U.S. patent application number 15/567524 was filed with the patent office on 2018-04-26 for preparations with dehydroabietic acid.
The applicant listed for this patent is Analyticon Discovery GmbH, B.R.A.I.N. Biotechnology Research Information Network AG. Invention is credited to Grit KLUGE, Michael KROHN, Katja RIEDEL, Karsten Siems.
Application Number | 20180110252 15/567524 |
Document ID | / |
Family ID | 55754308 |
Filed Date | 2018-04-26 |
United States Patent
Application |
20180110252 |
Kind Code |
A1 |
Siems; Karsten ; et
al. |
April 26, 2018 |
PREPARATIONS WITH DEHYDROABIETIC ACID
Abstract
Suggested are preparations, comprising or consisting of (a)
dehydroabietic acid, a salt of dehydroabietic acid and/or an
extract containing dehydroabietic acid, and (b1) at least one
sweetener that is different from saccharose and/or (b2) at least
one flavonoid and/or (b3) at least one inorganic salt or mineral
substance used in foods and/or (b4) at least one alkaloid present
in foods and/or (b5) at least one terpene present in foods.
Inventors: |
Siems; Karsten; (Michendorf,
DE) ; KLUGE; Grit; (Trebbin, DE) ; KROHN;
Michael; (Lorsch, DE) ; RIEDEL; Katja;
(Bensheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Analyticon Discovery GmbH
B.R.A.I.N. Biotechnology Research Information Network AG |
Potsdam
Zwingenberg |
|
DE
DE |
|
|
Family ID: |
55754308 |
Appl. No.: |
15/567524 |
Filed: |
April 16, 2016 |
PCT Filed: |
April 16, 2016 |
PCT NO: |
PCT/EP2016/058472 |
371 Date: |
October 18, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 27/86 20160801;
A23L 2/56 20130101; A23L 27/40 20160801; A23L 2/02 20130101; A23V
2002/00 20130101; A23F 3/405 20130101; A61K 33/06 20130101; A61K
47/12 20130101; A23L 27/33 20160801; A23L 27/84 20160801; A23L
27/2028 20160801; A61K 33/06 20130101; A61K 2300/00 20130101 |
International
Class: |
A23L 27/00 20060101
A23L027/00; A23L 27/20 20060101 A23L027/20; A23L 27/30 20060101
A23L027/30; A23L 2/56 20060101 A23L002/56; A23F 3/40 20060101
A23F003/40; A23L 2/02 20060101 A23L002/02; A23L 27/40 20060101
A23L027/40; A61K 47/12 20060101 A61K047/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 22, 2015 |
EP |
PCT/EP2015/058741 |
May 3, 2015 |
EP |
15166154.3 |
Claims
1. A preparation, comprising (a) dehydroabietic acid, a salt of
dehydroabietic acid and/or an extract containing dehydroabietic
acid, and (b1) at least one sweetener that is different from
saccharose, and/or (b2) at least one flavonoid, and/or (b3) at
least one inorganic salt or mineral substance used in foods, and/or
(b4) at least one alkaloid present in foods, and/or (b5) at least
one terpene present in foods.
2. The preparation of claim 1, wherein the extract has a content of
dehydroabietic acid of at least 20% by weight.
3. The preparation of claim 1, wherein the sweetener is selected
from the group consisting of trehalose, lactose, maltose,
melizitose, raffinose, palatinose, lactulose, D-fructose,
D-glucose, D-galactose, L-rhamnose, D-sorbose, D-mannose,
D-tagatose, D-arabinose, L-arabinose, D-ribose, D-glyceraldehyde or
maltodextrin. Plant-based preparations containing these substances
are also suitable, for example, on the basis of sugarbeet (Beta
vulgaris ssp., sugar fractions, sugar syrup, molasses), sugar cane
(Saccharum officinarum ssp., molasses, sugar cane syrup), maple
syrup (Acer ssp.) or agave (agave syrup), fruit concentrates (e.g.,
on the basis of apples or pears), erythritol, threitol, arabitol,
ribotol, xylitol, sorbitol, mannitol, dulcitol, lactitol),
miraculin, monellin, thaumatin, curculin, brazzein, magap, sodium
cyclamate, acesulfame-K, neohesperidin dihydrochalcone, naringin
dihydrochalcone, saccharine sodium salt, aspartame, superaspartame,
neotame, alitame, sucralose, stevioside, rebaudioside, lugduname,
carrelame, sucrononate, sucrooctate, monatin, phenylodulcin,
glycine, D-leucine, D-threonine, D-asparagine, D-phenylalanine,
D-tryptophan, L-proline, hernandulcin, phyllodulcin, dihydrochalcon
glycoside, glycerrhetinic acid and its derivatives and salts,
extracts of licorice (Glycyrrhiza ssp.), extracts of Lippia dulcis,
extracts of Momordica ssp., mogrosides and extracts of the plants
of the genus Rubus and mixtures thereof.
4. The preparation of claim 3, wherein the sweetener is selected
from the group consisting of saccharin, acesulfame-K, steviol
glycosides and stevia extracts and mixtures thereof.
5. The preparation of claim 1, wherein the flavonoid is selected
from the group consisting of catechin, epicatechin, gallocatechin,
gallocatechin gallate, epigallocatechin gallate, sinensetin,
sobiletin, naringin and mixtures thereof.
6. The preparation of claim 1, wherein the mineral substance is
selected from the group consisting of potassium and magnesium salts
and mixtures thereof.
7. The preparation of claim 1, wherein the alkaloid is
caffeine.
8. The preparation of claim 1, wherein terpene is selected from the
group consisting of menthol and limonene and mixtures thereof.
