U.S. patent application number 12/669393 was filed with the patent office on 2010-12-09 for mono- or bicyclic carboxylic acids as off-note blockers.
This patent application is currently assigned to GIVAUDAN SA. Invention is credited to Nicole Erna Irene Brune, Jenny Ellen Evans Pennimpede, Kimberley Gray, Christopher Todd Simons, Jay Patrick Slack, Ioana Maria Ungureanu.
Application Number | 20100311786 12/669393 |
Document ID | / |
Family ID | 40175106 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100311786 |
Kind Code |
A1 |
Ungureanu; Ioana Maria ; et
al. |
December 9, 2010 |
MONO- OR BICYCLIC CARBOXYLIC ACIDS AS OFF-NOTE BLOCKERS
Abstract
Disclosed are compounds that block off-notes in consumables and
methods of blocking off-notes in consumables including off-notes
provided by sweeteners such as stevioside, swingle extract,
glyccerhizin, perillartine, naringin dihydrochalcone,
neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus
extract, and rebaudioside A, and artificial sweeteners such as
aspartame, saccharin, acesulfame K (Acesulfame potassium),
sucralose and cyclamate.
Inventors: |
Ungureanu; Ioana Maria;
(Cincinnati, OH) ; Brune; Nicole Erna Irene; (Dan
Diego, CA) ; Slack; Jay Patrick; (Loveland, OH)
; Gray; Kimberley; (Loveland, OH) ; Simons;
Christopher Todd; (Wyoming, OH) ; Evans Pennimpede;
Jenny Ellen; (Cincinnati, OH) |
Correspondence
Address: |
CURATOLO SIDOTI CO., LPA
24500 CENTER RIDGE ROAD, SUITE 280
CLEVELAND
OH
44145
US
|
Assignee: |
; GIVAUDAN SA
Vernier
CH
|
Family ID: |
40175106 |
Appl. No.: |
12/669393 |
Filed: |
July 28, 2008 |
PCT Filed: |
July 28, 2008 |
PCT NO: |
PCT/CH2008/000332 |
371 Date: |
January 15, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60962514 |
Jul 30, 2007 |
|
|
|
Current U.S.
Class: |
514/289 ; 426/3;
426/442; 426/538; 426/72; 514/327; 514/784; 562/400; 562/503 |
Current CPC
Class: |
A61P 29/00 20180101;
A23L 27/30 20160801; A23L 27/84 20160801; A23L 27/86 20160801; A61P
11/14 20180101; A23V 2002/00 20130101 |
Class at
Publication: |
514/289 ;
562/503; 562/400; 514/784; 514/327; 426/538; 426/3; 426/72;
426/442 |
International
Class: |
A61K 31/4748 20060101
A61K031/4748; C07C 61/06 20060101 C07C061/06; C07C 61/08 20060101
C07C061/08; A61K 47/12 20060101 A61K047/12; A61K 31/445 20060101
A61K031/445; A61P 11/14 20060101 A61P011/14; A61P 29/00 20060101
A61P029/00; A23L 1/226 20060101 A23L001/226; A23G 4/06 20060101
A23G004/06; A23L 1/302 20060101 A23L001/302 |
Claims
1. An off-note blocking compound of Formula (I): ##STR00009##
wherein n is 0 or 1; wherein the ring residue together with the
dashed lines comprises a cyclopentane residue (for n=0), a
cyclohexane residue (for n=1), or a bicyclo[2.2.2]octane residue
(with the dashed lines forming single bonds); wherein for the
cyclopentane and cyclohexane ring residues, R1 comprises a C3 to
C10 alkane or arylalkane residue: wherein for the
bicyclo[2.2.2]octane residue. R1 comprises a C2 to C10 alkane or
arylalkane residue; and wherein the number of carbon atoms in the
residues of R1 and R2 together maximally is 12.
2. The off-note blocking compound of claim 1, wherein said R1
alkane or arylalkane residues is selected from propyl, isopropyl,
butyl, isobutyl, tertiary butyl, pentyl, isopentyl, hexyl,
isohexyl, 2-methyl-hexyl, heptyl, isoheptyl, 2-methyl-heptyl,
octyl, isooctyl, 2-methyl-octyl, nonyl, iononyl, 2-methyl-nonyl,
decyl, isodecyl, 2-methyl-decyl, phenyl, tolyl and benzyl; and
wherein the optional ring substituents R2, R2', R2'' and R2''' are
each independently selected from H and methyl, or alternatively,
instead of two of the methyl substituents, one cyclopropyl residue
formed of two of the methyl residues R2-R2''' and a ring atom is
present, or alternatively, instead of one of the methyl residues
R2-R2''', a cyclopropyl residue formed of one of the methyl
residues R2-R2''' and two ring carbon atoms is present.
3. The off-note blocking compound of claim 1, comprising one or
more of cis-4-propylcyclohexanecarboxylic acid,
cis-4-tert-butylcyclohexanecarboxylic acid,
cis-4-propylbicyclo[2.2.2]octane-1-carboxylic acid,
cis-4-sec-butylcyclohexanecarboxylic acid,
cis-4-(4-methylpentyl)cyclohexanecarboxylic acid,
cis-4-benzylcyclohexanecarboxylic acid, or
cis-4-phenylcyclohexanecarboxylic acid.
4. A flavor composition comprising an off-note providing consumable
ingredient and one or more off-note blocking compounds as defined
in claim 1.
5. A consumable comprising a) one or more ingredient in a
concentration sufficient to provide an off-note. b) one or more
off-note blocking compound as defined in claim 1.
6. The consumable of claim 5 wherein the one or more off-note
providing ingredient comprises one or more sweetener, artificial
sweetener, beverage, chewing gum, nutraceutical, or
pharmaceutical.
7. The consumable of claim 5 wherein the one or more off-note
providing ingredient comprises one or more artificial sweetener
selected from aspartame, acesulfame K, saccharin, sucralose, or
sodium cyclamate.
8. The consumable of claim 5 wherein the one or more off-note
providing ingredient comprises one or more sweetener selected from
stevioside, swingle extract, glyccerhizin, perillartine, naringin
dihydrochalcone, neohesperidine dihydrochalcone, mogroside V,
rubusoside, rubus extract, or rebaudioside A.
