CA1042437A - Mixtures of dipeptides and purines as flavouring products - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

These are disclosed flavouring compositions comprising mixtures of (A) a cyclo-dipeptide or mixture of cyclo-dipeptides and (B) a purine deri-vative of the formula:

wherein R1 and R2 are each methyl or one is methyl and the other is hydrogen.
The compositions are useful in the flavouring of foodstuffs in genera, parti-cularly for imparting to them a bitter taste.

Description

Much attention has been devoted in the past to the reproduction of the gustative notes which typically define the bitter character of certain foodstuffs. In the flavour industry there is in fact a need to provide flavouring ingredients, or compositions thereof, for improving the flavours of beverages or solid foodstuffs, particularly those comprising cocoa or a cocoa imitating substitute, by imparting to said products the distinct bitter-ness which characterizes the natural cocoa products. Sofar, however, no satisfactory solution has been found to this problem.
The term "bitterness", as used in the course of the present descrip-tion, is deemed to define the gustative property possessed by a given sub-stance of developing a bitter taste.
Most of these substances find their origin in the vegetable kingdom and they often occur in a variety of plants, such as those belonging to the families of Gentianaceae, Compositae and labiates.
Equally, some foodstuffs having a base of animal origin possess a bitter taste when subjected to certain treatments.
The properly so called "bitter substances" do not include however the alkaloid derivatives of purine, whose bitter taste is very often viewed as a side effect /K. Herrmann, Deutsche Lebensmittel Rundschau, 68, 105~1972)~.
Most of the bitter substances known hitherto belong either to the class of terpenoids possessing a lactonic or ketonic functional group or to that of phenol derivatives, such as for instance the compounds of the fla-vanone series found in citrus fruits. Peptides possessing a bitter taste are also known and their organoleptic properties are described in several scientific publications, viz. Zeit. fur Lebensmittel-Untersuch. Forsch., 147, 64 ~1971); idem, 149, 321 ~1972); Agr. Biol. Chem., 34, 729 (1970);
J. Food Sci., 35, 215 ~1970).
Certain amino acids /see e.g.: Greenstein and Winitz, Chemistry of ~he Aminoacids, vol. I, J. Wiley (1961) p. 150/ as well as certain oligo-peptides tAnnual Meeting of the Agr. Chem. Soc. Japan, 42 ~1972)7 develop bitter gustative notes.

~09L;~37 In the course of the past few years, several ~heories have been advanced in order to better define the relationships which may exist between the chemical structure of a given substance and its developed bitterness rsee: Nature, 223, 97-9(1969)_7. However, it has to be clearly stated that in the presen~ state of our knowledge, it is impossible to predict the organo-leptic properties of a compound on the sole base of its chemical structure.
Not surprisingly therefore one may observe that the discovery of new flavouring ingredients, in particular of bitter flavouring ingredients, occurs very of~en in a pure accidental way.
According to ~he present invention, there is provided a flavouring composition which comprises as active ingredients (A) a cyclo-dipeptide or a mixture of dipeptides including a cyclo-dipeptide and (B) a purine deriva-tive of the formula:

Rl _ N ~ 7 N - R2 wherein each of symbols Rl and R2 represents a methyl radical or one of them represents a hydrogen atom and the other a methyl radical.

' ~1 .

1~ 437 ; It has no~ bcen surprisin~ly discovered that by thc use of the said rlavourin~ compositio.n Or the invention, it was pos~ible to modiry, improve or enhance the or~anoleptic propcrties Or - roodsturrsJ animal reèds, beveragcs, pharmaceutical preparations S and tobacco products. ~'ore particularly, it has been found that by the use Or the composition of the invention, it lras possible to improve or confer a characteristic bitterness to the products to which it.was added.
. The present invention also relates to roodsturrs and beverages ~hich comprise having added thereto a small but rlavour modifying amount Or said composition. -~
More particularly, the present invention relates to the aromatiza-tion of cocoa, cocoa products or cocoa imitating substitutes, and generally to improving their organoleptic properties. .
t5 A further obj~c~ o~ the present ~.n~ention is ~ process for enhanc-ing, improving or n~odirying the taste and the flavour Or cocoa, : cocoa products or cocoa imitating substitutes, ~Ihich process compris- :
es adding thereto a rlavouring composition comprising as active ingredients compounds A and B,.said A compound being a member Or the group consisting Or an aminoacid and an oligopeptide, or any mixture Or oligopeptides, said B compound bein~ a purine derivative o~
. rormula I.
By such a process Or the invention, it is pos.sible to corr~ctly r.e-- produce the gustaki~ s~nsation, typically bitter and astrin~ent, at one time, ~enerated in the human perceptive system by the absorp-tion Or cocoa containing foodsturrs.