9. A food composition containing the preparation of claim 1.
10. The food composition of claim 9, which is a soft drink or a hot
drink or an instant drink.
11. A pharmaceutical composition containing the preparation of
claim 1.
12. The pharmaceutical composition of claim 11, which is a liquid
product.
13. The food composition of claim 10, containing dehydroabietic
acid or a salt of dehydroabietic acid in concentrations of about 1
to about 500 .mu.M.
14. A process for masking unpleasant, particularly bitter,
astringent and/or liquorice-like taste impressions of sweeteners
and/or flavonoids and/or inorganic salts and foods or
pharmaceutical preparations containing these substances, wherein a
quantity of dehydroabietic acid, a salt of dehydroabietic acid
and/or an extract containing dehydroabietic acid is added to
them.
15. (canceled)
16. The pharmaceutical composition of claim 12, containing
dehydroabietic acid or a salt of dehydroabietic acid in
concentrations of about 1 to about 500 .mu.M.
Description
FIELD OF THE INVENTION
[0001] The invention is in the food sector and relates to new
preparations containing dehydroabietic acid, substances containing
these, processes for taste modulation and corresponding uses of
these substances.
STATE OF THE ART
[0002] The purchasing and consuming behaviour of mostly younger
consumers has been changing significantly in a time when fitness
and health are increasingly gaining importance. Products are in
demand which do not contain any sugar but sweeteners, which are
synthetically produced such as, for example, saccharin or
acesulfame-K, or which are of plant origin such as, for example,
stevia, because they are calory-free and do not cause tooth decay
(caries). In addition, not only the classic tea varieties such as
Earl Grey have been enjoying great popularity, but also Chai teas,
Mate teas and the like. Both product groups, which are so
different, are sharing the same problem: synthetic or plant-based
sweeteners are free from the undesired effects of sugar, but they
do not convey the same taste experience. Teas, but also many fruit
juice preparations, contain large amounts of flavonoids,
specifically catechins and similar flavonoids, and other secondary
phytonutrients such as terpenes or polyphenols, which are said to
be beneficial for the human health and are therefore certainly
desirable as ingredients, but which convey an intensely bitter,
astringent and metallic taste, depending on their amount.
[0003] For this reason, there is an intensive demand in the market
for products which are capable of masking, suppressing or
modulating the unpleasant taste impressions of both sweeteners and,
if used together with sweeteners, also of flavonoids, alkaloids,
terpenes and other secondary phytonutrients, i.e. providing them
with a pleasant taste.
[0004] Taste-modulating natural substances, including,
specifically, the so-called "bitter blockers" are already known
from the state of the art. For example, DE 10 2012 214560 A1
suggests 1,3-enterodiol compounds for this purpose and EP 1258200A2
suggests flavanones, particularly eriodictyol derivatives. EP
2559346 A1 discloses oleanane triterpene glycosides for the same
purpose. The subject-matter of WO 2011 050955 A1 is antagonists and
agonists for the human bitter taste receptors TAS2R40, hTAS2R43,
hTAS2R44, hTAS2R46 and hTAS2R47, which are also terpene
derivatives. WO 2013 072332 A1 reports the use of Hardwickiid acid
to counteract the bitter taste.
[0005] Thus, the state of the art sufficiently describes the use of
natural substances, specifically of terpene derivatives, to
counteract, cover, or change undesired taste properties. However,
the disadvantages of the compositions mentioned are that they
require high quantities, and that they only have very specific
effects, particularly with respect to synthetic sweeteners and
flavonoids. In the quantities described, these substances also
exhibit a taste of their own which adulterates the desired taste
experience, usually with a deteriorating effect. In addition, the
effectiveness of these active agents strongly depends on the matrix
in which they are used. For producers of foods it is, therefore, of
great importance to be able to select from a large number of
alternative active agents in order to be able to determine the
optimal substance for the particular purpose of application.
[0006] The object of the present invention was, therefore, to
provide a natural substance which is capable of covering,
neutralising, or advantageously changing unpleasant taste
impressions both of sweeteners, specifically of saccharin and
acesulfame-K, as well as of secondary phytonutrients such as
inorganic salts, flavonoids, polyphenols, alkaloids and terpenes,
specifically those which are present in various tea varieties,
particularly fruit teas and herbal teas and beverages made of
citrus fruit, even when present in very small quantities.
DESCRIPTION OF THE INVENTION
[0007] A first subject-matter of the invention relates to
preparations, comprising or consisting of [0008] (a) dehydroabietic
acid, a salt of dehydroabietic acid and/or an extract containing
dehydroabietic acid, and [0009] (b1) at least one sweetener that is
different from saccharose and/or [0010] (b2) at least one flavonoid
and/or [0011] (b3) at least one inorganic salt or mineral used in
foods and/or [0012] (b4) at least one alkaloid present in foods
and/or [0013] (b5) at least one terpene present in foods.
[0014] Surprisingly, it was found that dehydroabietic acid fully
meets the complex profile of requirements described in the
beginning. It improves the taste impression in a wide range of
different species of both synthetic and natural sweeteners, and
also of various tea varieties and other products where unpleasant,
bitter, or astringent taste impressions are caused by flavonoids,
the alkaloid caffeine and the monoterpene menthol. It is a purely
plant-based product which is fully effective also in very small
concentrations of, for example, 25 .mu.M (7.5 mg/l), and which thus
proves to be superior to alternative prior-art substances.
[0015] Taste impressions, particularly unpleasant taste impressions
which are intended to be improved within the meaning of the
invention are understood as bitter and astringent taste sensations,
as are other types of tastes which are, for example, described as
similar to licorice. The latter are particularly found in the
context of the use of stevia extracts.
