy_AT", unless specified otherwise, refers to
.. sucrose synthase having amino-acid sequence "SEQ ID 1" as described in
Example 1.
As used hereinafter, the term "UGTS12", unless specified otherwise, refers to
UDP-glucosyltransferase having amino-acid sequence "SEQ ID 2" as described in
Example 1.
As used hereinafter, the term "UGT76G1", unless specified otherwise, refers to
UDP-glucosyltransferase having amino-acid sequence "SEQ ID 3" as described in
Example 1.
In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases
are produced in a microbial cell. The microbial cell may be, for example, E.
coli,
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Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.
etc. In another
embodiment, the UDP-glucosyltransferases are synthesized.
In one embodiment, the UDP-glucosyltransferase is selected from group
including
UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTS12, EUGT11 and UGTs having
substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%,
>95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these
polypeptides as
well as isolated nucleic acid molecules that code for these UGTs.
In one embodiment, steviol biosynthesis enzymes, UGTs and UDP-glucose
recycling system are present in one microorganism (microbial cell). The
microorganism
may be for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp.,
Bacillus sp.,
Yarrowia sp.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase
capable of adding at least one glucose unit to steviol or any starting steviol
glycoside
bearing an -OH functional group at C13 to give a target steviol glycoside
having an -0-
glucose beta glucopyranoside glycosidic linkage at C13. In a particular
embodiment, the
UDP-glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence
identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to steviol or
any starting
steviol glycoside bearing a -COOH functional group at C19 to give a target
steviol
glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at
C19. In a
particular embodiment, the UDP-glucosyltransferase is UGT74G1, or a UGT having
>85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to the
existing glucose at
C19 of any starting steviol glycoside to give a target steviol glycoside with
at least one
additional glucose bearing at least one beta 1-->2 glucopyranoside glycosidic
linkage(s) at
the newly formed glycosidic bond(s). In a
particular embodiment, the UDP-
glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence
identity with
UGTS12. In another particular embodiment, the UDP-glucosyltransferase is
EUGT11, or
a UGT having >85% amino-acid sequence identity with EUGT11. In yet another
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particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having
>85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to the
existing glucose at
C19 of any starting steviol glycoside to give a target steviol glycoside with
at least one
additional glucose bearing at least one beta 1¨>3 glucopyranoside glycosidic
linkage(s) at
the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-
glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence
identity
with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to the
existing glucose at
C13 of any starting steviol glycoside to give a target steviol glycoside with
at least one
additional glucose bearing at least one beta 1¨>2 glucopyranoside glycosidic
linkage(s) at
the newly formed glycosidic bond(s). In a particular embodiment, the UDP-
glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence
identity with
UGTS12. In another particular embodiment, the UDP-glucosyltransferase is
EUGT11, or a
UGT having >85% amino-acid sequence identity with EUGT11. In yet another
particular
embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-
acid sequence identity with UGT91D2.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase
capable of adding at least one glucose unit to steviol to form
steviolmonoside. In a
particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having
>85%
amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid
sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to steviol to
form
steviolmonoside A. In a particular embodiment, the UDP-glucosyltransferase is
UGT74G1
or a UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside A to
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form steviolbioside B. In a particular embodiment, the UDP-glucosyltransferase
is
UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside A to
form steviolbioside A. In a particular embodiment, the UDP-glucosyltransferase
is
UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In
another
particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having
>85%
amino-acid sequence identity with EUGT11. In yet another particular
embodiment, the
UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside A to
form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or
a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside to form
rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1
or a
UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside to form
steviolbioside.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside B to form
stevioside B. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or a
UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside B to form
stevioside C. In a particular embodiment, the UDP-glucosyltransferase is
UGTS12 or a
UGT having >85% amino-acid sequence identity with UGTS12. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
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acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside A to form
stevioside A. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or a
UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside A to form
stevioside C. In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1 or a
UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to rubusoside
to form
stevioside B. In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1 or a
UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to rubusoside
to form
stevioside A (rebaudioside KA). In a particular embodiment, the UDP-
glucosyltransferase
is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In
another
particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having
>85%
amino-acid sequence identity with EUGT11. In yet another particular
embodiment, the
UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to rubusoside
to form
stevioside.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside to form
stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1
or a
UGT having >85% amino-acid sequence identity with UGT74G1.
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In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
B to form
rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is
UGTS12 or a
UGT having >85% amino-acid sequence identity with UGTS12. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
B to form
rebaudioside E2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
A
(rebaudioside KA) to form rebaudioside E3. In a particular embodiment, the UDP-
glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence
identity
with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
A
(rebaudioside KA) to form rebaudioside E.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
C to form
rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or
a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
to form
rebaudioside E2. In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1 or
a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
to form
rebaudioside E. In a particular embodiment, the UDP-glucosyltransferase is
UGTSI2 or a
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UGT having >85% amino-acid sequence identity with UGTS12. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
rebaudioside E3 to form
rebaudioside AM
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
rebaudioside E2 to form
rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is
UGTS12 or
a UGT having >85% amino-acid sequence identity with UGTS12. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
rebaudioside E to form
rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1
or a UGT having >85% amino-acid sequence identity with UGT76G1.
Optionally, the method of the present invention further comprises using more
than
one UGT on a starting composition, to give a target steviol glycoside(s)
having more than
one glucose unit than the starting composition. In a particular embodiment,
the UDP-
glucosyltransferases are UGT74G1, UGT85C2, UGT76G1, UGTS12, EUGT11 and/or
UGT91D2 or any UGT having >85% amino-acid sequence identity with UGT74G1,
UGT85C2, UGT76G1, UGTS12, EUGT1 I and/or UGT91D2 or any combination thereof,
capable of adding more than one glucose unit to a starting composition to give
a steviol
glycoside(s) having more than one glucose unit than the starting composition.
In one embodiment, the UDP-glucosyltransferases are any UDP-
glucosyltransferases capable of adding overall two glucose unit to stevioside
to form
rebaudioside AM In a particular embodiment, the UDP-glucosyltransferases are
selected
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from UGTS12, EUGT11, UGT91D2, UGT76G1 or any UGT having >85% amino-acid
sequence identity with UGTS12, EUGT11, UGT91D2, UGT76G1 or any combination
thereof. In another particular embodiment, the UDP-glucosyltransferases are
UGTS12 and
UGT76G1.
Optionally, the method of the present invention further comprises recycling
UDP
to provide UDP-glucose. In one embodiment, the method comprises recycling UDP
by
providing a recycling catalyst and a recycling substrate, such that the
biotransformation of
steviol and/or the steviol glycoside substrate to the target steviol glycoside
is carried out
using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.
In one embodiment, the recycling catalyst is sucrose synthase SuSy At or a
sucrose synthase having >85% amino-acid sequence identity with SuSy_At.
In one embodiment, the recycling substrate is sucrose.
Optionally, the method of the present invention further comprises the use of
transglycosidases that use oligo- or poly-saccharides as the sugar donor to
modify
recipient target steviol glycoside molecules. Non-limiting examples include
cyclodextrin
glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase,
glucosucrase,
beta-h-fructosidase, beta-fructosidase, sucrase, fructosylinvertase, alkaline
invertase, acid
invertase, fructofuranosidase. In some embodiments, glucose and sugar(s) other
than
glucose, including but not limited to fructose, xylose, rhamnose, arabinose,
deoxyglucose,
galactose are transferred to the recipient target steviol glycosides. In one
embodiment, the
recipient steviol glycoside is rebaudioside AM.
Optionally, the method of the present invention further comprises separating
the
target steviol glycoside from the medium to provide a highly purified target
steviol
glycoside composition. The target steviol glycoside can be separated by at
least one
suitable method, such as, for example, crystallization, separation by
membranes,
centrifugation, extraction, chromatographic separation or a combination of
such methods.
In one embodiment, the target steviol glycoside can be produced within the
microorganism. In another embodiment, the target steviol glycoside can be
secreted out in
the medium. In one another embodiment, the released steviol glycoside can be
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continuously removed from the medium. In yet another embodiment, the target
steviol
glycoside is separated after the completion of the conversion reaction.
In one embodiment, separation produces a composition comprising greater than
about 80% by weight of the target steviol glycoside on an anhydrous basis,
i.e., a highly
purified steviol glycoside composition. In another embodiment, separation
produces a
composition comprising greater than about 90% by weight of the target steviol
glycoside.
In particular embodiments, the composition comprises greater than about 95% by
weight
of the target steviol glycoside. In other embodiments, the composition
comprises greater
than about 99% by weight of the target steviol glycoside.
The target steviol glycoside can be in any polymorphic or amorphous form,
including hydrates, solvates, anhydrous or combinations thereof.
Purified target steviol glycosides can be used in consumable products as a
sweetener, flavor modifier, flavor with modifying properties and/or foaming
suppressor.
Suitable consumable products include, but are not limited to, food, beverages,
pharmaceutical compositions, tobacco products, nutraceutical compositions,
oral hygiene
compositions, and cosmetic compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the chemical structure of rebaudioside AM.
FIG. 2 shows the pathways of producing rebaudioside AM and various steviol
glycosides from steviol.
FIG. 3 shows the biocatalytic production of rebaudioside AM from stevioside
using
the enzymes UGT512 and UGT76G1 and concomitant recycling of UDP to UDP-
glucose via sucrose synthase SuSy_At.
FIG. 4 shows the biocatalytic production of rebausioside AM from rebaudioside
E
using the enzyme UGT76G1 and concomitant recycling of UDP to UDP-glucose
via sucrose synthase SuSy_At.
FIG. 5 shows the HPLC chromatogram of stevioside. The peak with retention time
of 25.992 minutes corresponds to stevioside.
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FIG. 6 shows the HPLC chromatogram of the product of the biocatalytic
production of rebaudioside AM from stevioside. The peak with retention time of
10.636 minutes corresponds to rebaudioside AM.
FIG. 7 shows the HPLC chromatogram of rebaudioside E. The peak with retention
time of 10.835 minutes corresponds to rebaudioside E.
FIG. 8 shows the HPLC chromatogram of the product of the biocatalytic
production of rebaudioside AM from rebaudioside E. The peaks with retention
time
of 10.936 and 11.442 minutes correspond to rebaudioside E and rebaudioside AM
respectively.
FIG. 9 shows the HPLC chromatogram of rebaudioside AM after purification by
methanol crystallization. The peak with retention time of 10.336 minutes
corresponds to rebaudioside AM.
FIG. 10 shows the IHNMR spectrum of rebaudioside AM (500 MHz, pyridine-d5).
FIG. 11 shows the HSQC spectrum of rebaudioside AM (500 MHz, pyridine-d5).
FIG. 12 shows the H,H COSY spectrum of rebaudioside AM (500 MHz, pyridine-
d5).
FIG. 13 shows the 1-1MBC spectrum of rebaudioside AM (500 MHz, pyridine-d5).
FIG. 14 shows the HSQC-TOCSY spectrum of rebaudioside AM (500 MHz,
pyridine-d5).
FIG. 15a and FIG. 15b show the LC chromatogram and mass spectrum of
rebaudioside AM respectively.
FIG. 16 is a graph showing the effect of Reb AM on the flavor modification of
coconut water.
FIG. 17 is a graph showing the effect of Reb AM on the flavor modification of
a
chocolate protein shake.
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DETAILED DESCRIPTION
The present invention provides a process for preparing a composition
comprising a
target steviol glycoside by contacting a starting composition comprising an
organic
substrate with a microbial cell and/or enzyme preparation, thereby producing a
composition comprising a target steviol glycoside.
One object of the invention is to provide an efficient biocatalytic method for
preparing target steviol glycosides, particularly steviolmonoside,
steviolmonoside A,
steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3, rebaudioside AM or a synthetic steviol glycoside from various
starting
compositions.
As used herein, the abbreviation term "reb" refers to "rebaudioside". Both
terms
have the same meaning and may be used interchangeably.
As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural
or
genetically engineered biocatalysts, such as enzymes, or cells including
microorganisms,
comprising one or more enzyme, capable of single or multiple step chemical
transformations on organic compounds. Biocatalysis processes include
fermentation,
biosynthesis, bioconversion and biotransformation processes. Both isolated
enzyme, and
whole-cell biocatalysis methods are known in the art. Biocatalyst protein
enzymes can be
naturally occurring or recombinant proteins.
As used herein, the term "steviol glycoside(s)" refers to a glycoside of
steviol,
including, but not limited to, naturally occurring steviol glycosides, e.g.
steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3, rebaudioside AM, synthetic steviol
glycosides, e.g.
enzymatically glucosylated steviol glycosides and combinations thereof.
Starting Composition
As used herein, "starting composition" refers to any composition (generally an
aqueous solution) containing one or more organic compound comprising at least
one
carbon atom.
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In one embodiment, the starting composition is selected from the group
consisting
of steviol, steviol glycosides, polyols and various carbohydrates.
The starting composition steviol glycoside is selected from the group
consisting of
steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A,
steviolbioside B,
rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B,
stevioside C,
rebaudioside E, rebaudioside E2, rebaudioside E3 or other glycoside of steviol
occurring
in Stevia rebaudiana plant, synthetic steviol glycosides, e.g. enzymatically
glucosylated
steviol glycosides and combinations thereof
In one embodiment, the starting composition is steviol.
In another embodiment, the starting composition steviol glycoside is
steviolmonoside.
In yet another embodiment, the starting composition steviol glycoside is
steviolmonoside A.
In still another embodiment, the starting composition steviol glycoside is
rubusoside.
In yet another embodiment, the starting composition steviol glycoside is
steviolbioside.
In yet another embodiment, the starting composition steviol glycoside is
steviolbioside A.
In yet another embodiment, the starting composition steviol glycoside is
steviolbioside B.
