Taste Modulating Peptides from Overfermented Cocoa Beans

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Chemistry and Biology of Aroma and Taste

Taste Modulating Peptides from Overfermented Cocoa Beans Mathias Salger, Timo D. Stark, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00905 • Publication Date (Web): 27 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Journal of Agricultural and Food Chemistry

Taste Modulating Peptides from Overfermented Cocoa Beans

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Mathias Salger,$ Timo D. Stark$ and Thomas Hofmann$,§*

4 5

$Food

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Chemistry and Molecular and Sensory Science, Technische Universität

7

München, Lise-Meitner-Str. 34, 84354 Freising, Germany, and §Bavarian Center for

8

Biomolecular Mass Spectrometry, Gregor-Mendel-Straße 4,

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85354 Freising, Germany.

10 11 12 13 14 15 16 17 18 19 20

*

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PHONE

+49-8161/71-2902

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FAX

+49-8161/71-2949

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E-MAIL

[email protected]

To whom correspondence should be addressed

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ACS Paragon Plus Environment


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ABSTRACT

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Activity-guided fractionation of an aqueous extract of overfermented cocoa beans,

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which were recently found to be a rich source of previously unknown taste enhancing

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substances, revealed the presence of a series of taste modulating short peptides.

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Fractionation was achieved by means of sequential solvent extraction, MPLC as well

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as preparative HPLC and the taste-modulating activity was determined by means of

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matrix assisted taste dilution analysis. By means of UPLC-ToF-MS screening, LC-

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MS/MS methods and customized syntheses numerous short peptides could be

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identified in the taste modulating fractions. Sensory experiments of the target peptides

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showed umami enhancing and salt taste enhancing properties as well as kokumi

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effects when applied in a savory taste matrix. Evaluation of the taste threshold

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concentrations in model broth demonstrated a high taste modulating potential of 11 out

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of 13 identified peptides. Lowest threshold concentrations were determined for the salt

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taste enhancing tripeptide pEEE (55 µmol/L) and the kokumi active tripeptide VPA

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(90 µmol/L). Furthermore, a large number of dipeptides, either carrying a prolyl- or

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pyro-glutamyl moiety were located in the aqueous extract, exhibiting taste modulating

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properties and revealed a pH dependency of the taste modulating effect of the savory

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taste matrix. Additionally, synergistic effects of a mixture of five umami enhancing pyro-

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glutamyl dipeptides in the model matrix was demonstrated.

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Keywords: taste modulation, taste enhancer, overfermented cocoa, peptides, taste

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dilution analysis

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INTRODUCTION

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Taste and aroma are the key factors of consumers’ choice on food purchasing rather

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than prices or aspects of healthiness. To develop and produce highly desirable

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foodstuffs, which deliver a distinguishable and high palatability, taste modulating

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compounds gained more and more interest over the last decade. Furthermore, the

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availability of taste modulators enables the production of food products with an unique

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and consistent taste profile independent of raw material supply. At the same time,

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many consumers appreciate natural ingredients instead of artificial compounds.1

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Focusing on savory food products several umami and salt taste enhancing

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components as well as kokumi substances were identified in natural sources. Starting

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in 1967 with the characterization of purine-5’-ribonucleotides as umami enhancers

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many potent taste modulators were identified until today,2 like 5’-GMP derivatives in

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yeast extract,3 theogallin, L-theanine and succinic acid in matcha tea,4 rubemamine

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and rubescenamine and mono sodium L-/ and D-pyro-glutamate in potatoes.5,6,7

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Advanced investigations highlighted pyro-glutamyl peptides (pE-X) such as pEP, pEPS

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and pEPE in wheat gluten hydrolysates and pEG and pEQ in soy sauce as umami

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enhancers.8,9

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The potential adverse effect on humans’ health of a high intake of sodium chloride

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was one of the driving forces to search for salt taste enhancers.10 The added amount

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of sodium chloride to food products could be reduced while no change in salt taste

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intensity could be tasted. Next to synthetic substances a number of natural salt taste

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enhancing compounds were identified. Alapyridaine in beef boullion,11 several arginyl

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dipeptides in fermented fish sauce and in casein/lysozyme as well as the two pyro-

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glutamyl dipeptides, namely pEV and pEVL,12 were shown to be effective salt taste

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enhancers.13,14

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Next to umami and salty taste the so called kokumi perception, which can be

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described as mouthfulness, complexity or continuity, gained more and more attention

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in savory food products. A lot of naturally occurring compounds were identified to be

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responsible for this kind of taste modulation, e.g. alliin in onions,15 several γ-glutamyl

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peptides in edible beans and gouda cheese and the naturally occurring tripeptide

