EP0832103A1 - Cocoa flavour precursor peptides, dna encoding them, processes for producing the peptides, and their use for generating cocoa flavour - Google Patents

Cocoa flavour precursor peptides, dna encoding them, processes for producing the peptides, and their use for generating cocoa flavour

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Publication number
EP0832103A1
EP0832103A1 EP96917366A EP96917366A EP0832103A1 EP 0832103 A1 EP0832103 A1 EP 0832103A1 EP 96917366 A EP96917366 A EP 96917366A EP 96917366 A EP96917366 A EP 96917366A EP 0832103 A1 EP0832103 A1 EP 0832103A1
Authority
EP
European Patent Office
Prior art keywords
peptide
cocoa
peptides
flavour
cocoa flavour
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96917366A
Other languages
German (de)
English (en)
French (fr)
Inventor
Soren Rasmussen
Mogens Bach
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aarhus Oliefabrik AS
Original Assignee
Aarhus Oliefabrik AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aarhus Oliefabrik AS filed Critical Aarhus Oliefabrik AS
Publication of EP0832103A1 publication Critical patent/EP0832103A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
    • A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
    • A23G1/56—Liquid products; Solid products in the form of powders, flakes or granules for making liquid products, e.g. for making chocolate milk, drinks and the products for their preparation, pastes for spreading or milk crumb
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/20—Synthetic spices, flavouring agents or condiments
    • A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
    • A23L27/215—Synthetic spices, flavouring agents or condiments containing amino acids heated in the presence of reducing sugars, e.g. Maillard's non-enzymatic browning
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
    • A23G2200/00—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents
    • A23G2200/10—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF containing organic compounds, e.g. synthetic flavouring agents containing amino-acids, proteins, e.g. gelatine, peptides, polypeptides