9. The consumable of claim 5 wherein the one or more off-note
providing ingredient comprises a consumable selected from the group
consisting of cocoa, coffee, caffeine, theobromine,
diketopiperazines, vitamins, amino acids, vitamin B, casein, soy
protein, ibuprofen, salicylic acid, glucoronolactone,
acetaminophen, dextromethorphan, naringin, taurin, macrolide,
bioxin, erythromycin, paracetamol, acetylsalicylic acid,
cimetidine, ranitidine, amoxicillin, acetominophen,
cephalosporines, quassia, propylene glycol, triacetin, salts of
potassium, salts of zinc, loperamide, limonin, flavonoides,
isoflavones, genistein diadzein, polyphenol, catechin, epicatechin,
mint oil, D-menthol, hydrolysed vegetable protein, bitter peptides,
preservatives, benzoic acid, potassium sorbate, polysorbate 80,
sodium lactate, potassium lactate, sodium benzoate, citric acid,
quinine, urea, essential oils, thyme, sage, basil, mint, Maillard
reaction products, cyclic amines made from pyrrolidine/glucose,
alanine/xylose, proline/sucrose or alanine/xylose,
diketopiperazines, beer, hops, humulone, trans-isohumulone,
lupulone, and hulupone.
10. A method of blocking off-notes in a consumable comprising
admixing with the consumable a) one or more off-note providing
ingredient in a concentration sufficient to provide an off-note,
and b) one or more off-note blocking compound as defined in claim
1.
11. The method of claim 10 wherein the one or more off-note
providing ingredient comprises one or more sweetener, artificial
sweetener, beverage, chewing gum, nutraceutical, or
pharmaceutical.
12. The method of claim 10 wherein the one or more off-note
providing ingredient comprises one or more artificial sweetener
selected from aspartame, acesulfame K, saccharin, sucralose, or
sodium cyclamate.
13. The method of claim 10 wherein the one or more off-note
providing ingredient comprises one or more sweetener selected from
stevioside, swingle extract, glyccerhizin, perillartine, naringin
dihydrochalcone, neohesperidine dihydrochalcone, mogroside V,
rubusoside, rubus extract, or rebaudioside A.
14. The method of claim 10 wherein the one or more off-note
providing ingredient comprises a consumable selected from the group
consisting of cocoa, coffee, caffeine, theobromine,
diketopiperazines, vitamins, amino acids, vitamin B, casein, soy
protein, ibuprofen, salicylic acid, glucoronolactone,
acetaminophen, dextromethorphan, naringin, taurin, macrolide,
bioxin, erythromycin, paracetamol, acetolsalicilic acid,
cimetidine, ranitidine, amoxicillin, acetominophen,
cephalosporines, quassia, propylene glycol, triacetin, salts of
potassium, salts of zinc, loperamide, limonin, flavonoides,
isoflavones, genistein, diadzein, polyphenol, catechin,
epicatechin, mint oil, D-menthol, hydrolysed vegetable protein,
bitter peptides, preservatives, benzoic acid, potassium sorbate,
polysorbate 80, sodium lactate, and potassium lactate, sodium
benzoate, citric acid, quinine, urea, essential oils, thyme, sage,
basil, mint, Maillard reaction products, cyclic amines made from
pyrrolidine/glucose, alanine/xylose, proline/sucrose or
alanine/xylose, diketopiperazines, beer, hops, humulone,
trans-isohumulone, lupulone, and hulupone.
Description
TECHNICAL FIELD
[0001] Disclosed are compounds that allow to mask or block
undesirable off-notes in consumables and the method of blocking
off-notes employing said compounds in consumables.
SUMMARY
[0002] Provided are the following:
(1) An off-note blocking compound of Formula (I)
##STR00001##
wherein n is 0 or 1 (thereby forming cyclopentane or cyclohexane);
wherein the ring residue together with the dashed lines is selected
from a cyclopentane residue (for n=0), a cyclohexane residue (for
n=1), or a bicyclo[2.2.2]octane residue (with the dashed lines
forming single bonds); wherein for the cyclopentane and cyclohexane
ring residues, R1 comprise a C3 to C10 alkane or arylalkane
residue; wherein for the bicyclo[2.2.2]octane residue, R1 comprises
a C2 to C10 alkane or arylalkane residue; wherein said R1 alkane or
arylalkane residues are optionally selected from propyl, isopropyl,
butyl, isobutyl, tertiary butyl, pentyl, isopentyl, hexyl,
isohexyl, 2-methyl-hexyl, heptyl, isoheptyl, 2-methyl-heptyl,
octyl, isooctyl, 2-methyl-octyl, nonyl, isononyl, 2-methyl-nonyl,
decyl, isodecyl, 2-methyl-decyl, phenyl, tolyl and benzyl; wherein
the number of carbon atoms in the residues of R1 and R2 together
maximally is 12; and wherein the optional ring substituents R2,
R2', R2'' and R2''' are each independently selected from H and
methyl, or alternatively, instead of two of the methyl
substituents, one cyclopropyl residue formed of two of the methyl
residues R2-R2''' and a ring atom is present, or alternatively,
instead of one of the methyl residues R2-R2''', a cyclopropyl
residue formed of one of the methyl residues R2-R2''' and two ring
carbon atoms may be present. (2) The off-note blocking compound as
described herein selected from one or more of
cis-4-propylcyclohexanecarboxylic acid,
cis-4-tert-butylcyclohexanecarboxylic acid,
cis-4-propylbicyclo[2.2.2]octane-1-carboxylic acid,
cis-4-sec-butylcyclohexanecarboxylic acid,
cis-4-(4-methylpentyl)cyclohexanecarboxylic acid,
cis-4-benzylcyclohexanecarboxylic acid, and
cis-4-phenylcyclohexanecarboxylic acid. (3) A flavor composition
comprising an off-note providing consumable ingredient and one or
more off-note blocking compounds of Formula (I). (4) A consumable
comprising [0003] a) at least one ingredient in a concentration
sufficient to provide an off-note, [0004] b) one or more compounds
off-note blocking compounds of Formula (I). (5) A consumable as
herein described wherein the off-note providing ingredient
comprising one of sweeteners, artificial sweeteners, beverages,
chewing gums, nutraceuticals, and pharmaceuticals. (6) A consumable
of as herein described wherein the off-note providing ingredient
comprises one or more artificial sweetener selected from aspartame,
acesulfame K, saccharin, sucralose, and sodium cyclamate. (7) A
consumable as herein described wherein the off-note providing