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The cocoa rlavour has bccn thorou~hl~ studied in thc past and several solutions have bcen su~gested in or~lcr to recollsti~ute its natural aroma.
~or instance, US patent No.2,835,590 describes the preparation Or an artiricial chocolate rlavour by a process comprising treat-inS a saccharide with an oligopeptide containing 2 to 6 aminoacid units. Bitterness and astringency were in this case conrerred by the addition to the rlavour composition Or substances such as theobro~ine, carfeine, quinine or naringine, and tannins.
In the past, the bitter taste Or cocoa was attributed to the presence therein of theobromine and ca~feine ; more particularly, it was believed that its bitterness was dependent on the relative ~-~
proportions or the said purines ~H~rldbuch der Lebensmittelcnemie, vol. VI, Springer Verla~ (1970) p. 232 ; Deutsche LehensmittPl 15 Rundschau, 68, 140 (1972~
We have now discovered that the presence Or one or the other Or the said purines is a necessary, but insurricient condition ~or C reconstituting the cocoa rlavour. The presence Or ~ffr~br~n~
~ an oligopcptide; or a mixture Or oligopeptides, is equally ~-essential. Suitable oligopeptides include di-, tri-, tetra~
penta- and hexapeptides. Dipeptides are however prererred.
Among the variety o~ dipeptides which can be used as flavouring~
coingredients according to the invention, the rollowing open chai~ and cyclic peptides are of particular interest :
~ly-Ala, Gly-Yal,Gly-Leu, GlY.-Phe, Gly-Pro,Ala-Leu, Ala-Val, Ala-Phe,Ala-Pro, Val-Leu, Val-Phe,Val-Pro, ~', .

- - . ~.,,~
~ . ~ ., t ~ ~.~ .. - , . .. . .

Leu-Phe, Leu-Pro, Phe-Pro, Gly-Gly, Ala-Ala, Val-Val, Leu-Leu, Phe-Phe, as well as (Cyclo)-Gly-Ala- Ber., 39, 752 ~1906) ~Cyclo)-Gly-Val- Ann., 363, 136 ~1908) ~Cyclo)-Gly-Leu- Ber., _ , 2893 (1906) (Cyclo)-Gly-Phe- Ann., 363, 1 ~1908) ~Cyclo)-Gly-Pro- Ann., 363, 118 (1908) (Cyclo)-Ala-Leu- Ber., 39, 2893 (1906) (Cyclo)-Ala-Val- Ann., 363, 136 (1908) (Cyclo)-Ala-Phe- W. Schoeller, Dissertation Berlin (1906) ~Cyclo)-Ala-Pro- Ber., 37, 2842 (1904) (Cyclo)-Val-Leu- Ann., 363, 136 (1908) (Cyclo)-Val-Phe- Z. phys. Chem., 214, 63 (1933) (Cyclo)-Val-Pro- Hoppe-Seyler's Z. physiol. Chem., 132, 1 (1924) ~Cyclo)-Leu-Phe- Coll. C~ech. Chem. Comm., 32. 378 ~1967) ~Cyclo3-Leu-Pro- Ann., 363, 118 ~1908) ~Cyclo)-Phe-Pro- J. Biol. Chem., 155, 535 ~1944) ~Cyclo)-Pro-Asn- n.c.
~Cyclo)-Asn-Phe- Hoppe-Seyler's Z. physiol. Chem., 174, 76 ~1928) ~Cyclo)-Gly-Gly- J. Prakt. Chemie, 26, 175 ~1887) ~Cyclo)-Ala-Ala- Ann., 134, 372 ~1865) ~Cyclo)-Val-Val- Ann., 363, 136 ~1908) ~Cyclo)-Leu-Leu- Ann., 69 16 ~1849) ~Cyclo)-Phe-Phe- Ann., 219, 179 ~1883) -- 6 _ ;.. ~ .

