FORM - 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)
FLAVAN-3-OL CONTAINING FOODSTUFFS
HINDUSTAN UNILEVER LIMITED, a company incorporated under
the Indian Companies Act, 1913 and having its registered office
at 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India
The following specification particularly describes the invention and the manner in which it is to be performed

Flavan-3-ol Containing Foodstuffs
Technical field of the invention
The present invention is directed to compounds and compositions for the reduction or elimination of bitterness caused by flavan-3-ols.
Background to the invention
Excluding water, tea is the most popular beverage consumed by man. Tea is very refreshing, can be served either hot or cold, and has been made commercially available for many years. Lipton®, for example, is the world's leading brand of tea, made available in over 110 countries by Unilever,
Today, modern scientists are exploring the exciting potential of tea, which has a unique combination of natural antioxidants known as flavonoids that can include flavan-3-ols, flavonols and flavonol glucosides. Taken regularly, tea can help improve vascular function, combat fatigue, reduce cholesterol levels and increase feelings of vitality.
Studies have found that the f!avan-3-o!s, such as catechins, found in tea have many health benefits and epigallocatechin gallate, epicatechin, epicatechin gallate, and epigallocatechin have been shown to positively impact physiological activities, and their use as antiallergic agents and cerebral function activators has been proposed.
In view of the many positive contributions associated with flavan-3-ois, it is desirable to formulate consumable compositions, such as beverages and food, with elevated levels of flavan-3-ols. Unfortunately, increased amounts of flavan-3-ols can lead to end use products that can have poor organoleptic properties, in particular bitter taste.
US 2003/0096050 discloses a food composition containing catechins (i.e. flavan-3-ols) and gallic acid wherein the gallic acid is alleged to have a certain bitterness mitigating effect. There is therefore a need for consumable compositions with elevated levels of flavan-3-ols with improved bitterness reduction.

Definitions
Flavan-3-ols
As used herein the term "flavan-3-ols" is used as a generic term for catechins,
theaflavins, thearubigins, and mixtures thereof.
Catechins
As used herein the term "catechins" is used as a generic term for catechin, gallocatechin, catechin gallate, gallocatechin gallate, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and mixtures thereof.
Theaflavins
As used herein the term "theaflavins" is used as a generic term for theaflavin, isotheaflavin, neotheaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, theaflavin-3,3'-digallate, epitheaflavic acid, epitheaflavic acid-3'-galiate, theaflavic acid, theaflavic acid-3'-gailate and mixtures thereof, The structures of these compounds are well-known (see, for example, structures xi-xx in Chapter 17 of "Tea - Cultivation to consumption", K.C Willson and M.N. Clifford (Eds), 1992, Chapman & Hall, London, pp.555-601). The term theaflavins includes salt forms of these compounds. The preferred theaflavins are theaflavin, theaflavin-3-gallate, theaflavin-3'-gallate, theaflavin-3,3'-digal(ate and mixtures thereof, as these theaflavins are most abundant in natural sources, such as black tea.
Beverage
As used herein the term "beverage" refers to a substantially aqueous drinkable composition suitable for human consumption. Preferably the beverage comprises at least 85% water by weight of the beverage, more preferably at least 90% and most preferably from 95 to 99.9%.
Tea solids
As used herein, the term "tea solids" refers to dry material extractable from the leaves of the plant Camellia sinensis var. sinensis and/or Camellia sinensis var. assamica. The material may have been subjected to a so-called "fermentation" step wherein it is oxidised by certain endogenous enzymes that are released during the early stages of

"black tea" manufacture. This oxidation may even be supplemented by the action of exogenous enzymes such as oxidases, laccases and peroxidases. Alternatively the material may have been partially fermented ("oolong" tea) or substantially unfermented ("green tea").
Tea-based beverage
As used herein, the term "tea-based beverage" refers to a beverage comprising at least
0.01% by weight tea solids. Preferably the tea-based beverage comprises from 0.04 to
3% by weight tea solids, more preferably from 0.06 to 2%, most preferably from 0.1 to
1%.
Elevated levels of flavan-3-ols
As used herein, the term "elevated levels of flavan-3-ols" refers to compositions
comprising at least 0.01 wt% flavan-3-ols.
Effective amount
As used herein, the term "effective amount" refers to at least 0.01 wt% of the
compound or compounds for the reduction or elimination of bitterness caused by
flavan-3-ols.
Comprising
The term "comprising" is meant not to be limiting to any subsequently stated elements but rather to encompass non-specified elements of major or minor functional importance. In other words the listed steps, elerrients or options need not be exhaustive. Whenever the words "including" or "having" are used, these terms are meant to be equivalent to "comprising" as defined above.
Brief description of the invention
We have now found that bitterness in consumable compositions with elevated levels of flavan-3-o!s may be eliminated through the use of particular compounds. Accordingly, in a first aspect, the present invention provides the use of a compound of formula (I):


for the reduction or elimination of bitterness caused by f!avan-3 ols, wherein
Ri represents C2 - C3 saturated or unsaturated divalent hydrocarbon radical,
n is an integer from 0 to 3, and
each X is independently selected from C1- C3 alkyloxy and OH;
and wherein if n is 0 then R1 is C3 saturated divalent hydrocarbon radical or C2 - C3
unsaturated divalent hydrocarbon radical;
and wherein if n is greater than 0 then the compound has formula (II)