In still another embodiment, the starting composition steviol glycoside is
stevioside.
In yet another embodiment, the starting composition steviol glycoside is
stevioside
.. A, also known as rebaudioside K4.
In still another embodiment, the starting composition steviol glycoside is
stevioside
B.
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In still another embodiment, the starting composition steviol glycoside is
stevioside
C.
In another embodiment, the starting composition steviol glycoside is
rebaudioside
E.
In another embodiment, the starting composition steviol glycoside is
rebaudioside
E2.
In another embodiment, the starting composition steviol glycoside is
rebaudioside
E3.
The term "polyol" refers to a molecule that contains more than one hydroxyl
group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4
hydroxyl
groups, respectively. A polyol also may contain more than four hydroxyl
groups, such as a
pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl
groups,
respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric
alcohol, or
polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl
group
(aldehyde or ketone, reducing sugar) has been reduced to a primary or
secondary hydroxyl
group. Examples of polyols include, but are not limited to, erythritol,
maltitol, mannitol,
sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol,
threitol, galactitol,
hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-
oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup,
reduced
glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar
alcohols or any
other carbohydrates capable of being reduced.
The term "carbohydrate" refers to aldehyde or ketone compounds substituted
with
multiple hydroxyl groups, of the general formula (CH20)n, wherein n is 3-30,
as well as
their oligomers and polymers. The carbohydrates of the present invention can,
in addition,
be substituted or deoxygenated at one or more positions. Carbohydrates, as
used herein,
encompass unmodified carbohydrates, carbohydrate derivatives, substituted
carbohydrates,
and modified carbohydrates. As used herein, the phrases "carbohydrate
derivatives",
"substituted carbohydrate", and "modified carbohydrates" are synonymous.
Modified
carbohydrate means any carbohydrate wherein at least one atom has been added,
removed,
.. or substituted, or combinations thereof. Thus, carbohydrate derivatives or
substituted
carbohydrates include substituted and unsubstituted monosaccharides,
disaccharides,
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oligosaccharides, and polysaccharides. The carbohydrate derivatives or
substituted
carbohydrates optionally can be deoxygenated at any corresponding C-position,
and/or
substituted with one or more moieties such as hydrogen, halogen, haloalkyl,
carboxyl,
acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino,
arylamino,
alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl,
sulfinyl,
sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl,
phosphino,
thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or
any other
viable functional group provided the carbohydrate derivative or substituted
carbohydrate
functions to improve the sweet taste of the sweetener composition.
Examples of carbohydrates which may be used in accordance with this invention
include, but are not limited to, tagatose, trehalose, galactose, rhamnose,
various
cyclodextrins, cyclic oligosaccharides, various types of maltodextrins,
dextran, sucrose,
glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose,
altrose, mannose,
idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose,
isomaltulose, erythrose,
deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose,
cellobiose,
amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid,
glucono-
lactone, abequose, galactosamine, beet oligosaccharides, isomalto-
oligosaccharides
(isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides
(xylotriose,
xylobiose and the like), xylo-terminated oligosaccharides, gentio-
oligosaccharides
(gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero-
oligosaccharides,
palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the
like),
maltotetraol, maltotriol, malto-oligosaccharides
(maltotriose, maltotetraose,
maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin,
inulo-
oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid
sugars such as
high fructose corn syrups, coupling sugars, and soybean oligosaccharides.
Additionally,
the carbohydrates as used herein may be in either the D- or L-configuration.
The starting composition may be synthetic or purified (partially or entirely),
commercially available or prepared.
In one embodiment, the starting composition is glycerol.
In another embodiment, the starting composition is glucose.
In still another embodiment, the starting composition is sucrose.
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In yet another embodiment, the starting composition is starch.
In another embodiment, the starting composition is maltodextrin.
In yet another embodiment, the starting composition is cellulose.
In still another embodiment, the starting composition is amylose.
The organic compound(s) of starting composition serve as a substrate(s) for
the
production of the target steviol glycoside(s), as described herein.
Target Steviol Glycoside
The target steviol glycoside of the present method can be any steviol
glycoside that
can be prepared by the process disclosed herein. In one embodiment, the target
steviol
glycoside is selected from the group consisting of steviolmonoside,
steviolmonoside A,
steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3, rebaudioside AM or other glycoside of steviol occurring in
Stevia
rebaudiana plant, synthetic steviol glycosides, e.g. enzymatically
glucosylated steviol
glycosides and combinations thereof
In one embodiment, the target steviol glycoside is steviolmonoside.
In another embodiment, the target steviol glycoside is steviolmonoside A.
In another embodiment, the target steviol glycoside is steviolbioside.
In another embodiment, the target steviol glycoside is steviolbioside A.
In another embodiment, the target steviol glycoside is steviolbioside B.
In another embodiment, the target steviol glycoside is rubusoside.
In another embodiment, the target steviol glycoside is stevioside.
In another embodiment, the target steviol glycoside is stevioside A
(rebaudioside
KA).
In another embodiment, the target steviol glycoside is stevioside B.
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In another embodiment, the target steviol glycoside is stevioside C.
In another embodiment, the target steviol glycoside is rebaudioside E.
In another embodiment, the target steviol glycoside is rebaudioside E2.
In another embodiment, the target steviol glycoside is rebaudioside E3.
In another embodiment, the target steviol glycoside is rebaudioside AM
The target steviol glycoside can be in any polymorphic or amorphous form,
including hydrates, solvates, anhydrous or combinations thereof.
In one embodiment, the present invention is a biocatalytic process for the
production of steviolmonoside.
In one embodiment, the present invention is a biocatalytic process for the
production of steviolmonoside A.
In one embodiment, the present invention is a biocatalytic process for the
production of steviolbioside.
In one embodiment, the present invention is a biocatalytic process for the
production of steviolbioside A.
In one embodiment, the present invention is a biocatalytic process for the
production of steviolbioside B.
In one embodiment, the present invention is a biocatalytic process for the
production of rubusoside.
In one embodiment, the present invention is a biocatalytic process for the
production of stevioside.
In one embodiment, the present invention is a biocatalytic process for the
production of stevioside A (rebaudioside KA).
In one embodiment, the present invention is a biocatalytic process for the
.. production of stevioside B.
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In one embodiment, the present invention is a biocatalytic process for the
production of stevioside C.
In one embodiment, the present invention is a biocatalytic process for the
production of rebaudioside E.
In one embodiment, the present invention is a biocatalytic process for the
production of rebaudioside E2.
In one embodiment, the present invention is a biocatalytic process for the
production of rebaudioside E3.
In one embodiment, the present invention is a biocatalytic process for the
production of rebaudioside AM
In a particular embodiment, the present invention provides for the
biocatalytic
process for the production of rebaudioside AM from a starting composition
comprising
stevioside and UDP-glucose.
In another particular embodiment, the present invention provides for the
biocatalytic process for the production of rebaudioside AM from a starting
composition
comprising rebaudioside E and UDP-glucose.
Optionally, the method of the present invention further comprises separating
the
target steviol glycoside from the medium to provide a highly purified target
steviol
glycoside composition. The target steviol glycoside can be separated by any
suitable
method, such as, for example, crystallization, separation by membranes,
centrifugation,
extraction, chromatographic separation or a combination of such methods.
In particular embodiments, the process described herein results in a highly
purified
target steviol glycoside composition. The term "highly purified", as used
herein, refers to a
composition having greater than about 80% by weight of the target steviol
glycoside on an
anhydrous (dried) basis. In one embodiment, the highly purified target steviol
glycoside
composition contains greater than about 90% by weight of the target steviol
glycoside on
an anhydrous (dried) basis, such as, for example, greater than about 91%,
greater than
about 92%, greater than about 93%, greater than about 94%, greater than about
95%,
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greater than about 96%, greater than about 97%, greater than about 98% or
greater than
about 99% target steviol glycoside content on a dried basis.
In one embodiment, when the target steviol glycoside is reb AM, the process
described herein provides a composition having greater than about 90% reb AM
content by
weight on a dried basis. In another particular embodiment, when the target
steviol
glycoside is reb AM, the process described herein provides a composition
comprising
greater than about 95% reb AM content by weight on a dried basis.
Microorganisms and enzyme preparations
In one embodiment of present invention, a microorganism (microbial cell)
and/or
enzyme preparation is contacted with a medium containing the starting
composition to
produce target steviol glycosides.
The enzyme can be provided in the form of a whole cell suspension, a crude
lysate,
a purified enzyme or a combination thereof. In one embodiment, the biocatalyst
is a
purified enzyme capable of converting the starting composition to the target
steviol
glycoside. In another embodiment, the biocatalyst is a crude lysate comprising
at least one
enzyme capable of converting the starting composition to the target steviol
glycoside. In
still another embodiment, the biocatalyst is a whole cell suspension
comprising at least
one enzyme capable of converting the starting composition to the target
steviol glycoside.
In another embodiment, the biocatalyst is one or more microbial cells
comprising
enzyme(s) capable of converting the starting composition to the target steviol
glycoside.
The enzyme can be located on the surface of the cell, inside the cell or
located both on the
surface of the cell and inside the cell.
Suitable enzymes for converting the starting composition to target steviol
glycosides include, but are not limited to, the steviol biosynthesis enzymes
and UDP-
glucosyltransferases (UGTs). Optionally it may include UDP recycling
enzyme(s).
In one embodiment, the steviol biosynthesis enzymes include mevalonate (MVA)
pathway enzymes.
In another embodiment, the steviol biosynthesis enzymes include non-mevalonate
2-C-methyl-D-erythrito1-4-phosphate pathway (MEP/DOXP) enzymes.
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In one embodiment the steviol biosynthesis enzymes are selected from the group
including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase,
kaurene
synthase, kaurene oxidase, kaurenoic acid 13¨hydroxylase (KAH), steviol
synthetase,
deoxyxylulose 5 -phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate
reductoisomerase (DXR), 4-diphosphocytidy1-2-C-methyl-D-erythritol synthase
(CMS), 4-
diphosphocytidy1-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidy1-2-C-
methyl-D-erythritol 2,4- cyclodiphosphate synthase (MCS), 1-hydroxy-2-methy1-
2(E)-
butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-
diphosphate
reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase,
mevalonate
kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase,
cytochrome
P450 reductase etc.
The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of
adding at least one glucose unit to steviol and/or a steviol glycoside
substrate to provide
the target steviol glycoside.
In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases
are produced in a microbial cell. The microbial cell may be, for example, E.
coil,
Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.
etc. In another
embodiment, the UDP-glucosyltransferases are synthesized.
In one embodiment, the UDP-glucosyltransferase is selected from group
including
UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTS12, EUGT11 and UGTs having
substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%,
>95%, >96%,>97%, >98%, >99%) amino-acid sequence identity to these
polypeptides as
well as isolated nucleic acid molecules that code for these UGTs.
In one embodiment, steviol biosynthesis enzymes, UGTs and UDP-glucose
recycling system are present in one microorganism (microbial cell). The
microorganism
may be for example, E. coil, Saccharomyces sp., Aspergillus sp., Pichia sp.,
Bacillus sp.,
Yarrowia sp.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase
capable of adding at least one glucose unit to steviol or any starting steviol
glycoside
bearing an -OH functional group at C13 to give a target steviol glycoside
having an -0-
glucose beta glucopyranoside glycosidic linkage at C13. In a particular
embodiment, the
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UDP-glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence
identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to steviol or
any starting
steviol glycoside bearing a -COOH functional group at C19 to give a target
steviol
glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at
C19. In a
particular embodiment, the UDP-glucosyltransferase is UGT74G1, or a UGT having
>85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to the
existing glucose at
C19 of any starting steviol glycoside to give a target steviol glycoside with
at least one
additional glucose bearing at least one beta 1¨*2 glucopyranoside glycosidic
linkage(s) at
the newly formed glycosidic bond(s). In a
particular embodiment, the UDP-
glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence
identity with
UGTS12. In another particular embodiment, the UDP-glucosyltransferase is
EUGT11, or
a UGT having >85% amino-acid sequence identity with EUGT11. In yet another
particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having
>85% amino-acid sequence identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to the
existing glucose at
C19 of any starting steviol glycoside to give a target steviol glycoside with
at least one
additional glucose bearing at least one beta 1-->3 glucopyranoside glycosidic
linkage(s) at
the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-
glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence
identity
with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to the
existing glucose at
C13 of any starting steviol glycoside to give a target steviol glycoside with
at least one
additional glucose bearing at least one beta 1--->2 glucopyranoside glycosidic
linkage(s) at
the newly formed glycosidic bond(s). In a particular embodiment, the UDP-
glucosyltransferase is UGTSI2, or a UGT having >85% amino-acid sequence
identity with
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UGTS12. In another particular embodiment, the UDP-glucosyltransferase is
EUGT11, or a
UGT having >85% amino-acid sequence identity with EUGT11. In yet another
particular
embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-
acid sequence identity with UGT91D2.
In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase
capable of adding at least one glucose unit to steviol to form
steviolmonoside. In a
particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having
>85%
amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid
sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to steviol to
form
steviolmonoside A. In a particular embodiment, the UDP-glucosyltransferase is
UGT74G1
or a UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside A to
form steviolbioside B. In a particular embodiment, the UDP-glucosyltransferase
is
UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside A to
form steviolbioside A. In a particular embodiment, the UDP-glucosyltransferase
is
UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In
another
particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having
>85%
amino-acid sequence identity with EUGT11. In yet another particular
embodiment, the
UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside A to
form rubusoside. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or
a UGT having >85% amino-acid sequence identity with UGT85C2.