In the course of the present description the cyclic dipeptides, also known under the name of diketopiperazines, are designated by the current abbreviation preceded by the term "Cyclo". For example, the term "Ala-Ala"
designates alanyl-alanine, a compound of ~ormula CH3-CH-C-NH-CH-COOH also defined as H-Ala-Ala-OH, N~12 CH3 whereas the term "(Cyclo)-Ala-Ala-" designates 2,5-dimethyl-3,6-diketo-piperazine, a compound of formula U `
/ 6 \
H3C-IH l~H

~ ~ 3 / HC2-CH3 C : .
O - - " ~'' '"'~ ~."

In the hereinabove list of compounds, immediately following the chemical name of each member~ there is indicated the literature reference giving a method for its preparation. Some of the said compounds are commer-cially available and one of them is new and identified by the abbreviation n.c. Its preparation as well as that of the known compounds, can be achieved by the synthesis illustrated by the following reaction scheme: -..

. ~ .

~4~437 ~ base 7 Z-Nll-cll-c-oH ~ Cl-C-OC2H5~ ? Z-NII-CH-C-O-C-OC2H5 ¦ R4 O
base ~ + H2N-CH-C-OCH3.HCl R13 ~o IR4 1l H2/Pd 13 ~ IR4 ~o H2N-CH-C-NH-CH-C-OCH3 < 6-NH-CH-C-NH-CH-C-OCH ~CO ~C H OH

¦ ~, cyclisation R -CH NH

\ lCI ~

In the above given scheme~ symbol Z represents a carbobenzoxylic radical of formula I
~ CH2 -O-C-.. . .

", 43~ ~and R3 and R4 designate the characteristic substituents present in the mole-cule of the par~icular aminoacid employed as starting material. Thus, each of symbols R3 and R~ canJ for instance, represent a hydrogen atom or an alkyl radical such as methyl, ethyl, propyl, isopentyl, 2-methyl-butyl or 2-phenyl-ethyl.
To exemplify the preparation of the dipeptides of the invention, we indicate hereinbelow the method followed Lsee also: ~lelv. Chim. Acta, 34, 874 (1951) and J. Org. Chem~, 33, 864 (1968)/.
5 mM of the N-carbobenzoxylic derivative of the chosen aminoacid were dissolved in a solution of 7.5 mM of N-methyl-morpholine in 20 ml of -anhydrous tetrahydrofuran. The solution thus obtained was cooled to about -10C and at this temperature 5.2 mM of ethyl chlorocarbonate were added ~ -thereto; then~ the reaction mixture was left under stirring for 15 minutes.
A suspension of 5 m~ of the methyl ester of the aminoacid hydro-chloride chosen for the coupling and 7.5 mM of N-methyl-morpholine in 10 ml of dimethylformamide was added to the said mixture whose temperature was slow-ly increased up to about 20 - 25C while stirring.
By fil~ration and evaporation of the volatile portions under vacuum ~bath temperature of about 60C), there was obtained a residue, which was then dissolved in about 500 ml of ethyl acetate.
The resulting clear solution was washed twice with 10 ml of a 10%
aqueous citric acid solution, then with 15 ml of a 7% solution of sodium bi- -~
carbonate and finally with 3 portions of brine. After drying over Na2S04 and evaporation, the methyl ester of the N-carbobenzoxylic dipeptide was obtained as residue. This compound can be converted into its open chain or cyclic di-peptide according to one of the following methods:

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~34;~43~
A. Preparation of an open chain di~eptide The methyl ester of the N-carbobenzoxylic dipeptideJ prepared as indicated above, was saponified accordin~ to the method of Wieland et al.
/~ieb. Ann. 1973, 45/ by dissolving the raw product in 80 ml of dioxane and 6 ml of lN NaOll and keeping then the resulting clear solution at room tempera-ture under stirring for 1 hour.
The volatile portions were evaporated under reduced pressure at 20C and the obtained residue was dissolved in 100 ml of water and washed twice with two portions of 50 ml each of ethyl acetate.
The aqueous phase was acidified with 2N HCl to pH 1 and extracted then with 4 fractions of 100 ml each of ethyl acetate. After the usual treat-ments of drying over Na2SO4 and evaporation, there was obtained a residue which was then subjected to a catalytic hydrogenation in the presence of palladium on charcoal.
B. Preparation of cyclic dipeptides :
The methyl ester of the N-carbobenzoxylic dipeptide, prepared as indicated above, was dissolved in 150 ml of methanol and subjected to a catalytic hydrogenation in the presence of palladium on charcoal. Ten minutes were generally sufficient for achieving a complete conversion; the solutionJ
however, was kept under hydrogen atomosphere during 3 hours.
After filtration, an evaporation of the clear filtrate gave a resi-due mainly comprising the methyl ester of the open chain dipeptide which could then be cyclised according to the method of Nitecki et al.J J. Org. Chem.J
33, 864 (1968) by heating it in about 200 ml of a 96:4 mixture of toluene and sec.-butanol during 3 hours.
The cyclic dipeptide, obtained by evaporation of the volatile por-tions, was crystallized in methanol.

. ~ . , . . -. . .: , ~Z~37 ~Cyclo)-Pro-Asn-, which is a new compound, exhibits the following physical data:
m.p. 200 - 202 C ; _ a_~ O(H20) = -84.8 C
NMR ~DMSO) : 1.78 ~4H,m) ; 2.40 ~2H,m) ; 3.70-4.40 (4H,m) ;
6.75 ~lH, broad s) ; 7.28 ~lH, broad s) ;
7.80 ~lH,s) ~ ppm.
The compound can be represented by the following structural formula O '"`' ~ ". "

H

CH2-C-NH2 ~

O , ~3S-acetamido-1.4-dioxo-9S-2,5-Pyrrolopiperazine or ~Cyclo)-L-Pro-L-Asn). `-A diastereoisomer of the said dipeptide had ~he following physical characteristics:
m.p. 249 - Z50C ; r~ 20~H2o) = -61,7C
MMR ~DMS0) : 1.75 ~4H,m) ; 2.38 ~3H,m) ; 3.65-4.20 ~3H,m) ;
7.30 ~lH, broad s) ; 7.92 ~lH,d,J = 4 cps) ~ ppm. ~ ;
The following symmetrical cyclic dipeptides were prepared in accordance with the procedure described by E. ~ischer /see : Ber., 39, 453 ~1906)/ :
~Cyclo)-Gly-Gly-, ~Cyclo)-Ala-Ala-, ~Cyclo)-Val-Val-, ~Cyclo)-Leu-Leu-, ~Cyclo)-Phe-Phe-.

~34~37 Thus, the methyl esters of the chosen aminoacids were heated at about 110C for 3 hours. The reaction mixture solidified and the product was purified by crystallization in methanol.
Among the variety of useful oligopeptides in accordance with the invention, cyclic dipeptides are preferred.
We have observed, ~oreover, that the most interesting flavouring effects were obtained by the use of compositions containing a mixture com-prising at least two of the aforesaid cyclic dipeptides, the relative propor-~ions of which can vary within a wide range.
According to a preferred embodiment of the invention, theobromine is used as preferred purine.
A particular embodiment of the present invention is a flavouring composition comprising as ~ctive ingredient a cyclic dipeptide (diketopipera-zin~) in combination with theobromine.
A more particular embodiment of the present invention is a flavour-ing composition comprising as active ingredient at least two cyclic dipeptides in combination with theobromine.
The proportions of the compositions of the invention to be used to achieve an interesting flavouring effect can vary within wide limits. For instance, proportions of the order of 5 - 10 ppm by weight based on the total weight of the flavoured materials to which they are added, can already produce perceptible effects.
Preferably, however, these proportions are from about 50 to 500 ppm.
Higher proportions can be used whenever special effects are desired.