ingredient comprises one or more sweeteners selected from
stevioside, swingle extract, glyccerhizin, perillartine, naringin
dihydrochalcone, neohesperidine dihydrochalcone, mogroside V,
rubusoside, rubus extract, and rebaudioside A (8) A consumable as
herein described wherein the off-note providing ingredient
comprises a consumable selected from the group consisting of cocoa,
coffee, caffeine, theobromine, diketopiperazines, vitamins, amino
acids, vitamin B, casein, soy protein, ibuprofen, salicylic acid,
glucoronolactone, acetaminophen, dextromethorphan, naringin,
taurin, macrolide (including bioxin and erythomycin), paracetamol,
acetylsalicylic acid, cimetidine, ranitidine, amoxicillin,
acetominophen, cephalosporines, quassia, propylene glycol,
triacetin, salts of potassium, salts of zinc, loperamide, limonin,
flavonoides, isoflavones (including genistein and diadzein),
polyphenol (including catechin and epicatechin), mint oil,
D-menthol, hydrolysed vegetable protein, bitter peptides,
preservatives (including benzoic acid, potassium sorbate,
polysorbate 80, sodium and potassium lactate, sodium benzoate),
citric acid, quinine, urea (contained in chewing gums), essential
oils (including thyme, sage, basil, mint), Maillard reaction
products (including cyclic amines made from pyrrolidine/glucose,
alanine/xylose, proline/sucrose or alanine/xylose, for example
diketopiperazines), beer, hops, humulone, trans-isohumulone,
lupulone, and hulupone. (9) A method of blocking off-notes in
consumables comprising admixing with the consumable (a) at least
one off-note providing ingredient in a concentration sufficient to
provide an off-note, and (b) one or more off-note blocking
compounds of Formula (I). (10) A method as herein described wherein
the off-note providing ingredient comprises selected one of
sweeteners, artificial sweeteners, beverages, chewing gums,
nutraceuticals, and pharmaceuticals. (11) A method as herein
described wherein the off-note providing ingredient comprises one
or more artificial sweetener selected from aspartame, acesulfame K,
saccharin, sucralose, and sodium cyclamate. (12) A method of as
herein described wherein the off-note providing ingredient
comprises one or more sweetener selected from stevioside, swingle
extract, glyccerhizin, perillartine, naringin dihydrochalcone,
neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus
extract, and rebaudioside A. (13) A method as herein described
wherein the off-note providing ingredient comprises a consumable
selected from the group consisting of cocoa, coffee, caffeine,
theobromine, diketopiperazines, vitamins, amino acids, vitamin B,
casein, soy protein, ibuprofen, salicylic acid, glucoronolactone,
acetaminophen, dextromethorphan, naringin, taurin, macrolide
(including bioxin and erythomycin), paracetamol, acetolsalicilic
acid, cimetidine, ranitidine, amoxicillin, acetominophen,
cephalosporines, quassia, propylene glycol, triacetin, salts of
potassium, salts of zinc, loperamide, limonin, flavonoides,
isoflavones (including genistein and diadzein), polyphenol
(including catechin and epicatechin), mint oil, D-menthol,
hydrolysed vegetable protein, bitter peptides, preservatives
(including benzoic acid, potassium sorbate, polysorbate 80, sodium
and potassium lactate, sodium benzoate), citric acid, quinine, urea
(contained in chewing gums), essential oils (including thyme, sage,
basil, mint), Maillard reaction products (including cyclic amines
made from pyrrolidine/glucose, alanine/xylose, proline/sucrose or
alanine/xylose, for example diketopiperazines), beer, hops,
humulone, trans-isohumulone, lupulone, and hulupone.
DETAILED DESCRIPTION
[0005] The off-note blocking compound of Formula (I) provided
herein include, without limitation, the following off-note blocking
compounds: cis-4-propylcyclohexanecarboxylic acid,
cis-4-tert-butylcyclohexanecarboxylic acid,
cis-4-propylbicyclo[2.2.2]octane-1-carboxylic acid,
cis-4-sec-butylcyclohexanecarboxylic acid,
cis-4-(4-methylpentyl)cyclohexanecarboxylic acid,
cis-4-benzylcyclohexanecarboxylic acid, and
cis-4-phenylcyclohexanecarboxylic acid.
[0006] Their chemical structures are indicated below.
TABLE-US-00001 Structure Name ##STR00002##
cis-4-propylcyclohexanecarboxylic acid ##STR00003##
cis-4-tert-butylcyclohexanecarboxylic acid ##STR00004##
cis-4-propylbicyclo[2.2.2]octane-1-carboxylic acid ##STR00005##
cis-4-sec-butylcyclohexanecarboxylic acid ##STR00006##
cis-4-(4-methylpentyl)cyclohexanecarboxylic acid ##STR00007##
cis-4-benzylcyclohexanecarboxylic acid ##STR00008##
cis-4-phenylcyclohexanecarboxylic acid
[0007] cis-4-propylcyclohexanecarboxylic acid,
cis-4-tert-butylcyclohexanecarboxylic acid,
cis-4-propylbicyclo[2.2.2]octane-1-carboxylic acid,
cis-4-sec-butylcyclohexanecarboxylic acid,
cis-4-(4-methylpentyl)cyclohexanecarboxylic acid,
cis-4-benzylcyclohexanecarboxylic acid, and
cis-4-phenylcyclohexanecarboxylic acid are either available
commercially or can be synthesized as will be apparent to the
skilled person.
[0008] cis-4-propylcyclohexanecarboxylic acid,
cis-4-tert-butylcyclohexanecarboxylic acid and
cis-4-sec-butylcyclohexanecarboxylic acid can be synthesized by
hydrogenation of corresponding benzoic acid as will be apparent to
the skilled person. cis-4-tert-butylcyclohexanecarboxylic acid also
is commercially available from TCI America, Portland, Oreg.,
USA.
[0009] cis-4-propylbicyclo[2.2.2]octane-1-carboxylic acid is
commercially available from AsInEx, Moscow, Russia.
[0010] cis-4-(4-methylpentyl)cyclohexanecarboxylic acid is
commercially available from Sigma-Aldrich, St. Louis, Mo., USA.
[0011] cis-4-benzylcyclohexanecarboxylic acid can be synthesized by
reducing cis-4-benzoylcyclohexane-1-carboxylic acid, and this
starting material is available from Rieke Metals, Inc, Lincoln,
Nebr., USA.
[0012] cis-4-phenylcyclohexanecarboxylic acid commercially
available from ChemBridge, San Diego, Calif., USA.
[0013] All compounds of Formula (I) are either available
commercially or can be synthesized easily by methods well known in
the art, in particular hydrogenation, or as indicated herein.