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In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside to form
rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1
or a
UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolmonoside to form
steviolbioside.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside B to form
stevioside B. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or a
UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside B to form
stevioside C. In a particular embodiment, the UDP-glucosyltransferase is
UGTS12 or a
UGT having >85% amino-acid sequence identity with UGTS12. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside A to form
stevioside A. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or a
UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside A to form
stevioside C. In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1 or a
UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to rubusoside
to form
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stevioside B. In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1 or a
UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to rubusoside
to form
.. stevioside A (rebaudioside KA). In a particular embodiment, the UDP-
glucosyltransferase
is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In
another
particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having
>85%
amino-acid sequence identity with EUGT11. In yet another particular
embodiment, the
UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to rubusoside
to form
stevioside.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
steviolbioside to form
stevioside. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1
or a
UGT having >85% amino-acid sequence identity with UGT74G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
B to form
.. rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is
UGTS12 or a
UGT having >85% amino-acid sequence identity with UGTS12. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
B to form
rebaudioside E2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
A
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(rebaudioside KA) to form rebaudioside E3. In a particular embodiment, the UDP-
glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence
identity
with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
A
(rebaudioside KA) to form rebaudioside E.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
C to form
rebaudioside E3. In a particular embodiment, the UDP-glucosyltransferase is
UGT85C2 or
a UGT having >85% amino-acid sequence identity with UGT85C2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
to form
rebaudioside E2. In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1 or
a UGT having >85% amino-acid sequence identity with UGT76G1.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to stevioside
to form
rebaudioside E. In a particular embodiment, the UDP-glucosyltransferase is
UGTSI2 or a
UGT having >85% amino-acid sequence identity with UGTSI2. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
rebaudioside E3 to form
rebaudioside AM
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
rebaudioside E2 to form
rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is
UGTSI2 or
a UGT having >85% amino-acid sequence identity with UGTS12. In another
particular
embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino-
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acid sequence identity with EUGT11. In yet another particular embodiment, the
UDP-
glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence
identity
with UGT91D2.
In another embodiment, the UDP-glucosyltransferase is any UDP-
glucosyltransferase capable of adding at least one glucose unit to
rebaudioside E to form
rebaudioside AM In a particular embodiment, the UDP-glucosyltransferase is
UGT76G1
or a UGT having >85% amino-acid sequence identity with UGT76G1.
Optionally, the method of the present invention further comprises using more
than
one UGT on a starting composition, to give a target steviol glycoside(s)
having more than
one glucose unit than the starting composition. In a particular embodiment,
the UDP-
glucosyltransferases are UGT74G1, UGT85C2, UGT76G1, UGTS12, EUGT11 and/or
UGT91D2 or any UGT having >85% amino-acid sequence identity with UGT74G1,
UGT85C2, UGT76G1, UGTS12, EUGT11 and/or UGT91D2 or any combination thereof,
capable of adding more than one glucose unit to a starting composition to give
a steviol
glycoside(s) having more than one glucose unit than the starting composition.
In one embodiment, the UDP-glucosyltransferases are any UDP-
glucosyltransferases capable of adding overall two glucose unit to stevioside
to form
rebaudioside AM In a particular embodiment, the UDP-glucosyltransferases are
selected
from UGTS12, EUGT11, UGT91D2, UGT76G1 or any UGT having >85% amino-acid
sequence identity with UGTS12, EUGT11, UGT91D2, UGT76G1 or any combination
thereof In another particular embodiment, the UDP-glucosyltransferases are
UGTS12 and
UGT76G1.
Optionally, the method of the present invention further comprises recycling
UDP
to provide UDP-glucose. In one embodiment, the method comprises recycling UDP
by
providing a recycling catalyst and a recycling substrate, such that the
biotransformation of
steviol and/or the steviol glycoside substrate to the target steviol glycoside
is carried out
using catalytic amounts of UDP-glucosyltransferase and UDP-glucose. The UDP
recycling enzyme can be sucrose synthase SuSy_At or a sucrose synthase having
>85%
amino-acid sequence identity with SuSy_At and the recycling substrate can be
sucrose.
Optionally, the method of the present invention further comprises the use of
transglycosidases that use oligo- or poly-saccharides as the sugar donor to
modify
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recipient target steviol glycoside molecules. Non-limiting examples include
cyclodextrin
glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase,
glucosucrase,
beta-h-fructosidase, beta-fructosidase, sucrase, fructosylinvertase, alkaline
invertase, acid
invertase, fructofuranosidase. In some embodiments, glucose and sugar(s) other
than
glucose, including but not limited to fructose, xylose, rhamnose, arabinose,
deoxyglucose,
galactose are transferred to the recipient target steviol glycosides. In one
embodiment, the
recipient steviol glycoside is rebaudioside AM.
In another embodiment, the UDP-glucosyltransferase capable of adding at least
one glucose unit to starting composition steviol glycoside has >85% amino-acid
sequence
identity with UGTs selected from the following listing of GenInfo identifier
numbers,
preferably from the group presented in Table 1, and Table 2.
397567 30680413 115480946 147798902 218193594 225443294
454245 32816174 116310259 147811764 218193942 225444853
1359905 32816178 116310985 147827151 219885307 225449296
1685003 34393978 116788066 147836230 222615927 225449700
1685005 37993665 116788606 147839909 222619587 225454338
2191136 37993671 116789315 147846163 222623142 225454340
2501497 37993675 119394507 147855977 222625633 225454342
2911049 39104603 119640480 148905778 222625635 225454473
4218003 41469414 122209731 148905999 222636620 225454475
4314356 41469452 125526997 148906835 222636621 225458362
13492674 42566366 125534279 148907340 222636628 225461551
13492676 42570280 125534461 148908935 222636629 225461556
15217773 42572855 125540090 148909182 224053242 225461558
15217796 44890129 125541516 148909920 224053386 225469538
15223396 46806235 125545408 148910082 224055535 225469540
15223589 50284482 125547340 148910154 224056138 226316457
15227766 51090402 125547520 148910612 224056160 226492603
15230017 51090594 125554547 148910769 224067918 226494221
15231757 52839682 125557592 156138791 224072747 226495389
15234056 56550539 125557593 156138797 224080189 226495945
15234195 62734263 125557608 156138799 224091845 226502400
15234196 62857204 125559566 156138803 224094703 226507980
15238503 62857206 125563266 165972256 224100653 226531147
15239523 62857210 125571055 168016721 224100657 226532094
15239525 62857212 125579728 171674071 224101569 238477377
15239543 75265643 125588307 171906258 224103105 240254512
15239937 75285934 125589492 183013901 224103633 242032615
15240305 75288884 125599469 183013903 224103637 242032621
15240534 77550661 125601477 186478321 224109218 242038423
15982889 77556148 126635837 187373030 224114583 242043290
18086351 82791223 126635845 187373042 224116284 242044836
18418378 83778990 126635847 190692175 224120552 242051252
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18418380 89953335 126635863 194701936 224121288 242056217
18418382 110741436 126635867 195620060 224121296 242056219
19743740 110743955 126635883 209954691 224121300 242056663
19911201 115438196 126635887 209954719 224130358 242059339
20149064 115438785 133874210 209954725 224140703 242059341
20260654 115441237 133874212 209954733 224143404 242060922
21435782 115454819 145358033 210063105 224143406 242067411
21553613 115456047 147772508 210063107 224144306 242067413
21593514 115457492 147776893 212275846 224285244 242076258
22759895 115459312 147776894 216296854 225431707 242076396
23955910 115464719 147776895 217074506 225435532 242084750
26452040 115471069 147786916 218185693 225436321 242091005
28393204 115471071 147798900 218187075 225440041 242095206
30679796 115474009 147798901 218189427 225441116 242345159
242345161 297724601 326492035 356523945 357140904 359486938
255536859 297725463 326493430 356523957 357165849 359487055
255538228 297728331 326500410 356523959 357165852 359488135
255541676 297738632 326506816 356523961 357168415 359488708
255547075 297745347 326507826 356523963 357437837 359493630
255552620 297745348 326508394 356524387 357442755 359493632
255552622 297795735 326509445 356524403 357442757 359493634
255555343 297796253 326511261 356527181 357445729 359493636
255555361 297796257 326511866 356533209 357445731 359493815
255555363 297796261 326512412 356533852 357445733 359495856
255555365 297797587 326517673 356534718 357446799 359495858
255555369 297798502 326518800 356535480 357446805 359495869
255555373 297799226 326521124 356542996 357452779 359495871
255555377 297805988 326525567 356543136 357452781 359497638
255556812 297807499 326525957 356543932 357452783 359807261
255556818 297809125 326526607 356549841 357452787 374256637
255563008 297809127 326527141 356549843 357452789 377655465
255564074 297811403 326530093 356554358 357452791 378405177
255564531 297820040 326534036 356554360 357452797 378829085
255572878 297821483 326534312 356558606 357452799 387135070
255577901 297825217 332071132 356560333 357470367 387135072
255583249 297832276 339715876 356560599 357472193 387135078
255583253 297832280 342306012 356560749 357472195 387135092
255583255 297832518 342306016 356566018 357474295 387135094
255585664 297832520 343457675 356566169 357474493 387135098
255585666 297840825 343457677 356566173 357474497 387135100
255634688 297840827 350534960 356567761 357474499 387135134
255644801 297847402 356498085 356574704 357490035 387135136
255645821 297849372 356499771 356576401 357493567 387135174
255647456 300078590 356499777 356577660 357497139 387135176
255648275 300669727 356499779 357114993 357497581 387135184
260279126 302142947 356501328 357115447 357497671 387135186
260279128 302142948 356502523 357115451 357500579 387135188
261343326 302142950 356503180 357115453 357504663 387135190
283132367 302142951 356503184 357116080 357504691 387135192
283362112 302765302 356503295 357116928 357504699 387135194
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289188052 302796334 356504436 357117461 357504707 387135282
295841350 302811470 356504523 357117463 357505859 387135284
296088529 302821107 356504765 357117829 357510851 387135294
296090415 302821679 356511113 357117839 357516975 387135298
296090524 319759260 356515120 357125059 359477003 387135300
296090526 319759266 356517088 357126015 359477998 387135302
297599503 320148814 356520732 357134488 359478043 387135304
297601531 326489963 356522586 357135657 359478286 387135312
297611791 326490273 356522588 357138503 359484299 387135314
297722841 326491131 356522590 357139683 359486936 387135316
387135318 449440433 460376293 460413408 462423864 475546199
387135320 449445896 460378310 460416351 470101924 475556485
387135322 449446454 460380744 462394387 470102280 475559699
387135324 449447657 460381726 462394433 470102858 475578293
387135326 449449002 460382093 462394557 470104211 475591753
387135328 449449004 460382095 462395646 470104264 475593742
388493506 449449006 460382754 462395678 470104266 475612072
388495496 449451379 460384935 462396388 470106317 475622476
388498446 449451589 460384937 462396389 470106357 475622507
388499220 449451591 460385076 462396419 470115448 475623787
388502176 449451593 460385872 462396542 470130404 482550481
388517521 449453712 460386018 462397507 470131550 482550499
388519407 449453714 460389217 462399998 470136482 482550740
388521413 449453716 460394872 462400798 470136484 482550999
388827901 449453732 460396139 462401217 470136488 482552352
388827903 449457075 460397862 462402118 470136492 482554970
388827907 449467555 460397864 462402237 470137933 482555336
388827909 449468742 460398541 462402284 470137937 482555478
388827913 449495638 460403139 462402416 470140422 482556454
393887637 449495736 460403141 462404228 470140426 482557289
393887646 449499880 460403143 462406358 470140908 482558462
393887649 449502786 460403145 462408262 470141232 482558508
393990627 449503471 460405998 462409325 470142008 482558547
397746860 449503473 460407578 462409359 470142010 482561055
397789318 449515857 460407590 462409777 470142012 482561555
413924864 449518643 460409128 462411467 470143607 482562795
414590349 449519559 460409134 462414311 470143939 482562850
414590661 449522783 460409136 462414416 470145404 482565074
414591157 449524530 460409459 462414476 473923244 482566269
414879558 449524591 460409461 462415526 474114354 482566296
414879559 449528823 460409463 462415603 474143634 482566307
414879560 449528825 460409465 462415731 474202268 482568689
414888074 449534021 460409467 462416307 474299266 482570049
431812559 460365546 460410124 462416920 474363119 482570572
449432064 460366882 460410126 462416922 474366157 482575121
449432066 460369823 460410128 462416923 474429346
449433069 460369829 460410130 462416924 475432777
449436944 460369831 460410132 462417401 475473002
449438665 460369833 460410134 462419769 475489790
449438667 460370755 460410213 462420317 475511330
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449440431 460374714 460411200 462423366 475516200
Table 1
GI number Accession Origin
190692175 ACE87855.1 Stevia rebaudiana
41469452 AAS07253.1 Oryza saliva
62857204 BAD95881.1 Ipomoea nil
62857206 BAD95882.1 Ipomoea purperea
56550539 BAD77944.1 Bellis perennis
115454819 NP_001051010.1 Oryza saliva Japonica Group
115459312 NP_001053256.1 ayza saliva Japonica Group
115471069 NP_001059133.1 Oryza saliva Japonica Group
115471071 NP 001059134.1 Oryza saliva Japonica Group
116310985 CATI67920.1 Oryza saliva Indica Group
116788066 ABK24743.1 Picea sitchensis
122209731 Q2V6J9.1 Fragaria x ananassa
125534461 EAY81009.1 Oryza saliva Indica Group
125559566 EAZ05102.1 Oryza saliva Indica Group
125588307 EAZ28971.1 Oryza saliva Japonica Group
148907340 ABR16806.1 Picea sitchensis
148910082 ABR18123.1 Picea sitchensis
148910612 ABR18376.1 Picea sitchensis
15234195 NP 194486.1 Arabidopsis thaliana