:

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10~ 37 When the compositions of the invention are used to cr~ate novel flavouring compositions, these proportions can be as high as about 15%, or even moreJ but preferentially up to 1% of l:he weight of the composition.
The ratio between the respective proportions of each flavouring ingredient in the compositions of the invention can equally vary within wide limits.
Whenever a composition is constituted by a mixture comprising two dipeptides and theobromine, particularly suitable weight ratios of the respective pro- ~-portions of each ingredient are of between about 1:1:1 and 1:3:5. For ex-ample, the following standard compositions were prepared by mixing together the given ingredients in the proportions indicated as parts by weight:
1. (Cyclo)-Phe-Phe- 10 (Cyclo)-Phe-Val- 20 Theobromine 100

2. (Cyclo)-Phe-Gly- 30 ~Cyclo)-Ala-Val- 30 Theobromine 100 Owing to the presence of one or more asymmetrical carbon atoms in their molecule, the aminoacids or the polypeptides of the invention can occur in the form of different configurational isomers. Thus, for instance the term "Ala-Phe" or "(Cyclo~-Ala-Phe-" designates one of the following isomeric peptides: ~ -D-Ala-L-Phe and(Cyclo)-D-Ala-L-Phe-D-Ala-D-Phe (Cyclo)-D-Ala-D-Phe-L-Ala-D-Phe (Cyclo)-L-Ala-D-Phe-L-Ala-L-Phe (Cyclo)-L-Ala-L-Phe-, respectively.

~, ~ . . . . . .. . ~

~(~4;~43~ :
Owing to the similarity of their organoleptic characters, each o~
the said isomers can be replaced by one of the others, or even mixtures there-of, without noticeably affecting the achieved flavour.
The preparation of the different confi~urational isomers of cyclic dipeptides is effected in accordance with the same procedure as that given above - see reaction scheme -. For example, the preparation of the four isomers of ~Cyclo~-Ala-Phe- was carried out by coupling e~uimolecular amounts of the N-carbobenzoxylic derivative of L-alanine and the racemic methyl ester of phenylalanine hydrochloride on one hand, and the N-carbobenzoxylic deriva-tive of D-alanine and the racemic methyl ester of phenylalanine hydrochloride on the other hand. Two pairs of diastereoisomers were thus obtained which, upon separation by means of column chromatography on silica gel, gave the four compounds in their pure isomeric form.
The following table summarizes the different isomeric forms of two members of the above mentioned class of cyclic dipeptides, viz. (Cyclo)-Ala-Phe- and (Cyclo)-Ala-Val-. Together with their analytical data, mention has been made of the nature of the starting materials used for their prepara-tion.

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- ~ , , ~2~37 The invention is better illustrated but not limited by the follow-ing examples.
EXAMPLES
A. A flavouring composition was prepared by mixing together during 1 hour under vigourous stirring the hereinbelow given oligopeptides and theobromine in 100 ml of commercial cow mi:Lk.
To-the obtained solution, there were added 0.08 ml o a 10% alcoholic solution ~95% ethanol) of commercially available cocoa flavour composition without bitter taste (~irmenich SA, Geneva, Switzerland, No. 51490) and 2_g of soluble cocoa powder having a bland taste.
The taste of the beverage thus obtained was evaluated by comparison with a beverage prepared by dissolving 4 g of soluble cocoa powder in 100 ml of milk.
The flavour experts have unanimously stated that the beverage flavoured with the flavouring composition above described presented a natural cocoa flavour, par~icularly with respect to its bitter and astringent character.
Its aroma was in fact to a great extent similar to that developed by the bever-age prepared with soluble cocoa alone. -i~
- B. A second procedure o~ degustation consisted in evaluating the hereinbelow given compositions in mineral water ~type: ~vian ~ by comparison with solutions prepared by dissolving 2 g of soluble cocoa powder in 50 ml of mineral water. The proportions used were the following:

3 mg of oligopeptide 6 mg of theobromine 30 ml of mineral water :

Trademark , : . : ' :~ ::
.

1~4Z437 In the following table the different kasted compositions are clas-sified according to a relative scale of valuesJ viz. "strongly bitter", "bitter" and "slightly bitter".

.. . . .

, . ~am~lc Gomposit.~on Evalllation __ . .., ~ , 11 mg (Cyclo)-Pi~e--Phe-2 ~n~ (Cyclo)-PIle-Val stron~ly bitter . 10 mg theobromine _ _ _ - .
2 1.5 mg (Cyclo)-Phe-Phe-. . 3 mg (Cyclo)-Ala-Val- strongly bitter 10 m6 theobromlne .