[0014] A taste receptor screen and concentration-response analysis
was performed and from results inhibitory concentration (IC)
IC.sub.50 values can be calculated by nonlinear regression using
the function f(x)=(a-d)/(1+(x/C).sup.nh)+d; with a=minimum signal,
d=maximum signal, nh=hill coefficient, C=IC.sub.50 and
x=concentration of antagonist. IC.sub.50 is the molar concentration
of an antagonist which produces 50% of the maximum possible
inhibitory response for that antagonist. A more potent antagonist
will have a lower IC.sub.50 value.
[0015] Most of the off-note blocking compounds disclosed herein,
and in particular the compounds listed below, have an IC.sub.50 in
the range of about 0.1 to about 20 micromolar when tested with the
TAS2R44 bitter taste receptor: cis-4-propylcyclohexanecarboxylic
acid, cis-4-tert-butylcyclohexanecarboxylic acid,
cis-4-propylbicyclo[2.2.2]octane-1-carboxylic acid,
cis-4-sec-butylcyclohexanecarboxylic acid,
cis-4-(4-methylpentyl)cyclohexanecarboxylic acid,
cis-4-benzylcyclohexanecarboxylic acid, and
cis-4-phenylcyclohexanecarboxylic acid
[0016] For most food applications, a low IC.sub.50 [micro molar] of
about 0.05 to about 10 is desirable, however, IC.sub.50 of about 10
to about 25 are still good and above about 25 may also still
acceptable depending on the application.
[0017] Various food ingredients (including ingredients naturally
contained in food or additives admixed to food including flavor
ingredients) provide undesirable taste notes, so-called off-notes.
Particularly undesirable off-notes are the bitter off-notes,
metallic off-notes, lingering, licorice-type and astringent
off-notes. The term off-note refers to an unpleasant after taste
that develops over time after consumption of consumables.
[0018] Other particular examples are the bitter and/or metallic
and/or astringent and/or "artificial" off-notes and/or a cloyingly
sweet off-note (as opposed to the "cleaner" taste of sugar) that
are associated with a number of artificial sweeteners including
aspartame, acesulfame K, saccharin, sucralose, and sodium
cyclamate. Sometimes these off-notes of artificial sweeteners are
described collectively as bitter off-notes.
[0019] Further examples of off-note providing ingredients are
naturally occurring sweeteners including stevioside, swingle
extract, glyccerhizin, perillartine, naringin dihydrochalcone,
neohesperidine dihydrochalcone, mogroside V, rubusoside, rubus
extract, and rebaudioside A
[0020] Still further examples of off-note providing ingredients
include cocoa, coffee, caffeine, theobromine, diketopiperazines,
vitamins, amino acids, vitamin B, casein, soy protein, ibuprofen,
salicylic acid, glucoronolactone, acetaminophen, dextromethorphan,
naringin, taurin, macrolide (including bioxin and erythomycin),
paracetamol, acetolsalicilic acid, cimetidine, ranitidine,
amoxicillin, acetominophen, cephalosporines, quassia, propylene
glycol, triacetin, salts of potassium, salts of zinc, loperamide,
limonin, flavonoides, isoflavones (including genistein and
diadzein), polyphenol (including catechin and epicatechin), mint
oil, D-menthol, hydrolysed vegetable protein, bitter peptides,
preservatives (including benzoic acid, potassium sorbate,
polysorbate 80, sodium and potassium lactate, sodium benzoate),
citric acid, quinine, urea (contained in chewing gums), essential
oils (including thyme, sage, basil, mint), Maillard reaction
products (including cyclic amines made from pyrrolidine/glucose,
alanine/xylose, proline/sucrose or alanine/xylose, for example
diketopiperazines), beer, hops, humulone, trans-isohumulone,
lupulone, hulupone.
[0021] The addition of off-note blockers will block or mask the
off-notes and make them less apparent or unnoticeable. Artificial
sweeteners will thereby lose their bitter/metallic taste and/or
their cloyingly sweet lingering sweetness and instead taste more
like actual sugar (sucrose).
[0022] Aspartame is the name for aspartyl-phenylalanine-1-methyl
ester, a dipeptide. It is known under various trademark names
including Equal.RTM., and Canderel.RTM.. In the European Union, it
is also known under the E number (additive code) E951.
[0023] Acesulfame potassium (AceK) is the potassium salt of
6-methyl-1,2,3-oxathiazine-4(3H)-one 2,2-dioxide, an
N-sulfonylamide. It is also known as Acesulfame K or AceK, or under
various trademark names including Sunett.RTM. and Sweet One.RTM..
In the European Union it is also known under the E number (additive
code) E950.
[0024] Saccharin is the Na salt of 1,2-Benzisothiazol-3(2H)-one,
1,1-dioxide, an N-sulfonamide. It is also known under various
trademark names including Sweet'n Low.RTM..
[0025] Sucralose is the name for
6-dichloro-1,6-dideoxy-.beta.-D-fructo-furanosyl
4-chloro-4-deoxy-.alpha.-D-galactopyranoside, which is a
chlorodeoxysugar. It is also known by the trade name Splenda.RTM..
In the European Union, it is also known under the E number
(additive code) E955. Sucralose has an off-note (also designated
"aftertaste") that is a lingering liquorice-like off-note sometimes
also described as bitter.
[0026] The off-note blockers can be added to consumables to block
the undesirable off-notes of ingredients present in said
consumables or added to such consumables.
[0027] Flavor compositions for addition to consumables can be
formed that provide the off-note blockers and an off-note providing
ingredient for addition to consumables, and optionally food grade
excipients. Alternatively, the off note blockers can be directly
added to consumables.
[0028] In particular, the off-note blockers can be added to flavor
compositions or directly to consumables to block the undesirable
off-notes of off-note providing ingredients including natural and
artificial sweeteners added to such consumables.
[0029] Consumables include all food products, food additives,
nutraceuticals, pharmaceuticals and any product placed in the mouth
including chewing gum, oral care products, and oral hygiene
products including but not limited to, cereal products, rice
products, tapioca products, sago products, baker's products,
biscuit products, pastry products, bread products, confectionery
products, desert products, gums, chewing gums, mouthwash, dental
floss, flavored or flavor-coated straws, flavor or flavor-coated
food/beverage containers, chocolates, ices, honey products, treacle
products, yeast products, baking-powder, salt and spice products,
savory products, mustard products, vinegar products, sauces
(condiments), tobacco products, cigars, cigarettes, processed
foods, cooked fruits and vegetable products, meat and meat
products, jellies, jams, fruit sauces, egg products, milk and dairy
products, yoghurts, cheese products, butter and butter substitute
products, milk substitute products, soy products, edible oils and
fat products, medicaments, beverages, carbonated beverages,
alcoholic drinks, beers, soft drinks, mineral and aerated waters
and other non-alcoholic drinks, fruit drinks, fruit juices, coffee,
artificial coffee, tea, cocoa, including forms requiring
reconstitution, food extracts, plant extracts, meat extracts,
condiments, nutraceuticals, gelatins, pharmaceutical and
non-pharmaceutical gums, tablets, lozenges, drops, emulsions,
elixirs, syrups and other preparations for making beverages, and
combinations thereof.