15239523 NP 200210.1 Arabidopsis thaliana
15239937 NP 196793.1 Arabidopsis thaliana
1685005 AA-1336653.1 Nicotiana tabacum
183013903 ACC38471.1 Medicago truncatula
186478321 NP 172511.3 Arabidopsis thaliana
187373030 ACD03249.1 Avena strigosa
194701936 ACF85052.1 Zea mays
19743740 AAL92461.1 Solanum lycopersicum
212275846 NP 001131009.1 Zea mays
222619587 EEE-55719.1 Oryza saliva Japonica Group
224055535 XP_002298527.1 Populus trichocarpa
224101569 XP_002334266.1 Populus trichocarpa
224120552 XP_002318358.1 Populus trichocarpa
224121288 XP_002330790.1 Populus trichocarpa
225444853 XP_002281094 Vitis vinifera
225454342 XP_002275850.1 Vitis vinifera
225454475 XP_002280923.1 Vitis vinifera
225461556 XP_002285222 Vitis vinifera
225469540 XP_002270294.1 Vitis vinifera
226495389 NP_001148083.1 Zea mays
226502400 NP 001147674.1 Zea mays
238477377 AC-R.43489.1 Triticum aestivum
240254512 NP 565540.4 Arabidopsis thaliana
2501497 Q43-716.1 Petunia x hybrida
255555369 XP 002518721.1 Ricinus communis
26452040 BA-C43110.1 Arabidopsis thaliana
296088529 CBI37520.3 Vitis vinifera
297611791 NP 001067852.2 Oryza saliva Japonica Group
297795735 XP 002865752.1 Arabidopsis lyrata subsp. lyrata
297798502 XP 002867135.1 Arabidopsis lyrata subsp. lyrata
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297820040 XP_002877903.1 Arabidopsis lyrata subsp. lyrata
297832276 XP_002884020.1 Arabidopsis lyrata subsp. lyrata
302821107 XP_002992218.1 Selaginella moellendorffii
30680413 NP 179446.2 Arabidopsis thaliana
319759266 ADV71369.1 Pueraria montana var. lobata
326507826 BAJ86656.1 Hordeum vulgare subsp. Vulgare
343457675 AEM37036.1 Brass/ca rapa subsp. oleifera
350534960 NP_001234680.1 Solanum lycopersicum
356501328 XP_003519477.1 Glycine max
356522586 XP_003529927.1 Glycine max
356535480 XP_003536273.1 Glycine max
357445733 XP_003593144.1 Medicago truncatula
357452783 XP 003596668.1 Medicago truncatula
357474493 XP 003607531.1 Medicago truncatula
357500579 XP 003620578.1 Medicago truncatula
357504691 XP_003622634.1 Medicago truncatula
359477998 XP_003632051.1 Vitis vinifera
359487055 XP_002271587 Vitis vinifera
359495869 XP 003635104.1 Vitis vinifera
387135134 AFJ52948.1 Linum usitatissimum
387135176 AFJ52969.1 Linum usitatissimum
387135192 AFJ52977.1 Linum usitatissimum
387135282 AFJ53022.1 Linum usitatissimum
387135302 AFJ53032.1 Linum usitatissimum
387135312 AFJ53037.1 Linum usitatissimum
388519407 AFK47765.1 Medicago truncatula
393887646 AFN26668.1 Barbarea vulgaris subsp. arcuata
414888074 DAA64088.1 Zea mays
42572855 NP 974524.1 Arabidopsis thaliana
449440433 X13_004137989.1 Cucumis sativus
449446454 XP_004140986.1 Cucumis sativus
449449004 XP_004142255.1 Cucumis sativus
449451593 XP_004143546.1 Cucumis sativus
449515857 XP_004164964.1 Cucumis sativus
460382095 XP_004236775.1 Solanum lycopersicum
460409128 XP_004249992.1 Solanum lycopersicum
460409461 XP_004250157.1 Solanum lycopersicum
460409465 XP 004250159.1 Solanum lycopersicum
462396388 EMJ02187.1 Prunus persica
462402118 EMJ07675.1 Prunus persica
462409359 EMJ14693.1 Prunus persica
462416923 EMJ21660.1 Prunus persica
46806235 BAD17459.1 Otyza saliva Japonica Group
470104266 XP 004288529.1 Fragaria vesca subsp. vesca
470142008 XP 004306714.1 Fragaria vesca subsp. vesca
475432777 EM-T01232.1 Aegilops tauschii
51090402 BAD35324.1 Otyza sativa Japonica Group
Table 2
GI number Accession Origin Internal reference
460409128 XP.004249992.1 Solanum lycopersicum
UGTS1
460386018 XP.004238697.1 Solanum lycopersicum
-
460409134 XP.004249995.1 Solanum lycopersicum
-
460410132 XP.004250485.1 Solanum lycopersicum
UGTS12
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460410130 XP.004250484.1 Solanum lycopersicum
460410128 XP.004250483.1 Solanum lycopersicum
460378310 XP.004234916.1 Solanum lycopersicum
209954733 BAG80557.1 Lye/urn barbarum UGTLB
209954725 BAG80553.1 Lycium barbarum
One embodiment of the present invention is a microbial cell comprising an
enzyme, i.e. an enzyme capable of converting the starting composition to the
target steviol
glycoside. Accordingly, some embodiments of the present method include
contacting a
microorganism with a medium containing the starting composition to provide a
medium
comprising at least one target steviol glycoside.
The microorganism can be any microorganism possessing the necessary enzyme(s)
for converting the starting composition to target steviol glycoside(s). These
enzymes are
encoded within the microorganism's genome.
Suitable microoganisms include, but are not limited to, E.coli, Saccharomyces
sp.,
Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc.
In one embodiment, the microorganism is free when contacted with the starting
composition.
In another embodiment, the microorganism is immobilized when contacted with
the starting composition. For example, the microorganism may be immobilized to
a solid
support made from inorganic or organic materials. Non-limiting examples of
solid
supports suitable to immobilize the microorganism include derivatized
cellulose or glass,
ceramics, metal oxides or membranes. The microorganism may be immobilized to
the
solid support, for example, by covalent attachment, adsorption, cross-linking,
entrapment
or encapsulation.
In still another embodiment, the enzyme capable of converting the starting
composition to the target steviol glycoside is secreted out of the
microorganism and into
the reaction medium.
The target steviol glycoside is optionally purified. Purification of the
target steviol
glycoside from the reaction medium can be achieved by at least one suitable
method to
provide a highly purified target steviol glycoside composition. Suitable
methods include
crystallization, separation by membranes, centrifugation, extraction (liquid
or solid phase),
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chromatographic separation, HPLC (preparative or analytical) or a combination
of such
methods.
Uses
Highly purified target glycoside(s), particularly steviolmonoside,
steviolmonoside
A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM obtained according to this invention
can be used
"as-is" or in combination with other sweeteners, flavors, food ingredients and
combinations thereof.
Non-limiting examples of flavors include, but are not limited to, lime, lemon,
orange, fruit, banana, grape, pear, pineapple, mango, berry, bitter almond,
cola, cinnamon,
sugar, cotton candy, vanilla and combinations thereof.
Non-limiting examples of other food ingredients include, but are not limited
to,
acidulants, organic and amino acids, coloring agents, bulking agents, modified
starches,
gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers,
stabilizers, thickeners,
gelling agents and combinations thereof.
Highly purified target glycoside(s), particularly steviolmonoside,
steviolmonoside
A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM obtained according to this invention
can be
prepared in various polymorphic forms, including but not limited to hydrates,
solvates,
anhydrous, amorphous forms and combinations thereof.
Highly purified target glycoside(s) particularly, steviolmonoside,
steviolmonoside
A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM obtained according to this invention
may be
incorporated as a high intensity natural sweetener in foodstuffs, beverages,
pharmaceutical
compositions, cosmetics, chewing gums, table top products, cereals, dairy
products,
toothpastes and other oral cavity compositions, etc.
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Highly purified target glycoside(s) particularly, steviolmonoside,
steviolmonoside
A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM obtained according to this invention
may be
employed as a sweetening compound as the sole sweetener, or it may be used
together
with at least one naturally occurring high intensity sweeteners such as
rebaudioside A,
rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside
C2,
rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside
F3,
rebaudioside G, rebaudioside 11, rebaudioside I, rebaudioside 12, rebaudioside
13,
.. rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside L,
rebaudioside M,
rebaudioside M2, rebaudioside N, rebaudioside 0, rebaudioside 02, rebaudioside
Q,
rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside
T,
rebaudioside Ti, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside
W,
rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z/,
rebaudioside Z2,
dulcoside A, dulcoside C, stevioside D, stevioside E, stevioside E2,
stevioside F,
mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its
salts,
thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside,
phlomisoside-I,
dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin,
carnosiflosides,
cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin,
phillodulcin,
glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-
acetate,
neoastilibin, trans-cinnamaldehyde, monatin and its salts, selligueain A,
hematoxylin,
monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin,
miraculin,
curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener,
mogroside V,
siamenoside and combinations thereof
In a particular embodiment, steviolmonoside, steviolmonoside A,
steviolbioside,
steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A
(rebaudioside KA),
stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3
and/or
rebaudioside AM can be used in a sweetener composition comprising a compound
selected
from the group consisting of rebaudioside A, rebaudioside A2, rebaudioside A3,
rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside
D2,
rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside
11,
rebaudioside I, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside
K,
rebaudioside K2, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside
N,
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rebaudioside 0, rebaudioside 02, rebaudioside Q, rebaudioside Q2, rebaudioside
Q3,
rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside Ti, rebaudioside
U,
rebaudioside U2, rebaudioside V, rebaudioside W, rebaudioside W2, rebaudioside
W3,
rebaudioside Y, rebaudioside Z/, rebaudioside Z2, dulcoside A, dulcoside C,
stevioside D,
.. stevioside E, stevioside E2, stevioside F, NSF-02, Mogroside V, Luo Han
Guo, allulose,
allose, D-tagatose, erythritol and combinations thereof.
Highly purified target glycoside(s), particularly steviolmonoside,
steviolmonoside
A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM may also be used in combination with
synthetic
high intensity sweeteners such as sucralose, potassium acesulfame, aspartame,
alitame,
saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan
advantame,
salts thereof, and combinations thereof
Moreover, highly purified target steviol glycoside(s) particularly
steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside K4), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in
combination
with natural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin,
lactisole,
and others.
Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A,
.. steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA),
stevioside B,
stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or
rebaudioside AM
may also be combined with various umami taste enhancers.
Steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be mixed with
umami
tasting and sweet amino acids such as glutamate, aspartic acid, glycine,
alanine, threonine,
proline, serine, glutamate, lysine, tryptophan and combinations thereof
Highly purified target steviol glycoside(s) particularly, steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside K4), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used in
combination
with one or more additive selected from the group consisting of carbohydrates,
polyols,
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amino acids and their corresponding salts, poly-amino acids and their
corresponding salts,
sugar acids and their corresponding salts, nucleotides, organic acids,
inorganic acids,
organic salts including organic acid salts and organic base salts, inorganic
salts, bitter
compounds, flavorants and flavoring ingredients, astringent compounds,
proteins or
protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers
and
combinations thereof.
Highly purified target steviol glycoside(s) particularly, steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be combined with
polyols
or sugar alcohols. The term "polyol" refers to a molecule that contains more
than one
hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2,
3, and 4
hydroxyl groups, respectively. A polyol also may contain more than four
hydroxyl groups,
such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7
hydroxyl groups,
respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric
alcohol, or
polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl
group
(aldehyde or ketone, reducing sugar) has been reduced to a primary or
secondary hydroxyl
group. Examples of polyols include, but are not limited to, erythritol,
maltitol, mannitol,
sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol,
threitol, galactitol,
hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-
oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup,
reduced
glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar
alcohols or any
other carbohydrates capable of being reduced which do not adversely affect the
taste of the
sweetener composition.
Highly purified target steviol glycoside(s), particularly steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may be combined with
reduced
calorie sweeteners such as, for example, D-tagatose, L-sugars, L-sorbose, L-
arabinose and
combinations thereof.
Highly purified target steviol glycoside(s), particularly steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
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stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM may also be combined
with
various carbohydrates. The term "carbohydrate" generally refers to aldehyde or
ketone
compounds substituted with multiple hydroxyl groups, of the general formula
(CH20),,,
wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates
of the
present invention can, in addition, be substituted or deoxygenated at one or
more
positions. Carbohydrates, as used herein, encompass unmodified carbohydrates,
carbohydrate derivatives, substituted carbohydrates, and modified
carbohydrates. As used
herein, the phrases "carbohydrate derivatives", "substituted carbohydrate",
and "modified
.. carbohydrates" are synonymous. Modified carbohydrate means any carbohydrate
wherein
at least one atom has been added, removed, or substituted, or combinations
thereof Thus,
carbohydrate derivatives or substituted carbohydrates include substituted and
unsubstituted monosaccharides, disaccharides, oligosaccharides, and
polysaccharides. The
carbohydrate derivatives or substituted carbohydrates optionally can be
deoxygenated at
any corresponding C-position, and/or substituted with one or more moieties
such as
hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl
derivatives,
alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo,
mercapto,
imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido,
phosphonyl,
phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino,
carbamyl,
phospho, phosphonato, or any other viable functional group provided the
carbohydrate
derivative or substituted carbohydrate functions to improve the sweet taste of
the
sweetener composition.