[0016] Dehydroabietic Acid
[0017] The diterpene dehydroabietic acid (component (a)) is present
both in the needles and the bark, root and particularly the resin
of many conifers (among others, in the plant families of Pinaceae
and Juniperaceae, and here, in particular, in the genera Pinus,
Abies, Larix, Juniperus). It is a byproduct of paper production and
contained in what is referred to as tall oil.
[0018] Dehydroabietic acid has also been isolated from other plant
families (e.g., from the genera Illicium, Liquidambar, Styrax,
Callicarpa, Rosmarinus, Salvia, Commiphora, Boswellia).
Dehydroabietic acid is most frequently found in resins (e.g.,
Liquidambar, Styrax, Boswellia, Commiphora, Colophony, but also in
amber).
##STR00001##
[0019] Structural Formula of Dehydroabietic Acid (CAS
1740-19-8)
[0020] The content of dehydroabietic acid in pine resin is mostly
below 10%, but it may also be increased to a content of >50%,
using a method that is known by the skilled person as
disproportionation (e.g., according to the process described by
Song et al in JOURNAL OF WOOD CHEMISTRY AND TECHNOLOGY, 5(4),
535-542 (1985)).
[0021] Both enantiomers of dehydroabietic acid are known (CAS
1740-19-8 and CAS 6980-63-8). The term "dehydroabietic acid" is
understood as meaning the isomers and any optional enantiomeric
mixtures within the meaning of the present invention.
[0022] Although extracts containing dehydroabietic acid as well as
the commercially available technical dehydroabietic acid having a
purity of about 85% can be used, the use of high-purity products is
preferred, which have a content of dehydroabietic acid of at least
90% by weight, preferably at least 95% by weight, and particularly
preferably from about 95 to about 99% by weight. These high-purity
products are obtainable by the typical work-up procedures of
preparative organic chemistry so that the skilled person is not
required to make an inventive step. In the following, a
corresponding process is described, by way of example, in example
1.
[0023] Instead of using dehydroabietic acid itself, it is also
possible to use its salts, specifically its alkali and ammonium
salts, particularly the sodium salt.
[0024] The invention also comprises the use of extracts containing
dehydroabietic acid, the production of which also forms part of the
skilled person's tools, and which is described, by way of example,
in example 2 below. Preferably, these extracts contain at least 20%
by weight, more preferably at least 30% by weight, and particularly
preferably from about 40 to about 60% by weight dehydroabietic
acid.
[0025] Sweeteners
[0026] Suitable sweeteners or sweet-tasting additives forming group
(b) are, firstly, carbohydrates such as, for example, trehalose,
lactose, maltose, melizitose, raffinose, palatinose, lactulose,
D-fructose, D-glucose, D-galactose, L-rhamnose, D-sorbose,
D-mannose, D-tagatose, D-arabinose, L-arabinose, D-ribose,
D-glyceraldehyde or maltodextrin. Plant-based preparations
containing these substances are also suitable, for example, on the
basis of sugarbeet (Beta vulgaris ssp., sugar fractions, sugar
syrup, molasses), sugar cane (Saccharum officinarum ssp., molasses,
sugar cane syrup), maple syrup (Acer ssp.) or agave (agave
syrup).
[0027] Suitable are also synthetic, i.e. usually enzymatically
produced starch or sugar hydrolysates (invert sugar, fructose
syrup) as well as natural or synthetic sweet-tasting substances,
such as [0028] Fruit concentrates (e.g., on the basis of apples or
pears); [0029] Sugar alcohols (e.g., erythritol, threitol,
arabitol, ribotol, xylitol, sorbitol, mannitol, dulcitol,
lactitol); [0030] Proteins (e.g., miraculin, monellin, thaumatin,
curculin, brazzein); [0031] Sweeteners (e.g., magap, sodium
cyclamate, acesulfame-K, neohesperidin dihydrochalcone, saccharine
sodium salt, aspartame, superaspartame, neotame, alitame,
sucralose, stevioside, rebaudioside, lugduname, carrelame,
sucrononate, sucrooctate, monatin, phenylodulcin); [0032]
Sweet-tasting amino acids (e.g., glycine, D-leucine, D-threonine,
D-asparagine, D-phenylalanine, D-tryptophan, L-proline); [0033]
Further sweet-tasting low-molecular substances such, e.g.,
hernandulcin, phyllodulcin, dihydrochalcon glycoside, glycyrrhizin,
glycerrhetinic acid and its derivatives and salts, rubusosides,
mogrosides [0034] Extracts of sweet-tasting plants such as Stevia
rebaudiana, Glycyrrhiza ssp. (liquorice), Lippia dulcis, Momordica
grosvenori.
[0035] The sweeteners may also be plant extracts, as is described
by way of example in the following.
[0036] Rebaudiosides are among the steviosides, which are the main
components of the plant Stevia rebaudiana, which is also referred
to as sweet weed or honey weed.
##STR00002##
[0037] 10% of the dry matter of the leaves are constituted by the
diterpene glycoside stevioside, followed by rebaudioside A (2 to 4%
by weight) as well as by more than ten other steviol glycosides
such as dulcoside. By now, most countries have approved
rebaudiosides and stevia extracts for use as sweeteners; a daily
uptake of up to 4 mg stevioside per kilogramme of bodyweight is
considered harmless. Within the meaning of the invention,
individual rebaudiosides or the extracts of the stevia plant may be
used. Particularly preferred, however, is the use of rebaudioside
A, as this substance has a lower bitterness and the highest
sweetening power. The substance mixtures according to the invention
may contain components (a) and (b) in a weight ratio from about
1:99 to about 99:1, preferably from about 25:75 to about 75:25, and
particularly preferably from about 40:60 to about 60:40.