[0030] For example, in consumables containing salts of potassium,
the off-note blocker may be added to suppress the bitterness and
the metallic off-note associated with salts of potassium.
[0031] In coffee and cocoa products, the off-note blocker may be
added to suppress the bitterness associated with caffeine,
theobromine, and/or diketopiperazines present in said products.
[0032] In cheese products, in particular in enzyme-modified cheese
products, the off-note blocker may be added to suppress the
bitterness associated with bitter peptides present in said cheese
products.
[0033] In soy products, the off-note blocker may be added to
suppress the bitterness and beany off-notes associated with
peptides, isoflavones such as genistein and diadzein present in
said products.
[0034] In HVP (hydrolysed vegetable protein) products, the off-note
blocker may be added to suppress the bitterness associated with
bitter peptides present in said products.
[0035] In functional ingredients used in fortified foods, the
off-note blocker may be added to suppress the bitterness associated
with vitamins and amino acids present in said products.
[0036] In pharmaceuticals, the off-note blocker may be added to
suppress the bitterness associated with actives or bitter additives
present in said products.
[0037] In consumables containing solvents, the off-note blocker may
be added to suppress the bitterness associated with propylene
glycol, triacetin, or ethanol present in said products.
[0038] In citrus products, the off-note blocker may be added to
suppress the bitterness associated with naringin present in said
products.
[0039] In neutraceuticals and herb medicines, the off-note blocker
may be added to suppress the bitterness associated with actives or
additives present in said products.
[0040] In consumables containing polyphenols such as catechin and
epicatechin, the off-note blocker may be added to suppress the
bitterness associated with these ingredients.
[0041] In consumables containing preservatives such as potassium
sorbate, polysorbate 80, sodium and potassium lactate, sodium
benzoate, the off-note blocker may be added to suppress the
bitterness associated with said preservatives.
[0042] In consumables containing zinc and other mineral
supplements, the off-note blocker may be added to suppress the
bitterness and metallic off-notes associated with said these
mineral supplements.
[0043] In consumables containing mint oil or menthol (e.g.
D-menthol) and citric acid of above 7%, the off-note blocker may be
added to suppress the bitterness associated with this combination
of ingredients.
[0044] In consumables containing quinine, the off-note blocker may
be added to suppress the bitterness associated with quinine.
[0045] In consumables containing artificial sweeteners (e.g.
aspartame, saccharin, acesulfame K, sucralose, cyclamate), for
example beverages, a off-note blocker may be added to suppress the
bitterness associated with artificial sweeteners.
[0046] In chewing gums, particular dental-type chewing gums, the
off-note blocker may be added to suppress the bitterness associated
with urea contained in chewing gums.
[0047] In consumables containing essential oils (e.g. thyme, sage,
basil, mint), the off-note blocker may be added to suppress the
bitterness associated with these essential oils.
[0048] In consumables containing vegetables or herbs or their
extracts, the off-note blocker may be added to suppress the
bitterness associated with these ingredients.
[0049] In consumables containing Maillard reaction products (i.e.
cyclic amines made from proline/sucrose or alanine/xylose, e.g.
diketopiperazines), the off-note blocker may be added to suppress
the bitterness associated with Maillard reaction products.
[0050] In beer and consumables containing beer or hops, the
off-note blocker may be added to suppress the bitterness associated
with hops.
EXAMPLES
[0051] The following examples are set forth to describe the
off-note blocking compounds in further detail and to illustrate the
methods of employing the off-note blocking compounds to block or
otherwise mask off-notes in consumables. The examples are
illustrative and should not be construed as limiting the compounds,
consumables or methods in any manner.
Example 1
Sensorial Evaluation in Various Consumables
[0052] Off-note blockers as herein described are tested by panels
of 6 to 10 bitter sensitive panelists.
[0053] Panelists are asked to describe the differences in off-notes
and bitter notes between the product with 0.001% (wt/wt) off-note
blocker unless otherwise stated and a control without off-note
blocker.
A) Aspartame/Acesulfame-K Containing Diet Energy Drink
[0054] The diet energy drink contained taurin, acesulfame K,
aspartame, sucralose, glucuronolacton, caffeine. B-group vitamins
(Niacin, pantothenic acid, B6, B12), aroma, sucrose, glucose,
colours
[0055] The sample containing the off-note blocker is found to be
less bitter compared to the control.
B) Sucrose/Glucose-Sweetened Energy Drink
[0056] The diet energy drink contains taurin, glucuronolacton,
caffeine, B-group vitamins (Niacin, pantothenic acid, B6, B12),
aroma, sucrose, glucose, colours.
[0057] The sample containing the off-note blocker is found to have
less off-notes, to be less bitter, and less astringent compared to
the control.
C) Iced Low-Sugar Coffee
[0058] The sample containing the off-note blocker is found to be
less bitter, and less astringent compared to the control.
D) Commercial Vanilla Flavored Nutritional Drink
[0059] Vanilla flavored nutritional drink containing calcium
caseinate, soy protein isolate, sodium caseinate, vitamins and
minerals.
[0060] The sample containing the off-note blacker is found to be
less chalky, to have reduced protein/vitamin induced off-notes
notes, and to be less astringent compared to control.
E) Saccharin Sweetened Cola Soft Drink
[0061] The sample containing the off-note blocker is found to be
less bitter and to have a reduced after taste compared to the
control.
F) Loperamide Containing Mint-Flavored Pharmaceutical Syrup
[0062] The syrup contained 1 mg loperamide HCl per 7.5 ml serving.
The off-note blocker is used in a concentration of 0.004%
(wt/wt).
[0063] The sample containing the off-note blocker is found to be
less bitter with especially the lingering bitter after taste
reduced.
G) Daytime Cough Syrup
[0064] The daytime cough syrup contains 325 mg acetaminophen, 10 mg
dextromethorphan HBr, 5 mg phenylephrine HCl per 15 ml serving.
[0065] The sample containing the off-note blocker is found to be
less bitter.