Examples of carbohydrates which may be used in accordance with this invention
include, but are not limited to, psicose, turanose, allose, tagatose,
trehalose, galactose,
.. rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of
maltodextrins,
dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose,
lyxose, allose,
altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose,
neotrehalose,
isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose,
xylulose, psicose,
turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose,
glucuronic acid,
gluconic acid, glucono-lactone, abequose, galactosamine, beet
oligosaccharides, isomalto-
oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-
oligosaccharides
(xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio-
oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like),
sorbose, nigero-
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oligosaccharides, palatinose oligosaccharides, fructooligosaccharides
(kestose, nystose and
the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose,
maltotetraose,
maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin,
inulo-
oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid
sugars such as
high fructose corn syrups, coupling sugars, and soybean oligosaccharides.
Additionally,
the carbohydrates as used herein may be in either the D- or L-configuration.
Highly purified target steviol glycoside(s), particularly steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to
this
invention can be used in combination with various physiologically active
substances or
functional ingredients. Functional ingredients generally are classified into
categories such
as carotenoids, dietary fiber, fatty acids, saponins, antioxidants,
nutraceuticals, flavonoids,
isothiocyanates, phenols, plant sterols and stanols (phytosterols and
phytostanols);
polyols; prebiotics, probiotics; phytoestrogens; soy protein; sulfides/thiols;
amino acids;
proteins; vitamins; and minerals. Functional ingredients also may be
classified based on
their health benefits, such as cardiovascular, cholesterol-reducing, and anti-
inflammatory.
Exemplary functional ingredients are provided in W02013/096420, the contents
of which
is hereby incorporated by reference.
Highly purified target steviol glycoside(s), particularly steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to
this
invention may be applied as a high intensity sweetener to produce zero
calorie, reduced
calorie or diabetic beverages and food products with improved taste
characteristics. It may
also be used in drinks, foodstuffs, pharmaceuticals, and other products in
which sugar
cannot be used. In addition, highly purified target steviol glycoside(s),
particularly
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM can be used as a
sweetener not
only for drinks, foodstuffs, and other products dedicated for human
consumption, but also
in animal feed and fodder with improved characteristics.
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Highly purified target steviol glycoside(s), particularly steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to
this
invention may be applied as a flavor modifier to produce zero calorie, reduced
calorie or
diabetic beverages and food products with modified flavor. When used as a
flavor
modifier, or a flavor with modifying properties (FMP), the highly purified
target steviol
glycoside is used in a consumable product below the detection level of the
flavor modifier
or FMP. The flavor modifier or FMP therefore does not impart a detectable
taste or flavor
of its own to the consumable product, but instead serves to modify the
consumer's
detection of tastes and/or flavors of other ingredients in the consumable
product. One
example of taste and flavor modification is sweetness enhancement, in which
the flavor
modifier or FMP itself does not contribute to the sweetness of the consumable
product, but
enhances the quality of the sweetness tasted by the consumer.
Examples of consumable products in which highly purified target steviol
glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside
A,
steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA),
stevioside B,
stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or
rebaudioside AM
may be used as a flavor modifier or flavor with modifying properties include,
but are not
limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake,
etc.; natural
juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie
drinks; reduced
calorie drinks and foods; yogurt drinks; instant juices; instant coffee;
powdered types of
instant beverages; canned products; syrups; fermented soybean paste; soy
sauce; vinegar;
dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy
sauce;
powdered vinegar; types of biscuits; rice biscuit; crackers; bread;
chocolates; caramel;
candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh
cream; jam;
marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and
fruits packed
in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce;
agricultural vegetable food products; seafood; ham; sausage; fish ham; fish
sausage; fish
paste; deep fried fish products; dried seafood products; frozen food products;
preserved
seaweed; preserved meat; tobacco; medicinal products; and many others. In
principle it
can have unlimited applications.
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Highly purified target steviol glycoside(s), particularly steviolmonoside,
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM obtained according to
this
invention may be applied as a foaming suppressor to produce zero calorie,
reduced calorie
or diabetic beverages and food products.
Examples of consumable products in which highly purified target steviol
glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside
A,
steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA),
stevioside B,
stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and/or
rebaudioside AM
may be used as a sweetening compound include, but are not limited to,
alcoholic
beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices;
refreshing
drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced
calorie drinks and
foods; yogurt drinks; instant juices; instant coffee; powdered types of
instant beverages;
canned products; syrups; fermented soybean paste; soy sauce; vinegar;
dressings;
mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce;
powdered
vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates;
caramel; candy;
chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream;
jam;
marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and
fruits packed
in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce;
agricultural vegetable food products; seafood; ham; sausage; fish ham; fish
sausage; fish
paste; deep fried fish products; dried seafood products; frozen food products;
preserved
seaweed; preserved meat; tobacco; medicinal products; and many others. In
principle it
can have unlimited applications.
During the manufacturing of products such as foodstuffs, drinks,
pharmaceuticals,
cosmetics, table top products, and chewing gum, the conventional methods such
as
mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling,
atomizing,
infusing and other methods may be used.
Moreover, the highly purified target steviol glycoside(s) steviolmonoside A,
steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
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rebaudioside E3 and/or rebaudioside AM obtained in this invention may be used
in dry or
liquid forms.
The highly purified target steviol glycoside can be added before or after heat
treatment of food products. The amount of the highly purified target steviol
glycoside(s),
particularly steviolmonoside A, steviolbioside, steviolbioside A,
steviolbioside B,
rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B,
stevioside C,
rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM
depends on the
purpose of usage. As discussed above, it can be added alone or in combination
with other
compounds.
The present invention is also directed to sweetness enhancement in beverages
using steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM. Accordingly, the
present
invention provides a beverage comprising a sweetener and steviolmonoside A,
steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM as a sweetness enhancer, wherein
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM is present in a
concentration at
or below their respective sweetness recognition thresholds.
As used herein, the term "sweetness enhancer" refers to a compound capable of
enhancing or intensifying the perception of sweet taste in a composition, such
as a
beverage. The term "sweetness enhancer" is synonymous with the terms "sweet
taste
potentiator," "sweetness potentiator," "sweetness amplifier," and "sweetness
intensifier."
The term "sweetness recognition threshold concentration," as generally used
herein, is the lowest known concentration of a sweet compound that is
perceivable by the
human sense of taste, typically around 1.0% sucrose equivalence (1.0% SE).
Generally,
the sweetness enhancers may enhance or potentiate the sweet taste of
sweeteners without
providing any noticeable sweet taste by themselves when present at or below
the
sweetness recognition threshold concentration of a given sweetness enhancer;
however,
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the sweetness enhancers may themselves provide sweet taste at concentrations
above their
sweetness recognition threshold concentration. The sweetness recognition
threshold
concentration is specific for a particular enhancer and can vary based on the
beverage
matrix. The sweetness recognition threshold concentration can be easily
determined by
taste testing increasing concentrations of a given enhancer until greater than
1.0% sucrose
equivalence in a given beverage matrix is detected. The concentration that
provides about
1.0% sucrose equivalence is considered the sweetness recognition threshold.
In some embodiments, sweetener is present in the beverage in an amount from
about 0.5% to about 12% by weight, such as, for example, about 1.0% by weight,
about
1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by
weight,
about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0%
by
weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight,
about 7.0%
by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight,
about
9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by
weight,
about 11.0% by weight, about 11.5% by weight or about 12.0% by weight.
In a particular embodiment, the sweetener is present in the beverage in an
amount
from about 0.5% of about 10%, such as for example, from about 2% to about 8%,
from
about 3% to about 7% or from about 4% to about 6% by weight. In a particular
embodiment, the sweetener is present in the beverage in an amount from about
0.5% to
about 8% by weight. In another particular embodiment, the sweetener is present
in the
beverage in an amount from about 2% to about 8% by weight.
In one embodiment, the sweetener is a traditional caloric sweetener. Suitable
sweeteners include, but are not limited to, sucrose, fructose, glucose, high
fructose corn
syrup and high fructose starch syrup.
In another embodiment, the sweetener is erythritol.
In still another embodiment, the sweetener is a rare sugar. Suitable rare
sugars
include, but are not limited to, D-allose, D-psicose, D-ribose, D-tagatose, L-
glucose, L-
fucose, L-arabinose, D-turanose, D-leucrose and combinations thereof.
It is contemplated that a sweetener can be used alone, or in combination with
other
sweeteners.
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In one embodiment, the rare sugar is D-allose. In a more particular
embodiment,
D-allose is present in the beverage in an amount of about 0.5% to about 10% by
weight,
such as, for example, from about 2% to about 8%.
In another embodiment, the rare sugar is D-psicose. In a more particular
embodiment, D-psicose is present in the beverage in an amount of about 0.5% to
about
10% by weight, such as, for example, from about 2% to about 8%.
In still another embodiment, the rare sugar is D-ribose. In a more particular
embodiment, D-ribose is present in the beverage in an amount of about 0.5% to
about 10%
by weight, such as, for example, from about 2% to about 8%.
In yet another embodiment, the rare sugar is D-tagatose. In a more particular
embodiment, D-tagatose is present in the beverage in an amount of about 0.5%
to about
10% by weight, such as, for example, from about 2% to about 8%.
In a further embodiment, the rare sugar is L-glucose. In a more particular
embodiment, L-glucose is present in the beverage in an amount of about 0.5% to
about
10% by weight, such as, for example, from about 2% to about 8%.
In one embodiment, the rare sugar is L-fucose. In a more particular
embodiment,
L-fucose is present in the beverage in an amount of about 0.5% to about 10% by
weight,
such as, for example, from about 2% to about 8%.
In another embodiment, the rare sugar is L-arabinose. In a more particular
embodiment, L-arabinose is present in the beverage in an amount of about 0.5%
to about
10% by weight, such as, for example, from about 2% to about 8%.
In yet another embodiment, the rare sugar is D-turanose. In a more particular
embodiment, D-turanose is present in the beverage in an amount of about 0.5%
to about
10% by weight, such as, for example, from about 2% to about 8%.
In yet another embodiment, the rare sugar is D-Ieucrose. In a more particular
embodiment, D-leucrose is present in the beverage in an amount of about 0.5%
to about
10% by weight, such as, for example, from about 2% to about 8%.
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The addition of the sweetness enhancer at a concentration at or below its
sweetness
recognition threshold increases the detected sucrose equivalence of the
beverage
comprising the sweetener and the sweetness enhancer compared to a
corresponding
beverage in the absence of the sweetness enhancer. Moreover, sweetness can be
increased
by an amount more than the detectable sweetness of a solution containing the
same
concentration of the at least one sweetness enhancer in the absence of any
sweetener.
Accordingly, the present invention also provides a method for enhancing the
sweetness of a beverage comprising a sweetener comprising providing a beverage
comprising a sweetener and adding a sweetness enhancer selected from
steviolmonoside
A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM or a combination thereof, wherein
steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B,
rubusoside,
stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C,
rebaudioside E,
rebaudioside E2, rebaudioside E3 and/or rebaudioside AM are present in a
concentration
at or below their sweetness recognition thresholds.
Addition of steviolmonoside A, steviolbioside, steviolbioside A,
steviolbioside B,
rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B,
stevioside C,
rebaudioside E, rebaudioside E2, rebaudioside E3 and/or rebaudioside AM in a
concentration at or below the sweetness recognition threshold to a beverage
containing a
sweetener may increase the detected sucrose equivalence from about 1.0% to
about 5.0%,
such as, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about
3.0%, about
3.5%, about 4.0%, about 4.5% or about 5.0%.
The following examples illustrate preferred embodiments of the invention for
the
preparation of highly purified target steviol glycoside(s), particularly
steviolmonoside A,
steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside,
stevioside A
(rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside
E2,
rebaudioside E3 and/or rebaudioside AM. It will be understood that the
invention is not
limited to the materials, proportions, conditions and procedures set forth in
the examples,
which are only illustrative.