[0038] Also the dihydrochalcones represent flavonoids, in which
particularly the two representatives naringenin dihydrochalcone and
neohesperidin dihydrochalcone must be highlighted, which are known
as synthetic sweeteners:
##STR00003##
[0039] A group of cucurbitane glycosides is referred to as
mogrosides, which are known as a component of the natural sweetener
Luo Han Guo. Mogroside-V, which is 400 times sweeter than sugar, is
highlighted herein.
##STR00004##
[0040] Eventually, suitable sweeteners also include extracts of the
plants which are selected from the group consisting of Rubus
allegheniensis, Rubus arcticu, Rubus strigosus, Rubus armeniacus,
Rubus caesius, Rubus chamaemorus, Rubus corylifolius agg., Rubus
fruticosus agg., Rubus geoides, Rubus glaucus, Rubus gunnianus,
Rubus idaeus, Rubus illecebrosus, Rubus laciniatus, Rubus
leucodermis, Rubus loganobaccus, Rubus loxensis, Rubus nepalensis,
Rubus nessensis, Rubus nivalis, Rubus odoratus, Rubus pentalobus,
Rubus phoenicolasius, Rubus saxatilis, Rubus setchuenensis, Rubus
spectabilis and Rubus ulmifolius and their mixtures. These are
substantially extracts of various blackberry and raspberry
varieties having a content in rubosides. Extracts of Rubus
suavissimus are preferred.
[0041] A further active agent in this group is glycyrrhetinic acid,
or a corresponding salt, or an extract containing this
substance.
##STR00005##
[0042] Within the meaning of the invention, it is possible to use
the acid itself, its salts--for example, sodium, potassium, or
ammonium salt--or the extracts of the plant Glycyrrhiza glabra.
Mono ammonium glycyrrhizinate is particularly preferred.
[0043] Preferred sweeteners, the taste perception of which is to be
improved, are selected from the group consisting of saccharin,
acesulfame-K, steviol glycosides, particularly rebaudioside A, and
stevia extracts.
[0044] Flavonoids
[0045] The flavonoids forming component (1b) are mostly catechins,
which are mainly contained in tea in varying quantities and
compositions, and which are among the most powerful bitter
principles. Herein, the most important representatives are:
##STR00006##
as well as epicatechin and gallocatechin. In addition to the
flavonoids and their oligomers and oxidation products, also the
alkaloid caffeine contributes to the bitter taste of green and
black tea. In beverages made of citrus fruit, the bitter or
astringent taste is mainly caused by polymethoxylated flavones such
as sinensetin and nobiletin, glycosylated flavonoids, e.g.,
naringin, and by terpenes of the limonoid type, e.g., limonene.
##STR00007##
[0046] Mineral Substances
[0047] Magnesium is an important mineral substance. Under-supply
with magnesium is often not diagnosed, but it may appear as a side
effect in many diseases (e.g., diabetes, hypertension). Senior
citizens and pregnant women frequently have an increased demand for
magnesium. An increased demand for magnesium may be balanced by
mineral supplements or by adding magnesium to foods, e.g., bread
that is specifically produced for senior citizens. Magnesium may be
taken in the form of various salts, e.g., as magnesium chloride or
magnesium sulfate. Magnesium-containing salts taste bitter,
particularly magnesium sulfate, which is also referred to as
epsomite.
[0048] Potassium is also added to foods, but usually not with the
intention to balance a potassium deficiency, but as a substitute
for salt in order to reduce the sodium content in foods while
maintaining the taste of salt. As potassium chloride has a bitter
taste, only part of the sodium may be substituted by potassium. In
order to reduce the bitter taste of potassium chloride, for
example, ascorbic acid, fumaric acid and citric acid, or various
sugars (lactose, dextrose), or yeast extract may be added (cf.
DE3035518C2). However, these solutions are only suitable for some
foods, as they are not neutral with regard to their taste or may
lead to an increased uptake of calories, which is not desired in
sodium-chloride reduced foods which are, therefore, healthier.
[0049] Preparations
[0050] Preparations of the present invention may contain components
(a), based on component (b), in concentrations of about 1 to about
500 .mu.M, preferably, about 10 to about 250 .mu.M, particularly
preferably, about 20 to about 100 .mu.M.
[0051] A form of preparation which is intended for commercial
distribution and which is particularly suitable, for example, in
form of a sweetener tablet typically contains 1 to 40 mg
sweetener(s), e.g., saccharin or acesulfame-K, and 1 to 30 mg
dehydroabietic acid, based on a total weight of the tablet of 60 mg
where the difference in quantity is represented by tableting
additives, particularly binders. One of these tablets corresponds
to the sweetening power of one sugar cube and is completely
sufficient to sweeten, for example, a common cup of tea, completely
masking the unpleasant taste that is linked with the addition of a
sweetener in the process.
[0052] Foods
[0053] A further subject-matter of the present invention relates to
foods which contain the preparations according to the invention. In
this case it is possible to add the components individually or
together, where the addition of the mixture is preferred. With
respect to the taste improvement in flavonoids, component (a) can
be used alone or as a mixture of (a) and (b).