Definitions

  • Cocoa flavour precursor peptides DNA encoding them, processes for producing the peptides, and their use for generating cocoa flavour
  • This invention concerns peptides which are cocoa flavour precursors, DNA encoding these peptides, vectors contain ⁇ ing the DNA, host cells transformed therewith, and proc ⁇ esses for producing the peptides as well as their use for generating cocoa flavour.
  • the peptides are isolated and characterized from West African cocoa beans isolated from the cocoa tree (Theobroma cacao) .
  • Cocoa beans are seeds in cocoa pods which, after harvest ⁇ ing, are freed from the pods and subjected to a fermenta ⁇ tion process at or near the cultivation site, following which the greater part is exported for industrial proc- essing. Fermented cocoa beans are roasted, giving rise to the characteristic chocolate or cocoa flavour. The subse ⁇ quent grinding produces cocoa mass which is included as a main component in the chocolate production. Frequently, part of the cocoa mass is pressed, resulting in cocoa butter and cocoa powder, respectively.
  • the fermentation process generates heat, ethanol and in particular acetic acid, and the microorganisms as such participate only indirectly in the process.
  • the heat ac- tivates e.g. protein, oligosaccharide and polysaccharide cleaving endogenous enzymes, which are again inactivated in the last part of the fermentation process by rela ⁇ tively large amounts of acetic acid.
  • Acetic acid diffuses into the fermented beans and, in addition to direct in- fluence on the degradation pattern and the rate, also ex ⁇ erts an indirect influence. The latter effect consists in changed location of both storage protein and in lipid in the beans. The result of the fermentation is thus i.a.
  • peptides serve as flavour precursors .
  • Cocoa and chocolate may be considered to belong to the range of food products whose flavour cannot be character ⁇ ized by a single or a few flavour components. Other exam ⁇ ples are boiled, roasted and grilled meat, baked bread and roasted coffee. The base flavour appears in all these food products as the overall impression of a balanced composition of many components.
  • Cocoa flavour mainly consists of volatile components, but the sensory experience is a combination of taste and smell sensation. Mainly two groups of chemical compounds contributing to the flavour sensation are formed during roasting. These are aldehydes which are formed by oxida- tive deamination of amino acids, and pyrazines formed as Maillard reaction products.
  • the patent represents one of the earliest lit ⁇ erature references for cocoa flavour substitutes from other raw materials, and is drafted in very broad terms.
  • DDR Patent No. 205 815 published on January 11, 1984, which preferably concerns enzymatically produced protein hydrolysates of gelatine and wheat gluten.
  • coli TGI (Amersham) was transformed with the vector, and the fusion protein was expressed (Sikorski, R.S. & Hieter, P. (1989) Genetics 122. 19-27).
  • the fusion protein was isolated by means of a glutathione "Agarose”® affinity column. Fusion protein so isolated and containing blood factor Xa cleavage site was cleaved with factor Xa and applied to the affinity column once more, whereby glutathione-S-transferase was retained on the column, and the peptide of interest was eluted.
  • the eluate was gel-filtered on a "Superdex®75" column (Pharmacia) by means of Pharmacia FPLC equipment. The fraction containing the peptide was rechromatographed on reverse phase column, following which the identity of the peptide was confirmed by means of mass spectrometry and amino acid sequence determination by Edman degrada ⁇ tion.
  • the invention provides a cocoa flavour pre- cursor peptide selected from an isolated peptide with the amino acid sequence:
  • the peptide of the invention is selected from fragments of the peptide with the above-mentioned sequence containing 2-9 amino acid residues calculated from the alanine residue No. 2, and it is preferably a nonapeptide with the amino acid sequence:
  • the invention also comprises a DNA isolate comprising a DNA sequence encoding a peptide as stated above, which isolate, however, does not include the coding sequences of the 67 kD, 47 kD and 31 kD cocoa proteins.
  • the DNA isolate of the invention comprises in particular the DNA sequence: 5'-AAR-GCN-CCN-TM-TC _ CCN _ GGN _ GAy _ GTN _ ⁇ _ GTN _3, or parts thereof of at least two codons in reading frame from the 5 '-terminus, and preferably the DNA sequence:
  • a particularly useful DNA isolate of the invention which comprises the coding sequences of a blood coagulation factor Xa cleavage site and of the above-mentioned non- apeptide as well as various restriction sites, is useful for ligation in vectors which contain a gene encoding a larger protein, so that these express a fusion protein which is easier to purify, and from which the nonapeptide can easily be released by factor Xa.
  • This DNA isolate has the DNA sequence:
  • the invention moreover comprises vectors which contain the sequence of one of the above-mentioned DNA isolates, and in particular expression vectors which contain one or more copies of such a sequence operably linked to con ⁇ trol sequences which are recognized by a host cell trans ⁇ formed with the vector.
  • recombinant host cells transformed with these vec ⁇ tors are comprised by the invention.
  • Such host cells may be prokaryotes, e.g. Escherichia coli, or eukaryotes, e.g. yeast, mycelial fungi or cell lines of multi-cell organisms.
  • Yeast which is a well-known microorganism widely used in the food industry, must be considered par ⁇ ticularly useful for producing cocoa flavour precursor peptides of the invention.
  • the invention moreover comprises various processes for producing the peptides of the invention.
  • the one best suited for industrial production of the peptides viz. by cultivation of a culture of a recombinant host cell, as stated above, and isolation of the resulting peptide from the cultivation mixture.
  • the peptides of the invention may also be pro ⁇ quizzed by chemical synthesis from the individual amino ac ⁇ ids .
  • the use of the peptides of the invention for producing cocoa flavour is also comprised by the invention.
  • the invention comprises cocoa flavour produced by mixing of one or more peptides of the invention with predominantly reducing saccharides and amino acids and subsequent heat treatment of the mixture for 1-60 min at 100-200 °C, preferably for 5-15 min at 110-150 °C.