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EXAMPLES
EXAMPLE 1
Protein sequences of engineered enzymes used in the biocatalytic process
SEQ ID 1:
>SuSy_At, variant PM1-54-2-E05 (engineered sucrose synthase; source of WT
gene:
Arab idopsis thaliana)
MANAERMI TRVHS QRERLNET LVSERNEVLALLSRVEAKGKGI LQQNQ I I
AEFEAL PEQTRKKLEGGP FFDLLKS TQEAIVL P PWVALAVRPRPGVWEYL
RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEP FNAS I PRPT
LHKYI GNGVDFLNRHLSAKLFHDKES LL PLLDFLRLHSHQGKNLMLSEKI
QNLNTLQHTLRKAEEYLAELKSETLYEE FEAKFEE I GLERGWGDNAERVL
DMIRLLLDLLEAPDPSTLET FLGRVPMVFNVVI LS PHGYFAQDNVLGYPD
TGGQVVYILDQVRALE IEMLQRIKQQGLNIKPRI L I LTRLL PDAVGTT CG
ERLERVYDSEYCDI LRVP FRTEKGIVRKW I SRFEVWPYLETYTEDAAVEL
S KELNGKP DL I I GNYS DGNLVASLLAHKLGVTQCT IAHALEKTKYP DS DI
YWKKLDDKYHFSCQFTADI FAMNHT DFI IT S T FQEIAGSKETVGQYESHT
AFT LPGLYRVVHGI DVFDPKFNIVS PGADMS IYFPYTEEKRRLTKFHSE I
EELLYS DVENDEHLCVLKDKKKP I L FTMARL DRVKNLS GLVEWYGKNTRL
RELVNLVVVGGDRRKE S KDNEEKAEMKKMYDL I EEYKLNGQFRWI S SQMD
RVRNGE LYRY I CDTKGAFVQPALYEAFGLTVVEAMTCGL PT FATCKGGPA
El IVHGKSGFHI DPYHGDQAADLLADFFTKCKEDPSHWDE I SKGGLQRIE
EKYTWQIYSQRLLT LTGVYGFWKHVSNL DRLEHRRYLEMFYALKYRPLAQ
AVPLAQDD
SEQ ID 2:
>UGTS12 variant 0234 (engineered glucosyltransferase; source of WT gene:
Solanum
lycopersicum)
MATNLRVLMFPWLAYGHI S PFLNIAKQLADRGFL IYLCSTRINLES I IRK
I PEKYADS IHL IELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN
FSRILQNLKPDLL IYDVLQPWAEHVANEQGI PAGKLLVSCAAVFSYFFS F
RKNPGVE FP FPAI HL PEVEKVKI RE I LAKE PEEGGRLDEGNKQMMLMCT S
RT I EAKYI DYCTELCNWKVVPVGP P FQDL ITNDADNKEL I DWLGTKPENS
TVFVS FGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALP
EGFLERIGERGRVLDKFAPQPRI LNHPSTGGFI SHCGWNSVMES I DFGVP
I IAMP I HNDQP INAKLMVELGVAVEIVRDDDGKIHRGE IAEALKSVVTGE
T GE I LRAKVRE I SKNLKS IRDEEMDAVAEEL IQLCRNSNKSK
SEQ ID 3:
>UGT76G1 variant 0042 (engineered glucosyltransferase; source of WT gene:
Stevia
rebaudiana)
MENKTETTVRRRRRI I LFPVP FQGHINP I LQLANVLYSKGFAIT I LHTNFNKPKT SNYPH
FT FRFI LDNDPQDERI SNLPTHGPLAGMRI P I INEHGADELRRELELLMLASEEDEEVSC
L IT DALWYFAQDVADSLNLRRLVLMT S S L FN FHAHVSL PQFDELGYLDP DDKTRLEEQAS
GFPMLKVKDIKSAYSNWQI GKE I LGKMIKQTKAS SGVIWNS FKELEESELETVIRE I PAP
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S FL I PLPKHLTAS SSSLLDHDRTVFEWLDQQAPSSVLYVS FGSTSEVDEKDFLEIARGLV
DSGQS FLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWN
STLESVCEGVPMI FS S FGGDQPLNARYMS DVLRVGVYLENGWERGEVVNAIRRVMVDEEG
EYIRQNARVLKQKADVSLMKGGS SYESLESLVSYI S SL
EXAMPLE 2
Expression and formulation of SuSy_At variant of SEQ ID 1
The gene coding for the SuSy_At variant of SEQ ID 1 (EXAMPLE 1) was cloned
into the expression vector pLE1A17 (derivative of pRSF-lb, Novagen). The
resulting
plasmid was used for transformation of E.coli BL21(DE3) cells.
Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression
and Purification 41(2005) 207-234) supplemented with kanamycin (50 mg/1) at 37
C.
Expression of the genes was induced at logarithmic phase by IPTG (0.2 mM) and
carried
out at 30 C and 200 rpm for 16-18 hours.
Cells were harvested by centrifugation (3220 x g, 20 min, 4 C) and re-
suspended to
an optical density of 200 (measured at 600nm (0D600)) with cell lysis buffer
(100 mM
Tris-HC1 pH 7.0; 2 mM MgCl2, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells
were then disrupted by sonication and crude extracts were separated from cell
debris by
centrifugation (18000 x g 40 min, 4 C). The supernatant was sterilized by
filtration
through a 0.2 [tm filter and diluted 50:50 with distilled water, resulting in
an enzymatic
active preparation.
For enzymatic active preparations of SuSy_At, activity in Units is defined as
follows: 1 mU of SuSy_At turns over 1 nmol of sucrose into fructose in 1
minute.
Reaction conditions for the assay are 30 C, 50 mM potassium phosphate buffer
pH 7.0,
400 mM sucrose at to, 3 mM MgCl2, and 15 mM uridine diphosphate (UDP).
EXAMPLE 3
Expression and formulation of UGTS12 variant of SEQ ID 2
The gene coding for the UGTS12 variant of SEQ ID 2 (EXAMPLE 1) was cloned
into the expression vector pLE1A17 (derivative of pRSF-lb, Novagen). The
resulting
plasmid was used for transformation of E.coli BL21(DE3) cells.
Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression
and Purification 41(2005) 207-234) supplemented with kanamycin (50 mg/1) at 37
C.
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Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and
carried
out at 30 C and 200 rpm for 16-18 hours.
Cells were harvested by centrifugation (3220 x g, 20 min, 4 C) and re-
suspended to
an optical density of 200 (measured at 600nm (0D600)) with cell lysis buffer
(100 mM
Tris-HCI pH 7.0; 2 mM MgC12, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells
were then disrupted by sonication and crude extracts were separated from cell
debris by
centrifugation (18000 x g 40 min, 4 C). The supernatant was sterilized by
filtration
through a 0.2 p.m filter and diluted 50:50 with 1 M sucrose solution,
resulting in an
enzymatic active preparation.
For enzymatic active preparations of UGTS12, activity in Units is defined as
follows:
1 mU of UGTS12 turns over 1 nmol of rebaudioside A (RebA) into rebaudioside D
(Reb D)
in 1 minute. Reaction conditions for the assay are 30 C, 50 mM potassium
phosphate
buffer pH 7.0, 10 mM RebA at to, 500 mM sucrose, 3 mM MgCl2, 0.25 mM uridine
diphosphate (UDP) and 3 U/mL of SuSy_At.
EXAMPLE 4
Expression and formulation of UGT76G1 variant of SEQ ID 3
The gene coding for the UGT76G1 variant of SEQ ID 3 (EXAMPLE 1) was
cloned into the expression vector pLE1A17 (derivative of pRSF-lb, Novagen).
The
resulting plasmid was used for transformation of E.coli BL21(DE3) cells.
Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression
and Purification 41(2005) 207-234) supplemented with kanamycin (50 mg/1) at 37
C.
Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and
carried
out at 30 C and 200 rpm for 16-18 hours.
Cells were harvested by centrifugation (3220 x g, 20 min, 4 C) and re-
suspended
to an optical density of 200 (measured at 600nm (0D600)) with cell lysis
buffer (100 mM
Tris-HCl pH 7.0; 2 mM MgCl2, DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells
were then disrupted by sonication and crude extracts were separated from cell
debris by
centrifugation (18000 x g 40 min, 4 C). The supernatant was sterilized by
filtration
through a 0.2 j.tm filter and diluted 50:50 with 1 M sucrose solution,
resulting in an
enzymatic active preparation.
For enzymatic active preparations of UGT76G1, activity in Units is defined as
follows: 1 mU of UGT76G1 turns over 1 nmol of rebaudioside D (Reb D) into
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rebaudioside M (Reb Al) in 1 minute. Reaction conditions for the assay are 30
C, 50 mM
potassium phosphate buffer pH 7.0, 10 mM RebA at to, 500 mM sucrose, 3 mM
MgCl2,
0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.
EXAMPLE 5
Synthesis of rebaudioside AM from stevioside in a one-pot reaction, adding
UGTS12,
SuSy_At and UGT76G1 at the same time
Rebaudioside AM (reb AM) was synthesized directly from stevioside in a one-pot
reaction (Fig. 3), utilizing the three enzymes (see EXAMPLES 1, 2, 3 and 4):
UGT5I2
(variant of SEQ ID 2), SuSy_At-(variant of SEQ ID 1) and UGT76G1 (variant of
SEQ ID
3). The final reaction solution contained 105 U/L UGT512, 405 U/L SuSy_At, 3
U/L
UGT76G1, 5 mM stevioside, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM
MgCl2 and potassium phosphate buffer (pH 6.6). First, 207 mL of distilled
water were
mixed with 0.24 g MgC12.6H20, 103g sucrose, 9.9 mL of 1.5 M potassium
phosphate
buffer (pH 6.6) and 15g stevioside. After dissolving the components, the
temperature was
adjusted to 45 C and UGTS12, SuSy_At, UGT76G1 and 39 mg UDP were added. The
reaction mixture was incubated at 45 C shaker for 24 hrs. Additional 39 mg UDP
was
added at 8hrs and 18hours. The content of reb AM, reb E, stevioside, reb M,
reb B,
steviolbioside and reb I at several time points was analyzed by HPLC.
For analysis, biotransformation samples were inactivated by adjusting the
reaction
mixture to pH5.5 using 17% H3PO4 and then boiled for 10 minutes. Resulting
samples
were filtered, the filtrates were diluted 10 times and used as samples for
HPLC analysis.
HPLC assay was carried out on Agilent HP 1200 HPLC system, comprised of a
pump, a
column thermostat, an auto sampler, a UV detector capable of background
correction and
a data acquisition system. Analytes were separated using Agilent Poroshell 120
SB- C18,
4.6 mm x 150 mm, 2.7 um at 40 C. The mobile phase consisted of two premixes:
-
premix 1 containing 75% 10 mM phosphate buffer (pH2.6) and 25% acetonitrile,
and
- premix 2 containing 68% 10 mM phosphate buffer (pH2.6) and 32%
acetonitrile.
Elution gradient started with premix 1, changed to premix 2 to 50% at 12.5
minute,
changed to premix 2 to 100% at 13 minutes. Total run time was 45 minutes. The
column
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temperature was maintained at 40 C. The injection volume was 5 L.
Rebaudioside
species were detected by UV at 210 nm.
Table 3 shows for each time point the conversion of stevioside into identified
rebaudioside species (area percentage). The chromatograms of stevioside and
the reaction
mixture at 24 hours are shown in Fig. 5 and Fig. 6, respectively. Those with
skill in the art
will appreciate that retention times can occasionally vary with changes in
solvent and/or
equipment.
Table 3
Biotransformation of stevioside to reb AM
Time, % conversion from stevioside
hrs Reb E Reb AM Reb M Reb I Stevioside Reb B
Steviolbioside
0 0 0 0 0 100 0 0
6 1.9 35.9 1.3 1.7 58.7 0.0 0.4
18 0.9 96.7 1.3 0.6 0.0 0.0 0.4
24 0.3 96.4 2.1 0.7 0.0 0.2 0.4
EXAMPLE 6
Synthesis of rebaudioside AM from rebaudioside E in a one-pot reaction,
SuSy_At
and UGT76G1 at the same time
Rebaudioside AM (reb AM) was synthesized directly from rebaudioside E (reb E)
in
a one-pot reaction (Fig. 4), utilizing the two enzymes (see EXAMPLES 1, 2 and
4):
SuSy_At-(variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3). The final
reaction
solution contained 405 U/L SuSy_At, 3 U/L UGT76G1, 5 mM reb E, 0.25 mM uridine
diphosphate (UDP), 1 M sucrose, 4 mM MgC12.6H20 and potassium phosphate buffer
(pH
6.6). First, 37 mL of distilled water were mixed with 40.3 mg MgCl2, 17.12g
sucrose, 1.65
mL of 1.5 M potassium phosphate buffer (pH 6.6) and 5.04 g reb E. After
dissolving the
components, the temperature was adjusted to 45 C and SuSy_At, UGT76G1 and 6.5
mg
UDP were added. The reaction mixture was incubated at 45 C shaker for 24 hrs.
Additional 6.5 mg UDP was added at 8hrs and 18hours. The content of reb AM,
reb E,
stevioside, reb A, reb M, reb B, and steviolbioside at several time points was
analyzed by
HPLC.
For analysis, biotransformation samples were inactivated by adjusting the
reaction
mixture to pH5.5 using 17% H3PO4 and then boiled for 10 minutes. Resulting
samples
were filtered, the filtrates were diluted 10 times and used as samples for
HPLC analysis.
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HPLC assay was carried out on Agilent HP 1200 HPLC system, comprised of a
pump, a
column thermostat, an auto sampler, a UV detector capable of background
correction and
a data acquisition system. Analytes were separated using Agilent Poroshell 120
SB- C18,
4.6 mm x 150 mm, 2.7 um at 40 C. The mobile phase consisted of two premixes:
- premix 1
containing 75% 10 mM phosphate buffer (pH2.6) and 25% acetonitrile,
and
- premix 2 containing 68% 10 mM phosphate buffer (pH2.6) and 32%
acetonitrile.
Elution gradient started with premix 1, changed to premix 2 to 50% at 12.5
minute,
changed to premix 2 to 100% at 13 minutes. Total run time was 45 minutes. The
column
temperature was maintained at 40 C. The injection volume was 5 L.
Rebaudioside
species were detected by UV at 210 nm.
Table 4 shows for each time point the conversion of reb E into identified
rebaudioside species (area percentage). The chromatograms of reb E and the
reaction
mixture at 24 hours are shown in Fig. 7 and Fig. 8, respectively. Those with
skill in the art
will appreciate that retention times can occasionally vary with changes in
solvent and/or
equipment.
Table 4
Biotransformation of reb E to reb AM
Time, % conversion from Reb E
hrs Reb E Reb AM Reb M Reb A
Stevioside Reb B Steviolbioside
0 99.46 __ 0 0 0.54 0 0 0
4 40.75 57.92 0 0.59 0 0.73 0
7 24.79 73.92 0 0.58 0 0.71 0
24 4.38 94.33 0 0.59 0 0.70 0
EXAMPLE 7
Purification of rebaudioside AM
The reaction mixture of EXAMPLE 5, after 24 hrs, was inactivated by adjusting
the
pH to pH 5.5 with H3PO4 and then boiled for 10 minutes. After boiling the
reaction
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mixture was filtered and diluted with RU water to 5% solids content. The
diluted solution
was passed through 1 L column packed with YWDO3 macroporous adsorption resin
(Cangzhou Yuanwei, China). Adsorbed steviol glycosides were eluted with 5L 70%
ethanol. The obtained eluate was evaporated until dryness to yield 16 g of dry
powder
which was dissolved in 80 mL of 70% methanol. The solution was crystallized at
20 C for
3 days. The crystals were separated by filtration and dried in vacuum oven at
80 C for 18
hours to yield 10.4 g of pure reb AM crystals with 95.92% purity, determined
by HPLC
assay. The chromatogram of reb AM is shown in Fig. 9. Those with skill in the
art will
appreciate that retention times can occasionally vary with changes in solvent
and/or
equipment.