[0054] Examples for foods, in principle, comprise baked goods, e.g.
bread, dry biscuits, cakes, other baked products, confectionery
(e.g. chocolates, chocolate bar products, other bar products, fruit
gum, hard and soft caramels, chewing gum), alcoholic or
nonalcoholic beverages (for example, coffee, tea, iced tea, wine,
wine-containing beverages, beer, beer-containing beverages,
liqueurs, schnapps, brandies, fruit-containing (carbonated)
beverages, isotonic (carbonated) beverages, refreshing (carbonated)
beverages, nectars, spritzers, fruit and vegetable juices, fruit or
vegetable juice formulations), instant beverages (for example,
instant cocoa beverages, instant tea beverages, instant coffee
beverages, instant fruit beverages), meat products (for example,
ham, fresh sausage or uncooked sausage formulations, seasoned or
marinated fresh or salted meat products), eggs or egg products
(dried egg, egg white, egg yolk), cereal products (for example,
breakfast cereals, muesli bars, precooked ready-made rice
products), dairy products (for example, milk beverages, butter milk
beverages, milk ice cream, yoghurt, kefir, fresh cheese, soft
cheese, hard cheese, dried milk powder, whey, whey beverages,
butter, buttermilk, products containing partly or completely
hydrolysed milk protein), products from soy protein or other soy
bean fractions (for example, soy milk and products produced
therefrom, fruit beverages with soy protein, soy
lecithin-containing formulations, fermented products such as tofu
or tempeh or products produced therefrom, soy sauces), products
made of other plant-based protein sources, for example, oat protein
beverages, fruit formulations (for example, preserves, fruit ice
cream, fruit sauces, fruit fillings), vegetable formulations (for
example, ketchup, sauces, dried vegetables, frozen vegetables,
precooked vegetables, vegetables preserved in vinegar), snack
products (for example, baked or fried potato crisps/chips or
products based on potato dough, extrudates on a maize or peanut
basis), products on a fat and oil basis, or emulsions of the same
(for example, mayonnaise, remoulade, dressings), other ready meals
and soups (for example, dried soups, instant soups, pre-cooked
soups), condiments, seasoning mixtures and, particularly,
seasonings, which are used, for example, in the snack industry.
[0055] Preferably, however, it is soft drinks or hot drinks or
instant drinks such as, for example, teas or fruit juices.
[0056] Pharmaceutical Preparations
[0057] The taste problem described above does not only appear in
foods with added non-sugar sweeteners, but also in pharmaceutical
products in which the frequently repulsive taste of the active
agent needs to be masked particularly carefully. Therefore, a
further subject-matter of the invention also relates to
pharmaceutical preparations, containing the preparations according
to the invention. Also in this case, it is preferred to add
component (a) and (b) as a mixture. Preferably, the pharmaceutical
preparations are liquid products, particularly cough syrup,
antipyretic agents, or antibiotics. Liquid forms of medication are
particularly used in children and where the masking of the bitter
taste of the active agent is technologically impossible, for
example, when applying capsules, tablets or other solid forms of
pharmaceuticals.
[0058] Both the foods and the pharmaceutical preparations may
contain dehydroabietic acid or a salt of dehydroabietic acid in
concentrations from about 1 to about 500 .mu.M, preferably, about
10 to about 250 .mu.M, particularly preferably about 20 to about
100 .mu.M.
[0059] Industrial Application
[0060] A further subject-matter of the invention relates to a
process for masking unpleasant taste impressions of sweeteners
and/or flavonoids and/or terpenes and/or alkaloids and/or mineral
salts as well as foods or pharmaceutical preparations containing
these substances, characterised in that a quantity of a
dehydroabietic acid, a salt of dehydroabietic acid and/or an
extract containing dehydroabietic acid is added to them.
[0061] Preferably, dehydroabietic acid, a salt of dehydroabietic
acid and/or an extract containing dehydroabietic acid is added in
quantities such that the concentration of dehydroabietic acid in
the product is from about 1 to about 500 .mu.M, preferably about 10
to about 250 .mu.M, more preferably about 20 to about 150 .mu.M and
particularly preferably about 50 to about 100 .mu.M.
[0062] The use of dehydroabietic acid, a salt of dehydroabietic
acid and/or an extract of dehydroabietic acid is also claimed for
masking unpleasant taste impressions of sweeteners and/or
flavonoids and/or terpenes and/or alkaloids and/or mineral salts as
well as foods or pharmaceutical preparations containing these
substances, in the process of which dehydroabietic acid, a salt of
dehydroabietic acid and/or an extract containing dehydroabietic
acid is added to these such that the concentration of
dehydroabietic acid in the product is from about 1 to about 500
.mu.M, preferably about 10 to about 250 .mu.M, particularly
preferably about 20 to about 100 .mu.M.
EXAMPLES
Example 1
[0063] Purification of Dehydroabietic Acid
[0064] 2 g of commercially available dehydroabietic acid (CAS
1740-19-8, purchased from Interchim, 211 bis AVENUE KENNEDY, BP
1140, 03103 MONTLUCON CEDEX, France) with a content of ca. 85% was
purified by preparative HPLC chromatography as follows. [0065]
Separation number: H-2074-B [0066] Stationary phase:
LichrospherSelect B, 10 .mu.m, 250.times.50 mm [0067] Mobile phase
A: 5 mMol ammonium formate buffer, set to pH 3.0 with formic acid
[0068] Mobile phase B: methanol--acetonitril 1:1 (v/v) with 5 mMol
ammonium formate [0069] Gradient: from 62% to 81% B in 57 min
[0070] Flow rate: 80 ml/min [0071] Detection: ELSD
[0072] Fractions containing the product were combined, the solvent
was evaporated in a vacuum, and the isolated dehydroabietic acid
was analytically characterised by means of H-NMR spectroscopy and
LC-MS. The identity of the isolated dehydroabietic acid was
confirmed by the NMR and the molecular mass, the purity was at
>98%.