  • the quantitative proportion between peptide(s), saccharides and amino acids is usually peptide(s) 30-90% by weight saccharides 10-40% by weight amino acids 0-30% by weight and preferably peptide(s) 50-80% by weight saccharides 15-35% by weight amino acids 5-15% by weight based on the total amount of these ingredients .
  • the sac ⁇ charides in the mixture may practically consist of fruc ⁇ tose or glucose or mixtures thereof, preferably a mixture of fructose and glucose in a weight ratio from 3:1 to 1:3.
  • the invention additionally comprises food products, cos ⁇ metic products and pharmaceutical products which have added thereto or contain a cocoa flavour, as stated above.
  • the food products may be choco ⁇ late, confectionery, pastry or soft drinks.
  • a particular embodiment of such products has been achieved in that during production they have been mixed with one or more peptides of the invention and, if necessary, predomi ⁇ nantly reducing saccharides and amino acids and then sub- jected to a heat treatment for 1-60 min at 100-200 °C, preferably for 5-15 min at 110-150 °C.
  • Figure 1 shows a typical reverse phase chromatogram of an extract of defatted and autolyzed cocoa beans with 70% aqueous methanol .
  • the nonapeptide Ala-Pro-Leu-Ser-Pro- Gly-Asp-Val-Phe having a particularly high cocoa flavour potential is isolated from the peak of the chromatogram which is marked by an arrow.
  • Figure 2 shows a reverse phase chromatogram of said nona ⁇ peptide produced by a chemical synthesis.
  • Figure 3 shows a reverse phase chromatogram of a peptide material isolated from an E. coli strain which has been transformed with a plasmid containing the code of said nonapeptide sequence. The nonapeptide is detected in the peak of the chromatogram which is marked by an arrow.
  • MOBILE PHASE Acetonitrile gradient, 0-20% acetonitrile of 30 min followed by 20-100% acetonitrile of 10 min in 0.1% trifluoroacetic acid (TFA).
  • TFA trifluoroacetic acid
  • Ripe fresh cocoa pods from the Gold Coast, Ivory Coast were used for processing as described in this example. What was involved was a hybrid, widely distributed in the region, between two traditional cocoa tree types, Criollo and Amelonado, representing the most important African Forastero type.
  • cocoa pods were divided into two halves by a sterile scalpel . The pulp was removed, and the beans were frozen in liquid nitrogen before drying in a freeze drier.
  • the acetone residue was then washed with cold 0.05 M cit ⁇ rate buffer admixed with 0.15% thioglycolic acid and 10 mM EDTA at pH 4.0. The washing procedure was repeated with a large excess of buffer.
  • the acid washed acetone residue thus produced and containing the greater part of the proteins and protein-related compounds (including en- dogenic enzymes) occurring in the beans was incubated with stirring at 50 °C in 0.2 M citrate buffer admixed with 0.5% thiogycolic acid at pH 4.0.
  • PVPP polyvinyl polypyrrolidone
  • Peptides and amino acids in the extract were then bound to washed and equilibrated strong cation exchanger ( "Dowex® 50W” ) , about 2.5 ml of wet ion ex- changer per gram of defatted bean. Then washing was per ⁇ formed in sequence with 20 and 80% 2-propanol followed by water to remove residual alcohol.
  • the peptide fraction was liberated by basic elution (pH 10.7-11.0), and after the elution the pH value was lowered as quickly as possi- ble to about 7 by addition of HC1.
  • the fraction was de ⁇ salted by means of cation exchanger and analyzed by re ⁇ verse phase chromatography (RPC).
  • RPC re ⁇ verse phase chromatography
  • bound peptides were eluted with ammoniumhydroxide (pH 12), which was subsequently removed as well as possible either by placement in an incubator under vacuum at 40 °C overnight or by freeze-drying.
  • the peptide fraction obtained by 24 hour autoproteolysis and isolation as described above was analyzed by means of RPC. Use was made of PEP-RPC, HR 16/10 (Pharmacia) as a stationary phase and acetonitrile gradient (0-20% aceto ⁇ nitrile of 30 minutes and 20-100% of 10 minutes) in 0.1% TFA (trifluoroacetic acid) as a mobile phase with a flow rate of 7 ml/min, and UV detection (214 and 280 nm) .
  • the FPLC system of Pharmacia was used, and a typical chroma ⁇ togram of the said autolysate appears from figure 1.
  • the eluate was divided into three fractions, corre ⁇ sponding to hydrophilic and hydrophobic fractions and an intermediate fraction, respectively. It was found in roasting tests and subsequent sensory evaluation that the flavour potential was clearly best in the hydrophobic fraction and poorest in the hydrophilic fraction.
  • thin layer roasting was used, as described by Mohr (Mohr, W. (1970) Fette, Seifen, Anstrichstoff 8 . , 695-704) comprising heat treatment at 130 °C for 8 min ⁇ utes.
  • the peptide/protein fraction to be tested was roasted together with fructose, glucose and amino acid mixture in a weight ratio of 10:3:1:2. Samples of 20 mg were roasted (in some cases smaller amounts had to be roasted) , and to ensure good contact the samples were wetted and dried in vacuum at 40 °C before roasting.
  • the amino acid mixture consisted of (% by weight) Alanine 10
  • Fraction 38 was held to have a great flavour potential and was found to contain the above-mentioned nonapetide.
  • the two strands were purified by polyacrylamide gel elec- trophoresis (PAGE) on 6% acrylamide gels containing urea (Sambrook, J. , Fritsch, E.F., and Maniatis, T. , (1989) In: Molecular Cloning - A Laboratory Manual ' . 11.23-11.28. 2nd ed., Cold Spring Harbor Lab. Press). Purified oli ⁇ gonucleotide strands were annealed to form the following double strand:
  • the first five nucleotides consti ⁇ tute the greater part of the Bglll restriction site, which is AGATCT.
  • the subsequent T is inserted to provide a correct reading frame.
  • the two subsequent triplets, GGA and TCC, encode Gly and Ser, respectively, and constitute a BamHI restriction site which is inserted as a marker with a view to optional later PCR reaction.
  • the triplets No. 4 and No. 3 from the 3 '-end of this synthetic oli- gonucleotide, which encode Asp-Val, are selected from the genetic code so as to form a BsaHI restriction site, GACGTC.
  • the last triplet on this strand, TAG, is a stop codon.