H) Dark Chocolate
[0066] The sample containing the off-note blocker is found to be
less bitter.
I) Baking Chocolate (100% Cocoa, Unsweetened)
[0067] The off-note blocker is used in a concentration of 0.002%
(wt/wt).
[0068] The sample containing the off-note blocker is found to be
less metallic, less bitter, especially the alkaloid/caffeine-like
bitterness is reduced while the upfront, warm, woody bitterness was
retained.
J) Overcooked Coffee
[0069] Coffee is brewed and cooked on a burner for 3 hours. The
off-note blocker is used in a concentration of 0.0005% (wt/wt).
[0070] The sample containing the off-note blocker is found to be
less bitter.
K) Aspartame/Acesulfame-K Sweetened Plain Nonfat Yogurt
[0071] The yogurt contained 0.0193% (wt/wt) and acesulfame-K
0.0083% (wt/wt). The off-note blocker is used in a concentration of
0.00175% (wt/wt).
[0072] The sample containing the off-note blocker is found to have
less off-notes compared to control.
L) Aspartame/Acesulfame-K Sweetened Cola Soft Drink
[0073] The off-note blocker is used in a concentration of 0.0063%
(wt/wt).
TABLE-US-00002 Cola soft drink % (by weight) Sodium Benzoate 0.026
Aspartame 0.043 Acesulfame-K 0.017 Caffeine 0.011 Phosphoric Acid
(85%) 0.043 Citric Acid (50% cut in water) 0.017 Caramel Color
0.085 Water Balance to 100
[0074] The sample containing the off-note blocker is found to have
less off-notes than the control.
M) Sucralose Sweetened Cola Soft Drink
[0075] The off-note blocker is used in a concentration of 0.0035%
(wt/wt).
TABLE-US-00003 sucralose cola soft drink % (by weight) Sodium
Benzoate 0.03 Sucralose (25% cut in water) 0.06 Caffeine 0.01
Phosphoric Acid (85%) 0.08 Caramel Color 0.09 Water Balance to
100
[0076] The sample containing the off-note blocker is found to have
less off-notes and be less bitter than the control.
Example 2
TAS2R44 Bitter Taste Receptor Assay for IC 50 Determination
1) Generation of Human TAS2R44 Expression Vector
[0077] The full length gene of human TAS2R44 was amplified by
polymerase chain reaction (PCR) using gene-specific primers that
span the entire coding region as described in WO 2004/029087.
[0078] The TAS2R44 cDNA was subcloned into an expression cassette
based on either of the following plasmids/expression vectors:
pcDNA3.1Zeo (Invitrogen). These vectors contain within their
multiple cloning sites the nucleotide sequence coding for the first
45 amino acids of the rat somatostatin receptor subtype 3 (RSS tag)
to facilitate cell surface targeting of the transgene (SEQ ID #4)
and the nucleotide sequence coding for the herpes simplex virus
(HSV) glycoprotein D epitope (HSV epitope in aminoterminal to
carboxyterminal direction, HSV tag). (SEQ ID #3) for facilitating
immunocytochemical detection.
[0079] The TAS2R44 construct contains RSS tag, TAS2R44, and the HSV
tag which are fused in frame to allow translation into the receptor
protein and the resulting receptor cDNA.
[0080] This transfected expression vector is called
pcDNA3.1Zeo-TAS2R44 (SEQ ID #1) and allows for expression of the
TAS2R44 protein (SEQ ID #2).
2) Generation of a Cell Line Stably Expressing
G.alpha.16-Gustducin44 and TAS2R44
[0081] A cell line that stably expresses the human bitter taste
receptor (TAS2R44) was generated by transfecting
pcDNA3.1Zeo-TAS2R44 into HEK293T/G.alpha.16-gustducin 44 cells
(both formed as described in under 1) above). The host cell line
HEK-293T is commercially available from the American Tissue Culture
Collection (catalog #CRL-1573).
[0082] Transfection was performed as follows:
[0083] On day 0, the HEK293T G.alpha.16-gustducin44 cells were
seeded in a 6-well plate at a density of 900,000 cells per well and
grown over night in selective growth medium (DMEM with 10% (v/v)
heat-inactivated fetal bovine serum, 2 mM L-glutamine. 100 units/ml
penicillin, 100 .mu.g/ml streptomycin, 200 .mu.g/ml G418 and 200
.mu.g/ml zeocin).
[0084] On day 1, the medium was exchanged with 2 ml of
antibiotic-free and serum-free growth medium. 10 .mu.l
Lipofectamine 2000 was dissolved in 250 .mu.l DMEM and incubated
for 5 minutes at room temperature. In parallel, 4 .mu.g TAS2R44
vector DNA were dissolved in 250 .mu.l DMEM. These two resulting
solutions are mixed and incubated for 20 minutes at room
temperature before they are added to the cells into the cell
culture medium. After 4 hours, the medium is replaced with
antibiotic-free, serum-containing growth medium.
[0085] The cells were incubated in humidified atmosphere
(37.degree. C., 5% CO.sub.2).
[0086] After 24 hours, the cells were re-plated in selective growth
medium and were further incubated in a humidified atmosphere
(37.degree. C., 5% CO.sub.2).
[0087] After 2 to 4 weeks of culture (replacing medium as
necessary), zeocin-resistant colonies were selected and
expanded.
[0088] The selected clone was tested successfully for functional
expression of TAS2R44.
3) Fluo-4 Calcium Assay
[0089] Fluo-4_AM (Invitrogen) is a fluorescent indicator of
intracellular calcium dynamics (change in concentration) and allows
monitoring changes in the calcium concentration, particularly an
increase in response to receptor activation occurring after agonist
exposure.
[0090] At day 0, the HEK293T cell line stably expressing
G.alpha.16-gustducin44 and TAS2R44 formed as described under 2) was
seeded in antibiotic-free growth medium (standard DMEM with 10%
(v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine
standard DMEM with 10% (v/v) heat-inactivated fetal bovine serum, 2
mM L-glutamine. 100 units/ml penicillin, and 100 .mu.g/ml
streptomycin) into black wall/clear bottom 96-well plates, coated
with poly(ethylenimine) (0.005% v/v) at a concentration of 15,000
cells per well and incubated for 48 hours in humidified atmosphere
(37.degree. C., 5% CO.sub.2).
[0091] At the time of the assay, the growth medium was discarded
and the cells were further incubated in a humidified atmosphere
(37.degree. C., 5% CO.sub.2) for 1 hour with 50 .mu.l of loading
buffer consisting of 1.5 .mu.M Fluo-4 AM and 2.5 .mu.M probenicid
(Sigma-Aldrich) in DMEM.