[0073] The LC-MS method for analytical characterisation of the
isolated substance is summarised in the following Table 1:
TABLE-US-00001 TABLE 1 LC-MS method HPLC system PE Series 200 MS
system Applied Biosystems API 150 Data system Analyst 1.3
Stationary phase Merck Select B 250 .times. 4 mm, 5 .mu.m Flow rate
1 ml/min Detection (+/(-)-ESI, Fast-Switching-Mode ELSD (Sedex 75)
UV (Merck, 254 nm) Sample 10 mg/ml in DMSO concentration Injection
volume 30 .mu.l Mobile phase: A: 5 mM ammonium formate and 0.1%
formic acid B: acetonitrile/methanol = 1:1, 5 mM ammonium formate
and 0.1% formic acid (pH 3) Time [min] % A % B Gradient 00.0 85 15
30.0 0 100 35.0 0 100
Example 2
[0074] Production of an Extract containing Dehydroabietic Acid
[0075] 10 g commercially available colophony resin (purchased from
Alfred Galke GmbH, Am Bahnhof 1, 37539 Bad Grund, order number
36004) is extracted with a mixture of MTB ether and methanol (1:1
v/v), removing the solvent in a vacuum. The extract contains ca.
25% dehydroabietic acid.
Example 3
[0076] In-Vitro Assay
[0077] Human bitter receptor cells were established, and the
activation of these cells was quantified in order to detect, by
means of a cell-based test system, natural substances which reduce
the bitterness of bitter principles. The change of the
intracellular potassium level was measured for quantitation of the
activation of bitter receptor cells.
[0078] Establishment of the Cell-Based Measurement System
[0079] In order to detect substances with inhibitory activity, a
high-throughput cell-based in-vitro measurement system was
established. To this end, a human bitter receptor cell line was
used that was produced for this application based on Hochheimer,
A., Krohn, M., Rudert, K., Riedel, K., Becker, S., Thirion, C. and
Zinke, H. "Endogenous gustatory responses and gene expression
profile of stably proliferating human taste cells isolated from
fungiform papillae" Chem Senses, 39, 359-377 (2014).
[0080] Quantitation of the Intracellular Calcium Level in the
Cell-Based Measurement System
[0081] A human bitter receptor cell line having endogeneous bitter
receptors was used for a screening for antagonists in a
high-throughput format. The modulation of the intracellular
potassium level of the bitter receptor cells was quantitated by
means of the potassium-sensitive fluorescence dye Fluo-4 AM in a
microplate reader. The activation of the bitter receptor cells by
an agonist leads to an increase of the intracellular potassium
level. The quantity of mobilised calcium may be quantitated by
means of fluorescence, which increases in proportion with the
concentration of calcium. In case of inhibition by an antagonist,
the quantity of mobilised calcium is significantly lower, as is the
change of fluorescence induced by an agonist.
[0082] Performance of the process: Human bitter receptor cells were
cultivated in a cell cultivation medium in a steam-saturated
atmosphere at 37.degree. C. and with 5% CO.sub.2. The cells were
seeded at a density of 20,000 cells per well in a 96-well plate. 24
hours after seeding, the cells were dyed with 4 .mu.M of the
calcium-sensitive fluorescence dye Fluo-4 AM in HBSS (Hank's
Balanced Salt Solution) buffer, and the mobilisation of calcium in
living cells was measured in the microplate reader
(FlexStation.RTM.Systenn, Molecular Devices).
[0083] Screening for antagonists: Parallel to this, human bitter
receptor cells were stimulated with 1-5 mM of the bitter principle
saccharin (agonist) and with 10 .mu.M of the potential antagonists.
The mobilisation of calcium was measured in the microplate reader
at 37.degree. C.
[0084] Analysis: The mobilisation of calcium was quantitated as the
change of fluorescence (dF) after the addition of the substance,
taking into account the basal fluorescence (F0) as dF/F0. The
relative fluorescence units (dF/F0), measured after stimulation
with agonist and antagonist, were compared with the sole
stimulation by the agonist (positive control). In doing so, active
substances that are lowering the calcium level were identified.
Potential antagonists were repeatedly tested for their activity.
Data processing and analysis was performed with a software program
of the microplate reader (SoftMax.RTM.Pro Software, Molecular
Devices).
[0085] Determination of the Dose-Effect Relationship of
Antagonists
[0086] In order to determine the average inhibitory concentration
(IC50) of the antagonist, human bitter taste receptors were
stimulated with 0.1-25 .mu.M of the substance in the presence of
the agonist. A representative dose-effect relationship is
illustrated in FIG. 1. It indicates the dose-effect relationship of
dehydroabietic acid (BRA-C-00001371) in human taste cells when
stimulated with 1 mM saccharin (fluorometric detection) IC50
1.7+/-0.5 .mu.M.
Example 4
[0087] The Natural Taste of High-Purity Dehydroabietic Acid and of
Extracts containing Dehydroabietic Acid
[0088] The sensory evaluation of the samples was performed by a
panel of five experienced assessors. The sensory tests were
performed descriptively, randomized, blinded, and by means of a
"sip and spit" method. To this end, the sample is moved within the
mouth for a few seconds to evaluate its taste, and it is not
swallowed but expectorated. The results may be taken from the
following Table 2a:
TABLE-US-00002 TABLE 2a Tasting of high-purity dehydroabietic acid
Test method Descriptive evaluation, "sip and spit" method, blinded
and randomised samples, pH neutral Test sample Purified
dehydroabietic acid, purity >98% Panelists 3-5 experienced
assessors Preparation of the Three different concentrations (10
.mu.M, 50 .mu.M, samples and 100 .mu.M) dissolved in 0.5% ethanol,
comparative sample 0.5% ethanol in water Evaluation 10 .mu.M
neutral 50 .mu.M astringent, dry, slightly metallic taste 100 .mu.M
Slightly cooling feeling on the tongue, astringent, dry, slightly
bitter, not sweet, slightly masking the taste of ethanol
TABLE-US-00003 TABLE 2b Tasting of extracts containing
dehydroabietic acid Test method Descriptive evaluation, "sip and
spit" method, blinded and randomised samples, pH neutral Test
sample 1) purified dehydroabietic acid, purity >98% 2)
commercially available dehydroabietic acid, purity 85% (HPLC) 3)
extract with ca. 25% dehydroabietic acid Panelists 3-5 experienced
assessors Preparation of the Three different concentrations
(dissolved in samples 0.5% ethanol), comparison sample 0.5% ethanol
in water, final concentration of the ethanol below 0.5% Conc.