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glutathione.16-18

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To enable the characterization of taste compounds the application of the taste

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dilution analysis (TDA),19 a sensory-guided method of food analysis, was successfully

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applied and many taste compounds could be identified, e.g. in cocoa beans,20 gouda

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and parmesan cheese,17,21 cooked crustaceans and hazelnuts.22,23 But not only

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intrinsic taste compounds could be tracked, but also taste enhancing components

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could be traced by means of the modified TDA. Comparative TDA and matrix assisted

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TDA enabled the identification of alapyridain,11 γ-glutamyl peptides in edible beans or

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gouda and carnosine in traditional Pot-au-Feu by using a tasting matrix,16,17,24 like a

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binary mixture of sodium chloride and sodium glutamate, instead of blank water.

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A previous study revealed “overfermented” cocoa beans to be a rich source of taste

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modulating substances.12 The expression “overfermented” is used to describe cocoa

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beans being fermented for more than eight days which is the common maximum

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fermentation time. During fermentation different processes occur like the formation of

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acetic acid, enzymatic degradation of the pulpa and hydrolysis of proteins and

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peptides. Not only precursors of taste and aroma compounds are formed during this

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process but probably also taste modulating compounds.12,20,25

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Therefore, the objective of this study was to screen overfermented cocoa beans on

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potential umami, salt as well as kokumi taste modulating compounds by application of

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the matrix assisted TDA, and, consequently identify the active target compounds via

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modern techniques of mass spectrometry and characterize the sensory properties. 4 ACS Paragon Plus Environment

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MATERIALS AND METHODS

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Chemicals and Materials. The following chemicals were obtained commercially:

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acetone, dichloromethane, ethyl acetate, n-pentane (VWR prolabo chemicals, AnalaR

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Normapur, France); ammonium acetate solution (5 M), methyl acetate, sodium

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chloride, L-tyrosine, maltodextrine (Sigma-Aldrich, Steinheim, Germany); formic acid,

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potassium hydroxide, monosodium glutamate, n-butyl acetate (Merck, Darmstadt,

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Germany); prolyl-peptides and γ-glutamyl peptides were purchased from Bachem

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(Bubendorf, Switzerland), pyro-glutamyl peptides and further reference peptides from

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Peptides & Elephants (Henningsdorf, Germany). Solvents used for HPLC-analysis

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were of HPLC grade (Merck) and solvents used for LC-MS analysis were of LC-MS

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grade (Honeywell, Seelze, Germany). Deuterated solvents were supplied by Euriso-

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Top (Saarbrücken, Germany). Deionized water used for chromatography was

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prepared by the use of a MiliQ Advantage A10 Water Purification System (Milipore

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S.A.S., Molsheim France). For sensory analysis, bottled water (Evian, Danone Waters

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Deutschland, Frankfurt am Main, Germany) and a commercially available yeast extract

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(Gistex XII LS, DSM, Heerlen, Netherlands) were used.

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Cocoa beans were harvested in Brazil and fermented for 14 days in wooden boxes covered with banana leaves and only dried, not roasted.

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Solvent Extraction of Overfermented Cocoa Beans. An aliquot (100 g) of

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overfermented cocoa beans were peeled by hand and deeply frozen with liquid

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nitrogen. After grinding (4000 rpm for 30 s) using a GM 300 type mill (Retsch, Haan,

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Germany), the obtained cocoa powder was extracted five times with n-pentane (300

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ml, for 15 min) at room temperature. After centrifugation and removal of the solvent,

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the defatted cocoa material was extracted five times with acetone/water (7:3; v/v; 300

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ml, 15 min each) at room temperature. The extracts were combined, filtrated (Filter

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paper 615-1/4, Macherey-Nagel, Düren, Germany) and the acetone was evaporated 5 ACS Paragon Plus Environment


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under reduced pressure at 40 °C. Subsequently, the aqueous phase was extracted

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with dichloromethane, methyl, ethyl and n-butyl acetate (5 x 200 ml, each) to remove

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bitter and astringent components like alkaloids and (epi)catechin as well as

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procyanidines. After removal of the residual organic solvents under a vacuum at 40 °C

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the aqueous extract (AE) was freeze-dried (Gamma 1-20, Christ, Osterode, Germany)

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twice yielding a brown powder. The AE was evaluated by means of comparative taste

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profile analysis and stored at -20 °C until further analyses.