[0092] Afterwards, the 96-well plate was washed 5 times with 200
.mu.l of assay buffer (130 mM NaCl, 5 mM KCl, 10 mM HEPES, 2 mM
CaCl.sub.2, and 5 mM dextrose, pH 7.4) per well, using an automated
plate washer (BioTek). The plate was further incubated for 30
minutes at room temperature in the dark to allow for complete
de-esterification of the Fluo-4. Afterwards the plate was washed 5
times with 200 .mu.l of assay buffer per well, and reconstituted
with 180 .mu.l of assay buffer per well.
[0093] For assay reading, the plate was placed in a Fluorometric
Imaging Plate Reader (FLIPR) (FLIPR-TETRA.TM., Molecular Devices),
and receptor activation was initiated by addition of 20 .mu.l of a
tenfold concentrated agonist stock solution (to give the desired
agonist end concentration when added to the 180 microliter assay
buffer volume), which was prepared in assay buffer.
[0094] Fluorescence was continuously monitored for 20 seconds to
give a signal baseline (averaged to give F.sub.0) prior to agonist
addition and for 120 seconds after agonist addition. The change in
signal divided by F.sub.o gives .DELTA.F/F.sub.0 indicated in the
table, with .DELTA.F being the maximum signal occurring within the
120 seconds minus the minimum signal (occurring within the 120
seconds after agonist addition.
[0095] All data was collected from at least two independent
experiments each carried out in triplicate.
[0096] A concentration-response analysis was performed and
IC.sub.50 values were calculated by nonlinear regression using the
function f(x)=(a-d)/(1+(x/C).sup.nh)+d; with a=minimum signal,
d=maximum signal, nh=hill coefficient, C=IC.sub.50, and
x=antagonist concentration. IC.sub.50 is the molar concentration of
an antagonist which produces 50% of the maximum possible
effective/inhibitory response for that antagonist. A more potent
antagonist will have a lower IC.sub.50 value.
[0097] The obtained calcium signals were corrected for the response
of cells expressing only the G Protein .alpha. subunit
(G.alpha.16-gustducin44) and normalized to the fluorescence of
cells prior to the stimulus using .DELTA.F/F0 (Fmax-Fmin/F0).
Example 3
Determination of IC.sub.50 of Off-Note Blockers
[0098] The following off-note blockers were tested:
cis-4-propylcyclohexanecarboxylic acid,
cis-4-tert-butylcyclohexanecarboxylic acid.
[0099] The method was performed as described in example 2, using
saccharin as agonist.
[0100] The cells are exposed to a constant concentration of
saccharin (0.5 mM) and to a set of different concentrations of the
off-note blocker. A fluo-4 calcium assay was performed as described
above in example 2 and gave an IC.sub.50 [micro molar] within the
range of 0.05 to 25.
[0101] This means that the off-note blockers inhibited the response
of the TAS2R44 bitter taste receptor and will be useful to block
bitter taste.
Example 4
Determination of IC.sub.50 for Off-Note Blockers
[0102] The method was performed as described in example 3,
exchanging saccharin for Acesulfame K (0.8 mM) as agonist. An
IC.sub.50 within the same range was determined. This means that the
off-note blockers inhibited the response of the TAS2R44 bitter
taste receptor and will be useful to block bitter taste.
[0103] While the compounds, consumable and methods have been
described above in connection with illustrative embodiments, it is
to be understood that other similar embodiments may be used or
modifications and additions may be made to the described
embodiments for performing the same function without deviating
therefrom. Further, all embodiments disclosed are not necessarily
in the alternative, as various embodiments of the invention may be
combined to provide the desired characteristics. Variations can be
made by one having ordinary skill in the art without departing from
the spirit and scope of the invention. Therefore, the compounds,
consumables and methods should not be limited to any single
embodiment, but rather construed in breadth and scope in accordance
with the recitation of the attached claims.
Sequence CWU 1
1
411113DNAArtificialrecombinant sequence of rat/human/HSV 1atg gcc
gct gtt acc tat cct tca tcc gtg cct acg acc ttg gac cct 48Met Ala
Ala Val Thr Tyr Pro Ser Ser Val Pro Thr Thr Leu Asp Pro1 5 10 15ggg
aat gca tcc tca gcc tgg ccc ctg gac acg tcc ctg ggg aat gca 96Gly
Asn Ala Ser Ser Ala Trp Pro Leu Asp Thr Ser Leu Gly Asn Ala 20 25
30tct gct ggc act agc ctg gca gga ctg gct gtc agt ggc gaa ttc atg
144Ser Ala Gly Thr Ser Leu Ala Gly Leu Ala Val Ser Gly Glu Phe Met
35 40 45aca act ttt ata ccc atc att ttt tcc agt gtg gta gtg gtt cta
ttt 192Thr Thr Phe Ile Pro Ile Ile Phe Ser Ser Val Val Val Val Leu
Phe 50 55 60gtt att gga aat ttt gct aat ggc ttc ata gca ttg gta aat