Sample 1 Sample 2 Sample 3 Evaluation 3 mg/l neutral neutral
neutral 7.5 mg/l neutral neutral slightly bitter 30 mg/l slightly
slightly More bitter than bitter bitter samples 1 and 2,
resinous
Example 5
[0089] Taste of the Combination of Dehydroabietic Acid with
Saccharin
[0090] The sensory evaluation of the samples was performed by a
team of five experienced assessors. The results may be taken from
the following Table 3:
TABLE-US-00004 TABLE 3 Tasting of high-purity dehydroabietic acid +
saccharin Test method Descriptive and discriminative evaluation,
"sip and spit" method, blinded and randomised samples, pH neutral
Test sample Purified dehydroabietic acid, purity >98% Panelists
3-5 experienced assessors, trained to recognise the taste of
saccharin and acesulfame-K Preparation of the Three different
concentrations of dehydroabietic samples acid (10 .mu.M, 25 .mu.M,
and 50 .mu.M) with 80 ppm saccharin (corresponding to a 3%
saccharose- equivalent) dissolved in 0.5% ethanol, 8 ppm saccharin,
comparison sample 0.5% ethanol in water, final concentration of the
ethanol below 0.5% Evaluation 10 .mu.M Slightly bitter aftertaste
25 .mu.M Less bitter with a very clear taste profile, less dry 50
.mu.M Less bitter, significantly less aftertaste than in 10
.mu.M
Example 6
[0091] Taste of the Combination of Dehydroabietic Acid with
Acesulfame-K
[0092] The sensory evaluation of the samples was performed by a
team of five experienced assessors. The results may be taken from
the following Table 4:
TABLE-US-00005 TABLE 4 Tasting of high-purity dehydroabietic acid +
acesulfame-K Test method Descriptive and discriminative evaluation,
"sip and spit" method, blinded and randomised samples, pH neutral
Test sample Purified dehydroabietic acid, purity >98% Panelists
3-5 experienced assessors, trained to recognise the taste of
saccharin and acesulfame-K Preparation of the Three different
concentrations of dehydroabietic samples acid (10 .mu.M, 25 .mu.M,
and 50 .mu.M) with 150 ppm acesulfame-K (corresponding to a 3%
saccharose equivalent) dissolved in 0.5% ethanol, 150 ppm
acesulfame-K comparison sample 0.5% ethanol in water, final
concentration of the ethanol below 0.5% Evaluation 10 .mu.M Very
full taste impression, significantly less bitter, masking the
astringent and metallic taste 25 .mu.M No bitter taste present any
more 50 .mu.M Clear taste impression, delays the increasing
formation of a bitter and acid taste.
Example 7
[0093] Taste Modulation of Black Tea
[0094] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 5:
TABLE-US-00006 TABLE 5 Tasting of Black tea Test method Descriptive
and discriminative evaluation, "sip and spit" method, blinded and
randomised samples, pH neutral Test sample Purified dehydroabietic
acid, purity >98% Panelists 5 experienced assessors Preparation
of the Two different concentrations of dehydroabietic samples acid
(25 .mu.M and 50 .mu.M) dissolved in ethanol, 1 tea bag of Earl
Grey, allowed to steep in 100 ml water for 10 min, addition of the
stock solution at ca. 20.degree. C., end concentration of ethanol
below 0.5%, comparison sample 0.5% ethanol in tea Evaluation 25
.mu.M Slightly less bitter than the comparison example 50 .mu.M
Significantly less bitter, a little less astringent than the
comparison example
Example 8
[0095] Taste Modulation of Green Tea
[0096] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 6:
TABLE-US-00007 TABLE 6 Tasting of Green tea Test method Descriptive
and discriminative evaluation, "sip and spit" method, blinded and
randomised samples, pH neutral Test sample Purified dehydroabietic
acid, purity >98% Panelists 5 experienced assessors Preparation
of the Two different concentrations of dehydroabietic samples acid
(25 .mu.M and 50 .mu.M) dissolved in ethanol, 3 g of Green tea,
allowed to steep in 250 ml water for 5 min, addition of the stock
solution at ca. 20.degree. C., end concentration of ethanol below
0.5%, comparison sample 0.5% ethanol in tea Evaluation 25 .mu.M
Slightly bitter taste that quickly fades 50 .mu.M As in 25 .mu.M,
but less astringent
Example 9
[0097] Taste Modulation of Grapefruit Juice
[0098] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 7:
TABLE-US-00008 TABLE 7 Tasting of grapefruit juice Test method
Descriptive and discriminative evaluation, "sip and spit" method,
blinded and randomised samples, pH neutral Test sample Purified
dehydroabietic acid, purity >98% Panelists 5 experienced
assessors Preparation of the Two different concentrations of
dehydroabietic samples acid (25 .mu.M and 50 .mu.M) dissolved in
ethanol, grapefruit juice (grocery store, from concentrate),
addition of the stock solution at ca. 20.degree. C., end
concentration of the added ethanol below 0.5%, pH of the test
sample 3.6, comparison sample 0.5% ethanol in grapefruit juice
Evaluation 25 .mu.M Significantly less bitter than the comparison
sample, clearly more aromatic 50 .mu.M Significantly less bitter
than the comparison sample, no negative influence of the fruit
taste
Experiments involving freshly squeezed grapefruit juice also
exhibited a significant reduction of the bitter taste without
negatively influencing the fruit aroma and the refreshing
tartness.