EXAMPLE 8
Structure elucidation of rebaudioside AM
NMR experiments were performed on a Bruker 500 MHz spectrometer, with the
sample dissolved in pyridine-d5. Along with signals from the sample, signals
from
pyridine-d5 at 8c 123.5, 135.5, 149.9 ppm and 8H 7.19, 7.55, 8.71 ppm were
observed.
1H-NMR-spectrum of rebaudioside AM in pyridine-d5 reveal the excellent quality
of the sample (see Fig. 10). The HSQC (see Fig. 11) shows the presence of an
exo-
methylene group in the sugar region with a long-range coupling to C-15,
observable in the
H,H-COSY (Fig. 12). Other deep-fielded signals of the quaternary carbons (C-
13, C-16
and C-19) are detected by the HMBC (Fig. 13). Correlation of the signals in
the HSQC,
HMBC and H,H-COSY reveal the presence of steviol glycoside with the following
aglycone structure:
12
11 13 ORi
20 :16 17
1 3 9 14:
2
10u 8
51, 15
3 7
H 6
018
Correlation of HSQC and HMBC signals reveal five anomeric signals. The
coupling constant of the anomeric protons of about 8 Hz and the broad signals
of their
sugar linkage allows the identification of these five sugars as P-D-
glucopyranosides.
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The observation of the anomeric protons in combination with HSQC and HM13C
reveal the sugar linkage and the correlation to the aglycone. The assignment
of the sugar
sequence was confirmed by using the combination of HSQC-TOCSY (Fig. 14) and
HSQC.
The NMR experiments above were applied to assign the chemical shifts of the
protons and carbons, main coupling constants and main HMBC correlations (see
Table 5).
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Table 5
Chemical shifts of rebaudioside AM
LPosition Sc [ppm] Sri [ppm] J [Hz] HMBC (H --> C)
Aglycone moiety
1 39.9 t 0.68 m
1.64 m
2 19.4 t 1.39 m
2.08 m
3 37.4
1.05 m
t
2.80 m
4 44.2s
57.3d 0.95m I
6 21.7
1.90 m
t
2.12 m
7 41.0
1.26 m
t
1.38 m
8 41.9s
9 53.3 d 0.85 m
39.2s
11 20.1 t 1.59 m
1.61 m
12 36.9 t 1.65 m
1.92 m
13 85.9s
1.78 d 11.0
14 43.8 t
2.52 d 11.0
47.4
2.00 d 16.0
7 , 8, 9, 14
t
2.06 d 16.0
16 154.6s
5.03 br s
17 104.3 t 13, 15, 16
5.71 br s
18 .28.5q 1.40 s 3, 4, 5, 19
19 175.2 s
16.2 q 1.06s 1, 5, 9, 10
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Table 5 (continued)
Chemical shifts of rebaudioside AM
Position Sc [ppm] 8011 [ppm] J [Hz] _ HMBC (H->C)
Sugar moiety
Sugar I: /3-D-Glucopyranoside
11 97.5 d 5.13 d 7.7 13
2i 84.0 d 4.14 m
3i 77.6 d 4.20 111
4i 71.3 d 4.19 IN
5i 77.6 d 3.70 in
62.0 t 4.23m
6i
4.32 in '
Sugar II: /3-D-Glucopyranoside
li' 106.3 d 5.26 d 8.0 2i
76.8 d 4.13 m
311 77.3 d 4.21 m
71.6 d 4.18 m
77.9 d 3.91 in
62.4
4.29 m
t
4.41m i
Sugar III: /3-D-Glucopyranoside
l iii 92.9 d 6.20 d 8.1 19
77.0 d 4.46 m
3111 88.1 d 4.24 m
69.0 d 4.12 m
5iii 78.4 d 3.82 m
61.3
4.20 m
t
4.33m 1
Sugar IV: /3-D-Glucopyranoside
liv 103.4 d 5.73 d 1 7.7
2" 75.4 d 3.98 m 1 .
3,v 78.1 d 4.09 m I .=
41v 72.6 d 4.08 m 1 '
5iv 77.4 d 3.92 in 1 !
62.9
4.32 m 1 .
6iv t 1
4.51 m 1 I
Sugar V: /J-D-Glucopyranoside
p 104.4 d 5.29 d 8.1
2" 75.1 d 4.00 m
3,,, 78.2d 4.24m
' 4" 71.4 d 4.27 m
5,v 78.2 d 3.99 m
6v 619t 4.27m ,
4.48 m 1 .
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Correlation of all NMR data indicates rebaudioside AM having five [3-D-
glucopyranoses attached to a steviol aglycone, as depicted with the following
chemical
structure:
9H OH
HOitcy11
0
OH
0,, ,,OH
OH
12 0 0
11 3
HO HO 20
1 E. 9 141 16 17
2 40
HO,. 0110*. 0 15 H 8 15
V 3 7
194. LI 6
HO 0 0-18
t "
OH 6 0
0
'110H
HOI--1µ1
Ho* OH
The chemical formula of rebaudioside AM is C50H80028, which corresponds to a
calculated monoisotopic molecular mass of 1128.5. For LCMS analysis,
rebaudioside AM
was dissolved in methanol and analyzed using Shimadzu Nexera 2020 UFLC LCMS
instrument on a Cortecs UPLC C18 1.61.tm , 50 x 2.1 mm column. The observed
LCMS
(negative EST mode) result of 1127.3 (see Fig. 15a and Fig. 15b respectively)
is consistent
with rebaudioside AM and corresponds to the ion (M-H)-.
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Solubility, Sweetness and Flavor Modification Properties of Reb AM
EXAMPLE 9
Reb AM was evaluated for it solubility and solution stability properties.
Tables 6a and 6b,
below, show the composition of the test sample, with the total steviol
glycoside (TSG)
percentage shown in the final column of Table 6b.
Table 6a: Composition of Test Sample:
Assay, % (as dried)
Sample Reb Reb Reg Reb Reb Reb Reb Reb Reb
ID E AM 0 D N M H I A
Reb AM 0.23 99.30 0.00 0.00 0.00 0.00 0.00 0.05 0.00
Sample 1
Table 6b
Assay, % (as dried)
Sample Stev Reb F Reb C Dul. Rubu Reb 8 Sbio TSG
ID A
Reb AM 0.00 0.00 0.00 0.00 0.00 0.00 0.26 99.84
Sample 1
Table 7: Physical Properties of Reb AM:
Physical Material & Reb AM Results
Description Method
Form Visual Evaluation Powder
Appearance Visual Evaluation Very Fine
1
Odor Olfactory Odorless
Evaluation
Color I Visual Evaluation White
Moisture Content
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Solution Stability:
Solubility characteristics were measured as follows. Prepare the following
solutions in
water and stir at 700 rpm for each. Add heat if necessary at 2 minutes and 30
seconds of
stirring. Using a stopwatch, determine how long it takes all powder to
dissolve completely
and record the temperature at which it dissolves. The following table
summarizes the
solubility characteristics of Rebaudiosides D, M, and AM. Surprisingly, Reb AM
shows
significantly higher solubility than other minor and major steviol glycosides.
Table 8: Comparison of Solubility Characteristics
Product Test Dissolution Dissolution in Solution Solution
Comment
Conc. water water after 24 hrs after 48 on
(Ambient) (Heated) hrs solubility
Reb D 0.05% Added heat at 2 8 min 30 sec Clear Clear
Easily Soluble
minutes/30 temp: 39 deg. C
seconds.
Reb D 0.1% Added heat at 2 15 min 4 sec Clear Clear
Easily Soluble
minutes/30 temp: 72 deg. C
seconds.
Reb D 0.3% Added heat at 2 20 min 27 Precipitate in
Requires
minutes/30 seconds temp: less than 24
dispersion
seconds. 78.5 deg. C hrs agent
Reb M 0.1% 12 minutes of No heat needed Clear Clear
Easily Soluble
agitation
Reb M 0.3% Added heat at 2 Heated to 99 deg Clear
Slight Moderately
min 30 seconds. C with agitation precipitation Soluble
Reb M 0.5% Added heat at 2 Heated to 87 Moderate
Requires
min 30 sec deg. C with Precipitation dispersion
agitation. 16 min in 2 hours agent
12 seconds
Reb AM 1% Stirred for 3 min No heat needed Clear Clear
Easily Soluble
28 sec
Reb AM 5% Added heat at 2 15 min 32 sec at Clear Clear
Easily Soluble
minutes/30 temperature:
seconds. 45C
Reb AM 10% Added heat at 2 10 min 2 sec Clear Slight
Moderately
minutes/30 Temperature: precipitation soluble
seconds. 54C
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Table 9: Summary of Solution Stability of Major and Minor steviol glycosides:
SG/Property Reb A* Stevioside* Reb AM Reb D Reb M
Solubility <0.7% <0.7% 10% 0.1% 0.3%
* Solubility of Stevioside was slightly lower than Reb A in aqueous solution.
Ref: Celaya
et al (2016). Int. J. of Food Studies, V.5, p 158-166
EXAMPLE 10
Reb AM was evaluated for its sensory attributes.
Sensory Attributes
Steviol glycoside molecules are known for their varied sweetness profiles,
which are a
function of the sugar moieties present in their structures. Since steviol
glycosides contain
hydrophobic (steviol) and hydrophilic (sugar moieties), they can display
flavor
modification at a certain dosage level without contributing any significant
detectable
sweetness perception.
Isosweet Determination of Reb AM and other Steviol glycosides:
= Five concentration levels of Test sweetener were identified to match
2.5%, 5%,
7.5% and 10% sucrose-equivalent in acidified water (pH of 3.2), for which a
panel
of 40 participants was recruited to conduct two alternate forced choice (2-
AFC)
test at each concentration level.
= Samples were evaluated and isosweet point was determined at a point in
which
50% of the panelist selected sucrose sample as sweeter and 50% selected stevia
sample as sweeter
= A Beidler model was used to fit the concentration-response relationship
using the
four isosweet concentrations and their corresponding target sweetness values
as the
data.
= Sweetness potency is calculated as a ratio of sugar concentration to
sweetness
equivalent. As an example, Reb AM was evaluated.
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Table 10: Iso-sweet concentration (ppm) and Sweetness Potency (x sugar
equivalent) of
Reb AM and other steviol glycosides
Sweetness Equivalent (ppm) in Water (sweetened to Sweetness Potency in
Water (sweetened to
achieve designated % SE @ pH = 3.2) achieve x % SE @ pH = 3.2)
sugar concentration 2.5% 5.0% 7.5% 10.0% 2.5% 5.0% 7.5% 10.0%
Reb A 94 299 NA NA 266 167 NA NA
Delta ( Reb D) 62 212 500 926 403 236 150 108
PCS-4000 ( Reb M) 84 209 418 832 298 239 179 90
Reb AM (Reb AM) 150 365 869 1750 167 137 86
57
Effect of Reb AM on Taste & Flavor Profiles of Food and Beverage Applications
A series of experiments were performed to evaluate the effect of Reb AM on
taste and
flavor profile. The sweetness and taste/flavor modification can influence each
other in
food and beverage applications. To determine the influence of the taste and
flavor
modification in different applications, the FEMA (Flavor and Extract
Manufacturing
Association) prescribes a sensory method that determines the sweetness
perception
threshold determination presented in Experiment 1, which is discussed below.
Experiment 1 provides the estimate of Reb AM concentration in water that
barely
contributes to sweetness perception. The sweetness perception threshold
concentration
provides significantly less sweetness than 1.5% sugar aqueous solution. The
summary of
sweetness perception threshold for selected steviol glycosides is below in
Table 11.
Table 11
Steviol Sweetness Perception FEMA FEMA GRAS Publication
Glycosides Threshold GRAS Reference (FEMA Website)
Concentration No
Reb A 30 ppm 4601 GRAS Flavoring Substances 24
(2008)
Reb D 32.5 ppm 4921 GRAS Flavoring Substances 29
(2018)
Reb M 24 ppm 4922 GRAS Flavoring Substances 29
(2018)
Reb AM 50 ppm NA NA
Experiment 2, which is further discussed below, explores the effect of Reb AM
on the flavor
profile of a non-alcoholic beverage. A commercial Raspberry Watermelon Coconut
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sample was used without (control) and with Reb AM (test) to determine the
effect of Reb
AM on different taste attributes of the beverage. The results indicated the
test sample having
Reb AM had significantly higher mango peach flavor, coconut water flavor, and
overall
liking compared to the control samples (at 95% confidence).
Experiment 3, which is further discussed below, explores the effect Reb AM on
taste &
flavor profile of a sweetened dairy product. A sensory panel tested samples of
stevia (Reb
A) sweetened, no-sugar-added chocolate flavored dairy protein shake without
(control) and
with Reb AM. The panel found the test sample containing 50 ppm of Reb AM to be
significantly lower bitterness, metallic note, whey protein and lower bitter
aftertaste than the
control (at 95% confidence) and higher in cocoa flavor, dairy notes, vanilla
flavor, and
overall liking (at 95% confidence).
A group of trained and experienced taste panel members evaluated no-calorie
Lemon-lime
carbonated soft drink (CSD) sweetened with 500 ppm of Reb AM, Reb D, or Reb M
samples. The panel members found the CSD with Reb AM is less sweet but has
significantly
less bitterness and sweetness lingering compared to other samples, especially
the CSD
sweetened with Reb M.