We have conducted extensive research into the physiological origin of bitterness from flavan-3-ols. The human tongue perceives bitterness via a family of about 25 related receptors, each of which detects a different set of bitter tasting molecules. Without wishing to be bound by theory, we believe that the bitter taste of flavan-3-ols is caused by the activation of a specific receptor, TAS2R39, by f\avan-3-ols. Furthermore, we have found that compounds of formula (I) specifically bind to receptor TA52R39 and antagonise its activation. Hence compounds according to the invention block or reduce the activation of receptor TAS2R39 by flavan-3-ols and the bitter taste of the

flavan-3-ois is alleviated, More specifically, compounds according to formula (i) are more effective at blocking TAS2R39 than gallic acid.
In a second aspect, the present invention provides a composition comprising greater than 0.01 wt % flavan-3-ols and an effective amount of at least one compound of formula (I). An advantage of this aspect of the present invention is that such a composition will allow consumers to reach the required intake of flavan-3-ols with fewer servings of, for example, a high-flavan-3-ol tea without the aversive effects of enhanced bitterness that this would normally introduce,
In a third aspect, the present invention provides a method of manufacturing the composition, the method comprising the steps of: providing a first substance comprising flavan-3-ols and a second substance comprising the at least one compound of formula (I), combining the two substances, and recovering the composition.
In a final aspect, the present invention provides a method for the reduction or elimination of bitterness caused by flavan-3-ols comprising consuming a first substance and a second substance, simultaneously or sequentially, wherein the first substance comprises flavan-3-ols and the second substance comprises an effective amount of at least one compound of formula (I).
Detailed description of the invention
The present invention is directed to consumable compositions that contain high levels of flavan-3-ols in which the bitter taste of the flavan-3-ols is reduced.
The present invention provides for the use of a compound of formula (I) that is more effective at blocking flavan-3-ol bitterness. The compound is particularly effective at blocking the bitter taste of catechins and hence a preferred use is for blocking bitterness caused by catechins. Especially effective, and thus preferred compounds, are those wherein each X of formula (J) is independently selected from methoxy and OH. Alternatively preferred compounds are those wherein if n of formula (I) is 0 then R1 is C2 unsaturated divalent hydrocarbon radical or C3 saturated divalent hydrocarbon radical. Particularly preferred compounds are:

Cinnamic acid, 4-Phenylbutanoic acid, 3-(3,4,5-trimethoxyphenyl)propanoic acid, 3,4,5-trimethoxycinnamic acid, 3-(4-methoxyphenyl)propanoic acid, 4-methoxycinnamic acid, 2,4-dimethoxycinnamic acid, 3-(3,4-dimethoxyphenyl)propanoic acid, 3,4-dimethoxycinnamic acid, 4-hydroxycinnamic acid, 3-(3,4-dihydroxyphenyl)propanoicacid, 3,4-dihydroxycinnamic acid, 3-(3-methoxy-4-hydroxyphenyl)propanoic acid, 3-methoxy-4-hydroxycinnamic acid, 4-hydroxy-3,5-dimethoxycinnamic acid, and mixtures thereof.
Yet more preferred compounds are: 4-Phenylbutanoic acid, 3-(3,4,5-trimethoxyphenyl)propanoic acid, 3,4,5-trimethoxycinnamic acid, 3-(4-rnethoxyphenyl)propanofc acid, 4-methoxycinnamic acid, 2,4-dimethoxycinnamic acid, 3-(3,4-dimethoxyphenyl)propanoic acid, 3,4-dimethoxycinnamic acid, 3-(3,4-dihydroxyphenyl)propanoic acid, 3-(3-methoxy-4-hydroxyphenyl)propanoic acid, and mixtures thereof.
The present invention also provides for a composition comprising greater than 0,01 wt% flavan-3-o!s, preferably greater than 0.02 wt% flavan-3-ols, more preferably greater than 0.05 wt% flavan-3-ols and an effective amount of at least one compound