Example 10
[0099] Taste Modulation of a Mineral Salt Solution
[0100] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 8:
TABLE-US-00009 TABLE 8 Tasting of a mineral salt solution Test
method Descriptive and discriminative evaluation, "sip and spit"
method, blinded and randomised samples, pH neutral Test sample
Purified dehydroabietic acid, purity >98% Panelists 5
experienced assessors Preparation of the Two different
concentrations of dehydroabietic acid samples (25 .mu.M and 50
.mu.M) dissolved in ethanol, 2 g MgCl.sub.2 (Lohmann) dissolved in
100 ml water, addition of the stock solution at ca. 20.degree. C.,
end concentration of the ethanol below 0.5%, comparison sample:
mineral salt solution containing 0.5% ethanol Evaluation 25 .mu.M
Almost no difference to the comparison sample 50 .mu.M
Significantly less bitter than the comparison sample
Example 11
[0101] Taste Modulation of a Solution containing Potassium
Chloride
[0102] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 9:
TABLE-US-00010 TABLE 9 Tasting of a potassium chloride solution
Test method Descriptive and discriminative evaluation, "sip and
spit" method, blinded and randomised samples, pH neutral and 3.2,
comparison example: 0.5% ethanol in potassium chloride solution at
a corresponding pH value Test sample Purified dehydroabietic acid,
purity >98% Panelists 5 experienced assessors Preparation of the
Two different concentrations of dehydroabietic acid samples (25
.mu.M and 50 .mu.M) dissolved in ethanol, 1,500 mg KCl dissolved in
100 ml water, pH value was adjusted by citric acid, addition of the
stock solution at ca. 20.degree. C., end concentration of ethanol
below 0.5% Evaluation 25 .mu.M Almost no difference to the
comparison pH neutral sample (bitterness and saltiness) 25 .mu.M
Almost no difference to the comparison pH 3.2 sample (bitterness)
Saltier than the comparison sample More pronounced tartness than
the comparison sample 50 .mu.M Less bitter than the comparison
sample pH neutral
Example 12
[0103] Taste Modulation of a Stevia/Rebaudioside A Solution
[0104] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 10:
TABLE-US-00011 TABLE 10 Tasting of stevia/rebA solution Test method
Descriptive and discriminative evaluation, "sip and spit" method,
blinded and randomised samples, pH 3.2 and pH neutral Test sample
purified dehydroabietic acid, purity >98% Panelists 5
experienced assessors Preparation of the Dehydroabietic acid (25
.mu.M) dissolved in ethanol, samples 24 mg (stevia-rebA
commercially available, equivalent to 6% saccharose) dissolved in
100 ml water, pH value was adjusted by means of citric acid,
addition of the stock solution at ca. 20.degree. C., end
concentration of the ethanol below 0.5%, comparison example 0.5%
ethanol in stevia/rebA solution Evaluation 25 .mu.M Slightly less
bitter than the comparison pH neutral sample Sweeter and fuller
taste profile than the comparison sample 25 .mu.M Unpleasant taste
(bitterness and licorice pH 3.2 aroma) reduced, sweeter and fuller
taste profile than comparison sample
Example 13
[0105] Taste Modulation of a Naringin Solution
[0106] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 11:
TABLE-US-00012 TABLE 11 Tasting of the naringin solution Test
method Descriptive and discriminative evaluation, "sip and spit"
method, blinded and randomised samples, pH 3.2 and pH neutral Test
sample Purified dehydroabietic acid, purity >98% Panelists 5
experienced assessors Preparation of the Dehydroabietic acid (25
.mu.M) dissolved in ethanol, samples 300 mg naringin dissolved in
100 ml water, pH value was adjusted by means of citric acid,
addition of the stock solution at ca. 20.degree. C., end
concentration of ethanol below 0.5%, comparison sample 0.5% ethanol
in the naringin solution Evaluation 25 .mu.M More aromatic taste
profile than the pH neutral comparison example Unpleasant
aftertaste (astringency) clearly reduced 25 .mu.M Unpleasant
aftertaste reduced, less bitter pH 3.2 than comparison example,
same astringency as comparison example.
Example 14
[0107] Taste Modulation of Menthol in a Lozenge
[0108] The sensory evaluation of the samples was performed by a
team of five experienced assessors. Details may be taken from the
following Table 12:
TABLE-US-00013 TABLE 12 Tasting of menthol lozenges Test method
Descriptive and discriminative evaluation, "sip and spit" method,
blinded and randomised samples, pH 3.2 and pH neutral Test sample
Purified dehydroabietic acid, purity >98% Panelists 5
experienced assessors Preparation of the Dehydroabietic acid (25
.mu.M) dissolved in ethanol, samples 0.3% menthol were formulated
in a lozenge, pH value was adjusted by means of citric acid,
addition of the stock solution at >100.degree. C., end
concentration of the ethanol below 0.5%, comparison samples were
lozenges without dehydroabietic acid Evaluation 25 .mu.M Sweeter
and significantly lower pH neutral astringency than the comparative
example Fuller taste profile and better aroma than the comparative
example 25 .mu.M Astringency comparable, unpleasant pH 3.2
aftertaste reduced, less bitter than the comparative example
* * * * *