tcc att 240Val Ile Gly Asn Phe Ala Asn Gly Phe Ile Ala Leu Val Asn
Ser Ile65 70 75 80gag cgg gtc aag aga caa aag atc tct ttt gct gac
cag att ctc act 288Glu Arg Val Lys Arg Gln Lys Ile Ser Phe Ala Asp
Gln Ile Leu Thr 85 90 95gct ctg gcg gtc tcc aga gtt ggt ttg ctc tgg
gta tta tta tta aat 336Ala Leu Ala Val Ser Arg Val Gly Leu Leu Trp
Val Leu Leu Leu Asn 100 105 110tgg tat tca act gtg ttt aat cca gct
ttt tat agt gta gaa gta aga 384Trp Tyr Ser Thr Val Phe Asn Pro Ala
Phe Tyr Ser Val Glu Val Arg 115 120 125act act gct tat aat gtc tgg
gca gta acc ggc cat ttc agc aac tgg 432Thr Thr Ala Tyr Asn Val Trp
Ala Val Thr Gly His Phe Ser Asn Trp 130 135 140ctt gct act agc ctc
agc ata ttt tat ttg ctc aag att gcc aat ttc 480Leu Ala Thr Ser Leu
Ser Ile Phe Tyr Leu Leu Lys Ile Ala Asn Phe145 150 155 160tcc aac
ctt att ttt ctt cac tta aag agg aga gtt aag agt gtc att 528Ser Asn
Leu Ile Phe Leu His Leu Lys Arg Arg Val Lys Ser Val Ile 165 170
175ctg gtg atg ctg ttg ggg cct tta cta ttt ttg gct tgt caa ctt ttt
576Leu Val Met Leu Leu Gly Pro Leu Leu Phe Leu Ala Cys Gln Leu Phe
180 185 190gtg ata aac atg aaa gag att gta cgg aca aaa gaa tat gaa
gga aac 624Val Ile Asn Met Lys Glu Ile Val Arg Thr Lys Glu Tyr Glu
Gly Asn 195 200 205atg act tgg aag atc aaa ttg agg agt gca gtg tac
ctt tca gat gcg 672Met Thr Trp Lys Ile Lys Leu Arg Ser Ala Val Tyr
Leu Ser Asp Ala 210 215 220act gta acc acg cta gga aac tta gtg ccc
ttc act ctg acc ctg cta 720Thr Val Thr Thr Leu Gly Asn Leu Val Pro
Phe Thr Leu Thr Leu Leu225 230 235 240tgt ttt ttg ctg tta atc tgt
tct ctg tgt aaa cat ctc aag aag atg 768Cys Phe Leu Leu Leu Ile Cys
Ser Leu Cys Lys His Leu Lys Lys Met 245 250 255cag ctc cat ggt aaa
gga tct caa gat ccc agc acc aag gtc cac ata 816Gln Leu His Gly Lys
Gly Ser Gln Asp Pro Ser Thr Lys Val His Ile 260 265 270aaa gct ttg
caa act gtg atc ttt ttc ctc ttg tta tgt gcc gtt tac 864Lys Ala Leu
Gln Thr Val Ile Phe Phe Leu Leu Leu Cys Ala Val Tyr 275 280 285ttt
ctg tcc ata atg ata tca gtt tgg agt ttt ggg agt ctg gaa aac 912Phe
Leu Ser Ile Met Ile Ser Val Trp Ser Phe Gly Ser Leu Glu Asn 290 295
300aaa cct gtc ttc atg ttc tgc aaa gct att aga ttc agc tat cct tca
960Lys Pro Val Phe Met Phe Cys Lys Ala Ile Arg Phe Ser Tyr Pro
Ser305 310 315 320atc cac cca ttc atc ctg att tgg gga aac aag aag
cta aag cag act 1008Ile His Pro Phe Ile Leu Ile Trp Gly Asn Lys Lys
Leu Lys Gln Thr 325 330 335ttt ctt tca gtt ttg cgg caa gtg agg tac
tgg gtg aaa gga gag aag 1056Phe Leu Ser Val Leu Arg Gln Val Arg Tyr
Trp Val Lys Gly Glu Lys 340 345 350cct tca tct cca tgc ggc cgc cag
cct gaa ctc gct cct gaa gac ccg 1104Pro Ser Ser Pro Cys Gly Arg Gln
Pro Glu Leu Ala Pro Glu Asp Pro 355 360 365gaa gat taa 1113Glu Asp
3702370PRTArtificialSynthetic Construct 2Met Ala Ala Val Thr Tyr
Pro Ser Ser Val Pro Thr Thr Leu Asp Pro1 5 10 15Gly Asn Ala Ser Ser
Ala Trp Pro Leu Asp Thr Ser Leu Gly Asn Ala 20 25 30Ser Ala Gly Thr
Ser Leu Ala Gly Leu Ala Val Ser Gly Glu Phe Met 35 40 45Thr Thr Phe
Ile Pro Ile Ile Phe Ser Ser Val Val Val Val Leu Phe 50 55 60Val Ile
Gly Asn Phe Ala Asn Gly Phe Ile Ala Leu Val Asn Ser Ile65 70 75
80Glu Arg Val Lys Arg Gln Lys Ile Ser Phe Ala Asp Gln Ile Leu Thr
85 90 95Ala Leu Ala Val Ser Arg Val Gly Leu Leu Trp Val Leu Leu Leu
Asn 100 105 110Trp Tyr Ser Thr Val Phe Asn Pro Ala Phe Tyr Ser Val
Glu Val Arg 115 120 125Thr Thr Ala Tyr Asn Val Trp Ala Val Thr Gly
His Phe Ser Asn Trp 130 135 140Leu Ala Thr Ser Leu Ser Ile Phe Tyr
Leu Leu Lys Ile Ala Asn Phe145 150 155 160Ser Asn Leu Ile Phe Leu
His Leu Lys Arg Arg Val Lys Ser Val Ile 165 170 175Leu Val Met Leu
Leu Gly Pro Leu Leu Phe Leu Ala Cys Gln Leu Phe 180 185 190Val Ile
Asn Met Lys Glu Ile Val Arg Thr Lys Glu Tyr Glu Gly Asn 195 200
205Met Thr Trp Lys Ile Lys Leu Arg Ser Ala Val Tyr Leu Ser Asp Ala
210 215 220Thr Val Thr Thr Leu Gly Asn Leu Val Pro Phe Thr Leu Thr
Leu Leu225 230 235 240Cys Phe Leu Leu Leu Ile Cys Ser Leu Cys Lys
His Leu Lys Lys Met 245 250 255Gln Leu His Gly Lys Gly Ser Gln Asp
Pro Ser Thr Lys Val His Ile 260 265 270Lys Ala Leu Gln Thr Val Ile
Phe Phe Leu Leu Leu Cys Ala Val Tyr 275 280 285Phe Leu Ser Ile Met
Ile Ser Val Trp Ser Phe Gly Ser Leu Glu Asn 290 295 300Lys Pro Val
Phe Met Phe Cys Lys Ala Ile Arg Phe Ser Tyr Pro Ser305 310 315
320Ile His Pro Phe Ile Leu Ile Trp Gly Asn Lys Lys Leu Lys Gln Thr
325 330 335Phe Leu Ser Val Leu Arg Gln Val Arg Tyr Trp Val Lys Gly
Glu Lys 340 345 350Pro Ser Ser Pro Cys Gly Arg Gln Pro Glu Leu Ala
Pro Glu Asp Pro 355 360 365Glu Asp 370345DNAherpes simplex virus
3tgcggccgcc agcctgaact cgctcctgaa gacccggaag attaa 454144DNArat
4atggccgctg ttacctatcc ttcatccgtg cctacgacct tggaccctgg gaatgcatcc
60tcagcctggc ccctggacac gtccctgggg aatgcatctg ctggcactag cctggcagga
120ctggctgtca gtggcgaatt catg 144
* * * * *