EXPERIMENT 1 OF EXAMPLE 10:
Sweetness Perception Threshold With Reb AM
Application: Neutral Water
The sweetness perception of 1.5% sugar solution and different solutions of Reb
AM were
tested with a sensory panel and found that 50 ppm of Reb AM solution in water
provided sweetness perception significantly lower than that of 1.5% sugar
solution.
Therefore we selected 50 ppm of Reb AM as the recognition threshold
concentration.
METHODOLOGY
Table 12
= Nature of Participants: Trained panel
= Number of Sessions 1
= Number of Participants: 30
= Test Design: 2- AFC, Balanced, randomized
within pair. Blind
= Sensory Test Method: Intensity ratings
= Environmental
Standard booth lighting
Condition
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= Attributes and Scales: Which sample is sweeter?
= Statistical Analysis: Paired comparison Test
= Sample Size ¨.1.5 oz. in a clear capped plastic
cup
= Serving Temperature Room temperature (-70 F)
= Serving/Panelists Samples served simultaneously.
Panelists instructed to read
Instruction: ingredient statement, evaluate each sample.
The following table (Table 13) shows an evaluation of the recognition
threshold
concentration to follow the methodology outlined in section 1.4.2 of the
"Guidance for the
Sensory Testing of Flavorings with Modifying Properties within the FEMA GRASTM
Program", issued by FEMA (Flavor and Extract Manufacturers Association
https://vvww.femaflavor.org/).
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Table 13
DATA: n=30
Two-Tailed Analysis Table Report for Result Reb AM Binomial Distribution
Probability
Percent
1.5% 30 ppm Frequency
Sucrose Reb AM Sample 1 P-value Sig
PC 29 1 96.7% 0.0001 ***
% Frequency 96.7% 3.3%
DATA: n=30
Two-Tailed Analysis Table Report for Result Reb AM Binomial
Distribution Probability
Percent
1.5% 50 ppm Frequency
Sucrose Reb AM Sample 1 P-value Sig
PC 23 7 76.7% 0.01 ***
% Frequency 76.7% 23.3%
DATA: n=30
Two-Tailed Analysis Table Report for Result IS03026A Binomial
Distribution Probability
Percent
1.5% 70 ppm of Frequency
Sucrose Reb AM Sample 1 P-value Sig
PC 9 21 30.0% 0.05 ***
% Frequency 30% 70%
DATA: n=30
Two-Tailed Analysis Table Report for Result Reb AM Binomial
Distribution Probability
Percent
1.5% 100 ppm Frequency
Sucrose Reb AM Sample 1 P-value Sig
PC 3 27 10% 0.0001 ***
% Frequency 10% 90%
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EXPERIMENT 2 OF EXAMPLE 10:
Raspberry Watermelon Coconut Water With Reb AM
Application: Non-alcoholic Beverage
SUMMARY
Thirty panel members evaluated two samples of raspberry watermelon flavored
coconut
water for overall acceptance and attribute intensities (sweetness, Raspberry
flavor,
watermelon flavor, coconut water flavor, saltiness, bitterness, and sweet
aftertaste, bitter
aftertaste) in two sessions. In session one, the two samples included: 1)
store-bought
Raspberry Watermelon Coconut Water control sample and 2) store-bought
Raspberry
Watermelon Coconut Water test sample containing Reb AM. The objective of the
test was
to determine if the addition of Reb AM affects the flavor profile of a non-
alcoholic
beverage. The results indicated the test sample Reb AM had significantly
higher mango
peach flavor, coconut water flavor, and overall liking compared to the control
samples (at
95% confidence).
OBJECTIVE
The project objective is to assess if the addition of stevia extract solids
has an effect on
key flavor attributes in various beverage applications.
TEST OBJECTIVE
The test objective is to determine if the flavor profile and overall
acceptance of a Control
sample of flavored coconut water differs from a Test sample of the same
beverage
containing Reb AM.
METHODOLOGY
Table 14
= Nature of Participants: Trained panel
= Number of Sessions 1
= Number of Participants: 30
= Test Design: Balanced, randomized within pair.
Blind
= Sensory Test Method: Intensity and acceptance
ratings
= Environmental Condition Standard booth lighting
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= Attributes and Scales:
= Overall Acceptance on a 10-pt hedonic scale where 10 = Extremely Like and
0 =
Extremely Dislike
= Overall liking, sweetness, raspberry flavor, watermelon flavor, coconut
water flavor,
astringency, artificial chemical note, bitterness, and sweet aftertaste,
bitter aftertaste.
10-pt continuous intensity scale where 0 = Imperceptible and 10 = Extremely
Pronounced
= Statistical Analysis: ANOVA (by Block) with
Post Hoc Duncan's Test
= Sample Size ¨1.5 oz. in a clear capped plastic
cup
= Serving Temperature Refrigerated temperature (-
45 F)
= Serving/Panelists Samples served
simultaneously. Panelists instructed to read
Instruction: ingredient statement, evaluate each sample.
SAMPLES
Table 15
Beverage Type I, Non-alcoholic
Reference Reb AM
*Coconut water raspberry watermelon juice 100 99.995
Reb AM 0.005
Total (g) 100 100
* Vita Coco store brand
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RESULTS
Table 16 (below) summarizes the overall acceptance and mean attribute
intensity results
for each sample.
Table 16: Mean Scores Raspberry Watermelon Coconut Water
with 50 ppm Reb AM
Summary of Mean-Scores, P-Values, and Significance
Test Result Code: Coconut Water (raspberry/watermelon flavor) Reb AM at 50 ppm
This test was performed on 30 panelists.
Coconut water
Coconut water
Attribute with 50 ppm of P-Value
Sig
control
Reb AM
Sweet Intensity 4.38 4.44 0.6990
NS
Bitter Intensity 0.32 0.24 0.4267
NS
Astringency 1.04 1.10 0.4942
NS
Coconut Flavor 4.89 5.11 0.4372
NS
a
Watermelon Flavor 3.85 4.41 0.0221
***
Raspberry Flavor 0.68 0.95 0.2423
NS
Artificial/Chemical Note 2.94 2.55 0.2583
NS
a
Sweet Aftertaste 1.60 1.33 0.0905
**
Bitter Aftertaste 0.36 0.29 0.5409
NS
a
Overall Liking 4.49 5.04 0.0710 **
*= 80% CI, ** = 90% CI, ***¨ 95%CI
The results indicate the test sample Reb AM had significantly higher
watermelon flavor and
overall liking compared to the control samples (at 95% confidence). Test
sample Reb AM
had significantly lower sweet aftertaste intensity compared to the control
samples (at 90%
confidence).
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CONCLUSION
Thirty panelists evaluated two samples of Raspberry Watermelon flavored
coconut water
for overall acceptance and attribute intensities (sweetness, watermelon
flavor, raspberry
flavor, coconut water flavor, astringency, artificial/chemical note,
bitterness, and sweet
aftertaste, bitter aftertaste) in two sessions. In session one, the two
samples included: 1)
store-bought Raspberry Watermelon Coconut Water control sample and 2) store-
bought
Raspberry Watermelon Coconut Water test sample containing Reb AM. The
objective of
the test was to determine if the addition of Reb AM affects the flavor profile
of a non-
alcoholic beverage. The results indicated the test sample Reb AM had
significantly higher
watermelon flavor and overall liking compared to the control samples (at 95%
confidence). A graph of the results is shown in FIG. 16.
Test sample Reb AM had significantly lower sweet aftertaste intensity compared
to the
control samples (at 90% confidence).
EXPERIMENT 3 OF EXAMPLE 10:
Chocolate Protein Shake With Reb AM
Application: Milk/Dairy Product
SUMMARY
Thirty trained panelists evaluated two samples of chocolate flavored dairy
protein shake
for overall acceptance and attribute intensities (cocoa flavor, dairy note,
whey protein,
vanilla, metallic, sweetness, bitterness and aftertaste). The two samples
included: 1) no
sugar added "Control" sample containing 300 ppm PureCircle Reb A and 2) no
sugar
added "Test" sample containing 300 ppm PureCircle Reb A and 50 ppm Reb AM. The
objective of the test was to determine if the addition of Reb AM affects the
flavor
profile of a milk product. The panel found the test sample containing 50 ppm
of Reb
AM to be significantly lower bitterness, metallic note, whey protein and lower
bitter
aftertaste than the control (at 95% confidence) and higher in cocoa flavor,
dairy notes,
vanilla flavor, and overall liking (at 95% confidence). Further, there was no
significant
impact on sweetness intensity.
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OBJECTIVE
The project objective is to assess if the addition of stevia extract solids
has an effect on
key flavor attributes in various beverage applications.
TEST OBJECTIVE
The test objective is to determine if the flavor profile and overall
acceptance of a control
sample of dairy beverage application differs from a Test sample of the same
beverage
containing Reb AM.
METHODOLOGY
Table 17
= Nature of Participants: Trained panel
= Number of Sessions 1
= Number of Participants: 30
= Test Design: Balanced, randomized within pair.
Blind
= Sensory Test Method: Intensity and acceptance
ratings
= Environmental Condition Standard booth lighting
= Attributes and Scales:
= Overall Acceptance on a 10-pt hedonic scale where 10 = Extremely Like and
0 =
Extremely Dislike
= Overall Liking, sweetness, bitterness, cocoa flavor, dairy notes,
chocolate, whey
protein notes, metallic note, vanilla note, and Aftertaste. 10-pt continuous
intensity
scale where 0 = Imperceptible and 10 = Extremely Pronounced
= Statistical Analysis: ANOVA (by Block) with Post
Hoc Duncan's Test
= Sample Size ¨1.5 oz. in a clear capped plastic
cup
= Serving Temperature Refrigerated temperature (-45
F)
= Serving/Panelists Samples served simultaneously.
Panelists instructed to read
Instruction: ingredient statement, evaluate each sample.
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SAMPLES
Table 18
Ingredient list Sugar 50 ppm Reb
Reference AM
Milk, 2% 86.47 86.465
Whey Protein 90 Instant - Non GMO (Prod: 18618) 6.8250 6.8250
Non-Fat Dry Milk 4.6269 4.6269
Maltrin QD M585 1.1066 1.1066
Vitamin Blend - 0.0063 0.0063
Xanthan Gum (Cold dissolve) 0.0359 0.0359
Forbes 10/12 Cocoa powder 7113 0.7194 0.7194
Vanilla Flavor Powder 0.1799 0.1799
Reb A 0.0300 0.0300
Reb AM 0.0050
TOTAL 100 100
Sugar Contribution (grams) per 100 grams* Sugar 165 ppm
Reference Reb AM
Milk, 2% 4.08 4.15
Non-Fat Dry Milk 2.41 2.41
Maltrin QD M585 0.08 0.08
TOTAL 8.07 6.64
* Calculated with Genesis R&D version 11.4
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Table 19: Effect Reb AM on flavor modification of Chocolate Protein shake
Summary of Mean-Scores, P-Values, and Significance
Test Result Code: PROTEIN6
Test Description: Chocolate Vanilla Protein Dairy Shake: 50 ppm Reb AM
This test was performed on 30 panelists.
Control - NSA Test - NSA Protein
Attribute Protein Shake w/ Shake w Reb A &
P-Value Sig
Reb A 50 ppm Reb AM
Sweet Intensity 6.04 5.98 0.7329 NS
a
Bitterness 1.98 1.46 0.0138 ***
a
Metallic Note 1.93 1.48 0.0311 ***
a
Cocoa Flavor 4.06 4.55 0.0409 ***
a
Dairy Note 4.10 4.59 0.0515 **
a
Whey Protein Note 4.79 4.32 0.0460 ***
a
Vanilla Note 2.10 2.52 0.0174 ***
Sweet Aftertaste 1.82 1.65 0.2130 NS
a
Bitter Aftertaste 1.03 0.77 0.0495 ***
a
Overall Liking 4.80 5.59 0.0001 ***
*= 80% Cl, "" = 90% Cl, *"*= 95%Cl
The panel found the test sample containing 50 ppm of Reb AM to be
significantly lower
bitterness, metallic note, whey protein and lower bitter aftertaste than the
control (at 95%
confidence).
The panel found the test sample containing 50 ppm of Reb AM to be
significantly higher
in cocoa flavor, dairy notes, vanilla flavor, and overall liking (at 95%
confidence).
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CONCLUSION
Thirty panelists evaluated two samples of chocolate flavored dairy protein
shake for
overall acceptance and attribute intensities (cocoa flavor, dairy note, whey
protein,
vanilla, metallic, sweetness, bitterness and aftertaste). The two samples
included: 1) no
sugar added "Control" sample containing 300 ppm PureCircle Reb A and 2) no
sugar
added "Test" sample containing 300 ppm PureCircle Reb A and 50 ppm Reb AM. The
objective of the test was to determine if the addition of Reb AM affects the
flavor
profile of a milk product. The panel found the test sample containing 50 ppm
of Reb
AM to be significantly lower bitterness, metallic note, whey protein and lower
bitter
aftertaste than the control (at 95% confidence) and higher in cocoa flavor,
dairy notes,
vanilla flavor, and overall liking (at 95% confidence), Further, there was no
significant
impact on sweetness intensity. A graph of the results is shown in FIG. 17.
Although the invention and its advantages have been described in detail, it
should be
understood that various changes, substitutions and alterations can be made
herein without
departing from the spirit and scope of the invention as defined by the
appended claims.
Moreover, the scope of the application is not intended to be limited to the
particular
embodiments of the invention described in the specification. As one of
ordinary skill in
the art will readily appreciate from the disclosure of the invention, the
compositions,
processes, methods, and steps, presently existing or later to be developed
that perform
substantially the same function or achieve substantially the same result as
the
corresponding embodiments described herein may be utilized according to the
invention.
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