. _. _ . .. __ . ~
- . 3 3 mg (Cyclo)-Phe-G]y 3 mg (Cyclo)-Ala Val- strongly bitter 10 mg theobromine .
. _ . _ ..
. . 4 3 mg (Cyclo)-Pro-Ala- .
: 3 mg (Cyclo~-Gly-Gly- bitter . . lO mg theobromine .
. ~ .
5 3 mg (Cyclo)-Ala-Gly- . - . :
. 3 mg (Cyclo)-Leu-Leu- bitter . 10 mg theobromine- .
. . , ..... _ - -63 mg (Cyclo)-Phe-Pro- .
. 3 mg (Cyclo)-Gly-Gly- slightly bitter 10 mg theobromine .
.~ ,, ,~
7 3 mg (Cyclo)-Phe-Pro-3 mg (Cyclo)-Leu-I,eu- bitter 10 mg theobromine .~ ,_.. _ __ _ . _ 1"'-,, .. . 1~ ~

" ' ' ' , , :

...................................
___ ... . ... __ . ..... .
83 mg (Cyclo)-Leu-Cly- .
3 ~G (c~clo)-pro-Asp- bittcr 10 mg theobromine . . . ... _._.. __ .
93 mg (Cyclo)-Val-Gly- . .
3 mg (Cyolo)-Phe-Leu-slightly bitter . 10 mg theobromine -~
. .................. . . .
~ . .. _ . ....._ ~ ___ .
103 mg (Cyclo)-Ala-Pro- .
. 3 mg (Cyclo)-Phe-Val-strongly bitter . 10 mg theobromine . .
~ ... .... .
11 5.5 mg (Cyclo)-Phe-Val- . .
. 10 nig theobromlne strongly bitter - ,.,,, . .
?5~5 mg (Cyclo)-Leu-Gly- . .
. . 10 mg theobromine slightly bitter .................. ,.... . . . . ~, , ~, .. . . . - . . -13~ 11 mg Val-Phe- : .
.~ 15 .10 mg theobromine slig}tly bitter . .
., .. _ . ~ . ~ ~.
14 3 mg (Cyclo)-Phe Gly~ .
. ~ 6 me theobromine bitter :
,.. ., . . ~
. ~ 15 3 mg (Cyclo)-Phe-Ala- :
6 mg theobromine strongly bitter . , ... ._ . . ....... _ . ~
'' ` ~ :

.
r lG4;~3~
_ ~ . ......... .. ~ .
16 3 m~ (Cyclo)-rhc-Leu-. 6 mg theobromine strongly bitter ............. '........... .
r _ ~ __ ._ __ 17 3 ~ (cyclo~-rh~-val- .
6 me theobromine strongly bitter ~ .. _ .
518 3 mg (Cyclo)-Phe-Pro-. 6 mg theobromine slightly bitter .. , ,.,_ _ .
. 19 3 mg (Cyclo)-Phe-Phe-- . . 6 mg theobromine strongly bitter _. _ - ._ . _ . .
. 20 3 me (Cyclo)-Leu-Leu- . . :
6 Dlg theobromine bittcr . .. .. -............... . . -~
_. ------- - . ._ 21 3 mg (Cyclo)-Leu-Val-- . 6 mg theGbromine . bitter ~ :
. . . ................................... . ~ :
-~ ... _ ~_ -.. __ . _ _ 22 3 mg (Cyclo)-Leu-Ala- .
~ mg theobromine bi~ter : .
_ .. ....... ~:
15 23 3 mg (Cyclo)-Leu-Pro- . ~ :
. 6 mg theobromine sli~htly bitter ' . . . . .
, ~ . ___ _. . . _ . _ . . _ , . 24 3 mg (Cyclo)-L-Val-L--Ala-_ 6 m theobromine bitter :.