  • the TTAA sequence at the 5 '-end on the other strand constitutes four of the six nucleotides of the EcoRI restriction site.
  • EcoRI and Bglll restriction was used for ligation into the pGEX-1 vector (Smith, D.B. & Johnson, K.S. (1988) Gene. 31-40), which contains a gene encoding glutathione- S-transferase localized so that expression in transformed bacteria causes synthesization of a fusion product be ⁇ tween this protein and the nonapeptide, which i.a. fa ⁇ cilitates purification and control of the expression product.
  • the ligated plasmid was used for transformation of E. coli strain TGI supplied by Amersham (Hanahan, D. (1983), J. Mol. Biol. 166, 557-580).
  • the above-mentioned BsaHI restriction site was introduced into the synthetic oli ⁇ gonucleotide to enable control of recombinant plasmid preparations by restriction mapping (Sambrook, J. et al. (1989), Molecular Cloning).
  • the correct recombinant plas- mids were sequenced by the dideoxy method (Sanger, F., Nicklen, S., and Coulson, A.R., (1977) Proc. Natl . Acad. Sci. USA 24., 5463-5467; Sambrook, J. et al . (1989) Mo- lecular Cloning) .
  • Transformed E. coli was cultivated in shaking bottles to AgQo of O- 7 " 1 - 0 at 28 °C, following which IPTG was added to a concentration of 0.1 mM for induction of the tac promoter. The cultures were cultivated for another 3-5 hours and then harvested.
  • lyse buffer 50 mM Tris HCl, pH 8.0, 0.2 mg/ml of lysozyme, 1 mM EDTA
  • the supernatant was applied to a glutathione "Agarose”® affinity column equilibrated in 50 mM Tris HCl, pH 8.0 at 8 °C.
  • the column was washed with a buffer, and the fusion protein was eluted with a buffer admixed with 5 mM re- **d glutathione, dialyzed and analyzed by SDS poly- acrylamide gel electrophoresis on an 18% polyacrylamide gel.
  • the protein concentration was determined by means of the Bradford method.
  • the yield of fusion protein was de ⁇ termined to be about 12 mg per g of E. coli cells (wet weight) .
  • Factor Xa cleavage (Nagai, K. & Th ⁇ gersen, H.C. (1984) Nature 309. 810-812) was performed as described by Knud- sen et al. (Knudsen, C.R., Clark, B.F.C., Degn, B., and Wiborg, 0., (1992) Biochem. Int. 2_, 352-362) with a few modifications. The weight ratio of protease to substrate was constantly kept at 1:200. After cleavage, affinity chromatography was again performed on the glutathione "Agarose”® affinity column, and pure nonapeptide was collected from the eluate.
  • affinity chromatography was again performed on the glutathione "Agarose”® affinity column, and pure nonapeptide was collected from the eluate.
  • the mass of the fusion protein and of the glutathione-S-transferase part of cleaved fusion protein was determined to 27 311 and 26 409, respectively, which, in view of the uncertainty of the method, corresponds to a difference that might be ascribed to the nonapeptide.
  • FPLC analyses showed that a gel filtration ( "Superdex® 75") was necessary to remove various contaminants from the nonapeptide. Then the same elution profile was re- vealed under RPC (figure 3) as for nonapeptide isolated from cocoa beans and for nonapeptide produced by chemical synthesis. Plasma desorption mass spectrometry verified the identity of the microbially synthesized peptide.
  • the sequence of the identified nonapeptide may be found as the amino acid residues Nos . 457 to 465 in the amino acid sequence of 67 kD cocoa seed storage protein precur ⁇ sor derived from the cDNA sequence ( International Patent Application No. WO 91/19801) and in the amino acid se ⁇ quence of cocoa seed vicilin derived from the gene se ⁇ quence (McHenry, L. _ Fritz, P.J. (1992), Plant Mol. Biol. 18., 1173-1176) .
  • the nonapeptide isolated from cocoa beans is generated by endogenic enzyme activity and thus represents naturally produced peptides.
  • the cleavage pattern reflects the en- dogenic enzyme activities under the given physical cir ⁇ cumstances, and, of course, it is conceivable that slightly changed conditions might give rise to new pep ⁇ tides that might have a unique flavour potential.
  • the present study comprised studying the flavour potential of the nonapeptide extended by the next N-term- inal amino acid, lysine, and the next C-terminal amino acid, valine, occurring in the cocoa storage protein.
  • a plurality of minor peptides was studied, whose identities are set forth below.
  • the peptides were synthesized by chemical methods and then purified by HPLC prior to tests in roasting experiments. Nomenclature/peptide identity
  • Ala- -2 Ala-Pro
  • Ala- -6 Ala-Pro-Leu-Ser-Pro-Gly
  • Ala- -7 Ala-Pro-Leu-Ser-Pro-Gly-Asp
  • Ala- -8 Ala-Pro-Leu-Ser-Pro-Gly-Asp-Val
  • Ala- -9 Ala-Pro-Leu-Ser-Pro-Gly-Asp-Val-Phe
  • Pro- -7 Pro-Leu-Ser-Pro-Gly-Asp-Val
  • Pro- -8 Pro-Leu-Ser-Pro-Gly-Asp-Val-Phe
  • Lys- - 10 Lys-Ala-Pro-Leu-Ser-Pro-Gly-Asp-Val-Phe
  • the nonapeptide was thus given a very positive evaluation when alanine was N-terminal, and thus confirmed the ob ⁇ servations from the cocoa bean isolate. Most of the minor peptides exhibited a not inconsiderable flavour poten ⁇ tial, and thus confirmed previous observations with frac ⁇ tionated cocoa bean isolate. In all experiments, the no ⁇ napeptide Ala-9 was evaluated as the clearly best one and being unique.
  • endogenic enzymes which are responsible for the formation of the nonapeptide dur ⁇ ing incubation of cocoa beans, may have a great resemb ⁇ lance to trypsin and chymotrypsin and/or pepsin, respec ⁇ tively. This in order to be able to generate the correct terminal amino acids, alanine and phenylalanine.
  • MOLECULE TYPE DNA (gen nic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (gencmic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Nutrition Science (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
EP96917366A 1995-06-01 1996-05-31 Cocoa flavour precursor peptides, dna encoding them, processes for producing the peptides, and their use for generating cocoa flavour Withdrawn EP0832103A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK061695A DK61695A (da) 1995-06-01 1995-06-01 Kakaoflavouraktive peptider, DNA som koder for dem, fremgangsmåder til fremstilling af peptiderne og deres anvendelse til frembringelse af kakaoflavour
DK61695 1995-06-01
PCT/DK1996/000230 WO1996038472A1 (en) 1995-06-01 1996-05-31 Cocoa flavour precursor peptides, dna encoding them, processes for producing the peptides, and their use for generating cocoa flavour