of formula ((). Preferably, the fiavan-3-ols are one or more catechins. The amount of the compound is typically at least 0.05 wt%, more preferably at least 0.075 wt%, most preferably at least 0.1 wt%. The amount of the compound is typically at most 1.5 wt%, more preferably at most 0.75 wt%, most preferably at most 0.25 wt%. The preferred form of the composition is a food, more preferably a beverage, most preferably a tea-based beverage, Preferably the compound is one or more of: Cinnamic acid, 4-Phenylbutanoic acid, 3-(3,4,5-trimethoxyphenyl)propanoic acid, 3,4,5-trirnethoxycinnamic acid, 3-(4-methoxyphenyl)propanoic acid, 4-methoxycinnamic acid, 2,4-dimethoxycinnamic acid, 3-(3,4-dimethoxyphenyl)propanoic acid, 3,4-dimethoxycinnamic acid, 4-hydroxycinnamic acid, 3-(3,4-dihydroxyphenyl)propanoic acid, 3,4-dihydroxycinnamic acid, 3-(3-methoxy-4-hydroxyphenyl)propanoic acid, 3-methoxy-4-hydroxycinnamic acid, and 4-hydroxy-3,5-dimethoxycinnamic acid. More preferably the compound is one or more of: 4-Phenylbutanoic acid, 3-{3,4,5-trimethoxypheny)prop.anok acid, 3,4,5-trimethoxycinnamic acid, 3-(4-methoxyphenyl)propanoic acid, 4-methoxycinnamic acid, 2,4-dimethoxycinnamic acid, 3-(3,4-dimethoxyphenyl)propanoic acid, 3,4-dimethoxycinnamic acid, 3-{3,4-dihydroxyphenyl)propanoic acid, and 3-(3-methoxy-4-hydroxyphenyl)propanoic acid.
The present invention further provides a method of manufacturing the composition, the method comprising the steps of: providing a first substance comprising flavan-3-ols and a second substance comprising the at least one compound of formula (I), combining the two substances, and recovering the composition. Preferably, the first substance comprises catechins.
The present invention finally provides a method for the reduction or elimination of bitterness caused by flavan-3-ols comprising consuming a first substance and a second substance wherein the first substance comprises flavan-3-ols and the second substance comprises an effective amount of at least one compound of formula (I). Preferably, the first substance comprises catechins. Preferably the first and second substances are consumed simultaneously as part of the same meal, more preferably the first and second substances are consumed simultaneously in the same composition. Alternatively the first and second substances are consumed sequentially, most

preferably when consumed sequentially, the first substance is consumed after the second substance.
Example 1
Evaluation of TSA2R39 inhibitors
Activation of the bitter receptor TAS2R39 causes the release of calcium from the endoplasmic reticulum of those cells containing the receptor. Therefore, changes in calcium levels within cells expressing TAS2R39 serves as a proxy marker for the activity of the receptor. An assay was used to assess the stimulation of TAS2R39 by the flavan-3-ol epicatechin gallate (ECG) and to assess the inhibition of this stimulation of TAS2R39 through the use of various compounds as follows:
HEK293 cells expressing the human bitter receptor TAS2R39 and a G-protein that allows coupling to a calcium readout system were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing DMEM and 10% tetracycline-free fetal bovine serum (Cambrex) supplemented with blasticidin (5 µg/ml) geneticin (400 µg/ml) and hygromycin (100 µg/ml). Cells were grown and maintained at 37°C / 5% C02.
Ceils were cultured in a 75 cm2 flask, harvested by trypsinisation and resuspended in DMEM medium. 100 ul of cells per well were added to a poly-L-lysine coated uCiear 96-well plate, and incubated overnight. Expression of the TAS2R39 gene was then induced by the addition of 100 pi DMEM medium containing 0.5 pg/ml doxycydin. After another 24-30 hrs the medium was removed and the cells were loaded for 1 hour with 50 µl Tyrodes buffer (140 mM NaCl, 5 mM KG, 10 mM glucose, 1 mM CaG2. 2 H2Or 1 mM MgCI2. 6 H20, 20 mM HEPES pH 7.4 (Sigma P8761)) containing 2.5 µM of the fluorescent marker of calcium, Fluo-4-AM, followed by a 1-hr incubation at 37°C. The cells were then washed with Tyrodes buffer and finally taken up in 100 pi Tyrodes buffer,
Test solutions containing 0.5mM ECG and 10mM of the test compounds, dissolved in Tyrodes buffer, were prepared and 100 µl of each test solution was added to the wells of the 96-well plate containing the cells. A comparative solution containing only

0.5mM ECG dissolved in Tyrodes buffer was also prepared and 100 µl was added to a well of the 96-well plate containing the ceils.
Intracellular calcium levels were monitored using a Flexstation II 384. The 96-well plate was loaded onto the Flexstation II 384 and fluorescence measurements were carried out at 37°C at an excitation wavelength of 485nm and an emission wavelength of 520ntn. Activity of TAS2R39 was measured as the change in fluorescence (AF) which was calculated by subtracting the maximum fluorescence after the addition of the test or comparative solution from the baseline fluorescence measured before addition of the test or comparative solution.
Inhibition of TAS2R39 activity was measured as the percentage reduction of AF in the presence of ECG alone, compared to AF in the presence of ECG plus a test compound. The test compounds, their structures and the percentage inhibition of TAS2R39 activity is given in the following table. 3,4,5-trihydroxybenzoic acid (gallic acid) was included in the selection of the test compounds.