~ .
r '

4~37 .
.. _. _ ........ ,. . ~
: 25 3 mg (Cyclo)-J.-'Jal-L~aly--6 m&, theobrom;neslightly bitter .. _ .... .. ~ . . _ ._ 26 3 mg (Cyclo)-I.-Ala-L-~la-6 me theobromineslightly bitter . . ~ . ~ .
27 3 mg (Cyclo)-L-Ala-L-Gly-.
. 6 mg theobromineslightly bitter -- .... .. _. ~ . _~ _ .... .. ~ ~
8 3 mg (Cyclo)-L Ala-L-Pro- :
6 mg theobromine slightly bitter ............. .... . . _ .
- . 29 3 mg (Cyclo)-L-Gly-L-Gly-6 me theobromine .slightly bitter .... _ ...... ~ ~ . ............ ;~ ~
3o 3 mg (Cyclo)-L-Gly-L-Pro- ~ :
. . 6 m~ theobromine slightly ~itter . . ... ............ .
31 3 mg (Cyclo)-L-Pro-L-Asn- . : .
.- 6 mg tbeobromine bitter ._... .. ~ .... _ 32 3 mg (Cyclo)-L-Pro-L-Asp-- . . 6 mg theobromine slightly bitter . . _ . . ._ . ._ ... __ . . .
33 3 mg (Cyclo)-D-Phe-Val-. ' ~ 6 mg theobromine strongly bitter ...........................
. , _ _ .~ :

- ~

: _ 21 r . .... ~ . . .. . . . .,~.. . ~ . .. .

~4~37 __ __ - . . . _ . I
31~ 3 mg (Cyclo)-l'hc-V-Val-6 me theobromine strongly bitter .
r __ _ _ _ __ .
3 mG (Cyclo)-D-I"lc-D-Val- .
. 6 mg theobrornille ~trongly bitter _ . _ , ~, 36 3 mg (Cyclo)-D-Val-Ala-. . 6 mg theobromine bitter ~ ,..... .... . .
37 3 mg (Cyclo)-Val-D-Ala- .
. 6 mg theobromlne . bitter ~ :
. , ~
38 3 m~ (Cyclo)-D-Val-D-Ala- : :
. 10 . 6 me theobromine bitter :
., . .................. . -_ ~ _ . 39 3 m~ (Cyclo)-Pro-D-Asn- . .
- . 6 mg theobromine bitter . . .. ,.,,.,........ _ . ~
. 40 3 mg (Cyclo3-Asn-Phe- .
. _ 5 m6 theobromine bitter ~.

,, ~ ' .

~ _ 22 .
., ' - ~ I

t~ ~;

Claims (11)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A flavouring composition which comprises as active ingredients (A) a cyclo-dipeptide or a mixture of dipeptides including a cyclo-dipeptide and (B) a purine derivative of the formula:

I

wherein each of symbols R1 and R2 represents a methyl radical or one of them represents a hydrogen atom and the other a methyl radical.

2. A flavouring composition according to claim 1 wherein the mixture of dipeptides comprises two cyclic dipeptides.

3. A flavouring composition according to claim 1 wherein the purine derivative is theobromine.

4. A flavouring composition according to claim 1, 2 or 3, wherein the cyclic dipeptide is a member selected from the group consisting of (Cyclo)-Phe-Val-, (Cyclo)-Phe-Leu-, (Cyclo)-Phe-Ala-, and (Cyclo)-Phe-Phe.

5. A flavouring composition according to claim 2 wherein the mixture comprising two cyclic dipeptides is selected from:

a. (Cyclo)-Phe-Phe-(Cyclo)-Phe-Val-b. (Cyclo)-Phe-Phe-(Cyclo)-Ala-Val-c. (Cyclo)-Phe-Gly-(Cyclo)-Ala-Val-d. (Cyclo)-Ala-Pro-(Cyclo)-Phe-Val-.

6. A foodstuff or a beverage having added thereto a flavouring composi-tion as defined in claim 1, 2 or 3.

7. Cocoa, cocoa product or cocoa imitating substitute having added thereto a flavouring composition as defined in claim 1.

8. Cocoa, cocoa product or cocoa imitating substitute according to

claim 8 wherein the flavouring composition is added in an amount of from about 50 to 500 ppm by weight based on the weight of the flavoured product.

9. A process for modifying, improving or enhancing the organoleptic properties of foodstuffs, animal feeds, beverages, pharmaceutical preparations and tobacco products, which comprises adding thereto a flavouring effective amount of a composition as defined in claim 1.

10. A process for modifying, improving or enhancing the organoleptic properties of cocoa, cocoa products or cocoa imitating substitutes which comprises adding thereto a flavouring effective amount of a composition as defined in claim 1.

11. 3-Acetamido-1,4-dioxo-2,5-pyrrolopiperazine.