Publications (1)

Publication Number Publication Date
EP0832103A1 true EP0832103A1 (en) 1998-04-01

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Application Number Title Priority Date Filing Date
EP96917366A Withdrawn EP0832103A1 (en) 1995-06-01 1996-05-31 Cocoa flavour precursor peptides, dna encoding them, processes for producing the peptides, and their use for generating cocoa flavour

Country Status (4)

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EP (1) EP0832103A1 (da)
AU (1) AU5997096A (da)
DK (1) DK61695A (da)
WO (1) WO1996038472A1 (da)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1235919B1 (en) * 1999-11-18 2004-06-30 Societe Des Produits Nestle S.A. Recombinant oleosins from cacao and their use as flavoring or emulsifying agents
EP1253200A1 (en) * 2001-04-25 2002-10-30 Société des Produits Nestlé S.A. Cocoa polypeptides and their use in the production of cocoa and chocolate flavour
EP1298210A1 (en) * 2001-10-01 2003-04-02 Societe Des Produits Nestle S.A. Cocoa flavour precursor peptides
EP1297753B1 (en) 2001-10-01 2009-08-26 Societe Des Produits Nestle S.A. Flavour-active peptides
FR2883873B1 (fr) 2005-03-31 2009-07-10 Pharmamens Sarl Inhibiteurs d'age
WO2011076954A1 (es) * 2009-12-23 2011-06-30 Biopolis S.L. Obtencion de productos bioactivos procedentes del cacao con actividad inhibidora de la enzima pep y actividad antioxidante y/o antineurodegenerativa
CA2896246C (en) 2013-01-22 2021-07-06 Mars, Incorporated Flavor composition and edible compositions containing same
WO2015177006A1 (en) * 2014-05-19 2015-11-26 Nestec S.A. Mechanical generation of flavour compositions

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9013016D0 (en) * 1990-06-11 1990-08-01 Mars Uk Ltd Compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9638472A1 *

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WO1996038472A1 (en) 1996-12-05
DK61695A (da) 1996-12-02

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