It can clearly be seen that although the test compounds share similar basic moieties, their ability to inhibit the bitter receptor TAS2R39 is varied. Only those compounds according to formula (I) are better than gallic acid. In particular, the results above demonstrate that compounds according to formula I, such as Cinnamic acid, 4-Phenylb u t a n o i c acid, 3-(3A5-trimethoxypheny)propanoic acid, 3,4,5-trimethoxycinnamic acid, 3-(4-methoxyphenyl)propanoic acid, 4-methoxycinnamic acid, 2,4-dimethoxycinnamic acid, 3-(3,4-dimethoxyphenyl)propanoic acid, 3,4-dimethoxycinnamic acid, 4-hydroxycinnamic acid, 3-(3,4-dihydroxyphenyl)propanoic acid, 3,4-dihydroxycinnamic acid, 3-(3-methoxy-4-hydroxyphenyl)propanoic acid, 3-methoxy-4-hydroxycinnamk acid and 4-hydroxy-3,5-dimethoxycinnamic acid, are more effective inhibitors of TAS2R39 than gallic acid.

Claims
1. Use of a compound wherein the compound is one or more of: 4-Phenylbutanok acid, 3-{3,4,5-trimethoxypheny!)prop3notc acid, 3,4,5-trimethoxydnnamk acid, 3-{4-methoxyphenyl)propanok acid, 4-methoxycinnamic acid, 2,4-dimethoxycinnamk acid, 3-(3,4-dimethoxyphenyl)propanok acid, 3,4-dimethoxycinnamic acid, 3-(3,4-dihydroxyphenyl)propanok acid, and 3-(3-methoxy-4-hydroxyphenyl)proparioic acid, for the reduction or elimination of bitterness caused by flavan-3-ols.
2. Use according to claim 1 wherein the bitterness is caused by catechins.
3. A composition comprising greater than 0.01 wt % fiavan-3-ols and an effective amount of at least one compound wherein the at least one compound is one or more of;
4-Phenylbutartoic acid, 3-{3,4,5-trimethoxyphenyl)propanoic acid, 3,4,5-trimethoxycinnamic acid, 3-(4-rnethoxyphenyl)propanok acid, 4-methoxycinnamic acid, 2,4-dimethoxycinnamk acid, 3-(3,4-dimethoxyphenyl)propanoic acid, 3,4-dimethoxycinnamic acid, 3-{3,4-dihydroxyphenyl)propanoic acid, and 3-{3-methoxy-4-hydroxypheny0prppanro acid.
4, A composition according to claim 3 comprising at least 0,05 wt % flavan-3-ols.

5. A composition according to claim 3 or claim 4 wherein the fiavan-3-ols are one or more catechins.
6. A composition according to any one of claims 3 to 5 wherein the amount of the at least one compound is from 0,05 to 1.5 wt%.
7. A composition according to any one of claims 3 to 6 wherein the composition is a beverage, preferably a tea-based beverage.
8. A method of manufacturing a composition according to any one of claims 3 to 7, the method comprising the steps of: providing a first substance comprising ffavan-3-o!s and a second substance comprising the at least one compound, combining the two substances, and recovering the composition.
9. A method according to claim 8 wherein the first substance comprises one or more catechins.
10. A method for the reduction or elimination of bitterness caused by flavan-3-ols
comprising consuming a first substance and a second substance, simultaneously
or sequentially, wherein the first substance comprises flavan-3-ols and the
second substance comprises an effective amount of at least one compound
wherein the at least one compound is one or more of:
4-Phenylbutanoic acid, 3-{3,4,5-trimethoxyphenyl)propanoic acid, 3,4,5-trimethoxydnnamic acid, 3-(4-methoxyphenyl)propanoic acid, 4-methoxycinnamlc acid, 2,4-dimethoxycihnarnic acid, 3-(3,4-dimethoxyphenyl)propanoic acid, 3,4-dimethoxycinnamic acid, 3-(3,4-dthydroxypheny!)propanoic acid, and 3-(3-methoxy-4-hydroxypheny!)propanoic acid.

11. A method according to claim 10 wherein the first substance comprises one or more catechins.