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Separation of the Aqueous Extract by Means of Medium Pressure Liquid

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Chromatography (MPLC). Aliquots (1 g) of the lyophilized AE were dissolved in water

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(15 ml) and separated via medium pressure liquid chromatography (MPLC) (Büchi,

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Flawil, Switzerland) using a polypropylene cartridge (150 x 40 mm) filled with

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LiChroprep RP-18 material (25-40 µm, Merck, Darmstadt, Germany). Signal detection

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was performed by an Evaporative Light Scattering Detector (ELSD) Sedex LT Model

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80 (Sedere, Alfortville, France) and automatic fraction collection was done using a

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fraction collector C-660 (Büchi). For chromatographic separation aqueous formic acid

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(0.1%) as eluent A and formic acid in acetonitrile (0.1%) as eluent B were used and

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the flow rate was set to 40 ml/min (binary pump module C-605, Büchi).

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Chromatography was conducted with the following gradient: starting with 0% B for 7

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min, increasing eluent B to 100% within 20 min and keeping at 100% B for 10 min,

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finally. The individual fractions were collected in glass tubes and combined to give 10

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fractions, namely M1 to M10. Organic solvent was evaporated under reduced pressure

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at 40 °C, residues were taken up in deionized water und finally lyophilized twice.

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Fractions were kept at -20 °C until they were used for taste dilution analysis (TDA) and

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further fractionation.

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Screening for Known Taste Modulators in MPLC-Fractions M1 to M10 by

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Means of Liquid Chromatography Mass Spectrometry (LC-MS/MS). Amino acids, 6 ACS Paragon Plus Environment

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nucleotides and γ-glutamyl-peptides. Aliquots (1 mg) of lyophilized and homogenized

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MPLC fractions M1 to M10 were dissolved in deionized water (1 ml), membrane

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filtrated (0.45 µm) and screened with established in-house methods by means of LC-

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MS/MS on known amino acids, taste modulating nucleotides and γ-glutamyl-

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peptides.17,21,22

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HPLC Sub-Fractionation of MPLC Fraction M4. A portion (50 mg) of fraction M4

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was dissolved in water/acetonitrile (95:5; v/v; 5 ml), membrane-filtered (0.45 µm) and

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aliquots (1 ml) were injected into a HPLC system (Jasco, Groß-Umstadt, Germany)

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equipped with a preparative HPLC column (Hyperclone ODS C-18, 250 x 21.2 mm,

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5 µm; Phenomenex, Aschaffenburg, Germany). As solvents, 0.1% formic acid in water

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(A) and 0.1% formic acid in acetonitrile (B) were used, while chromatography

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(18 ml/min) started with 5% B for 3 min and ELSD (Sedex 85, Sedere). The amount of

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B was increased to 35% in 15 min and to 100% in the following 3 min, which was kept

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for 3 min. Afterwards, the acetonitrile content was decreased to 5% in 2 min and finally

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held for 3 min at 5% B. Individual fractions of several runs were collected and combined

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to give seven HPLC fractions, namely M4-H1 to -H7, freed from organic solvents under

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reduced pressure at 40 °C and freeze-dried twice to be used for sensory experiments

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and further analyses.

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HPLC Sub-Fractionation of MPLC Fraction M5. A portion (50 mg) of fraction M5

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was dissolved in water/acetonitrile (9:1; v/v; 5 ml), membrane-filtered (0.45 µm), and

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aliquots were analyzed via the aforementioned HPLC system. Chromatographic

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separation was performed using the preparative HPLC column, corresponding eluents,

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detector and flow rate mentioned above and the following gradient: initially 10% B for

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3 min, increased to 18% B in 2 min and held for 3 min. Thereafter, eluent B was

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increased within 8 min to 40% and further to 100% in 3 min and kept for 3 min. Finally,

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eluent B was decreased to 10% in 2 min and kept for 3 min. Eight fractions, namely 7 ACS Paragon Plus Environment


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M5-H1 to M5-H8, were successively collected, freed from solvents under reduced

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pressure at 40 °C, freeze-dried twice, yielding a brownish dry powder which was stored

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at -20 °C until further analysis.

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Ultra Performance Liquid Chromatography (UPLC)-Time-of-Flight (ToF)-MS

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of Fractions M4-H2, M4-H7, M5-H3, M5-H6, M5-H8. Aliquots (1 mg) of lyophilized

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fractions

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acetonitrile/water (1 ml, each) in a ratio corresponding to UPLC starting conditions (see

188

below). After membrane filtration (0.45 µm), aliquots (2 µL) were analyzed via an

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Acquity UPLC core system (Waters, Milford, MA, USA) connected to a Synapt G2-Si

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HDMS (Waters, Manchester, UK) operating in high-resolution mode. The flow rate was

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set to 0.4 ml/min at 40 °C using 0.1% formic acid in water (A) and 0.1% formic acid in

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acetonitrile (B) as solvents using the following conditions:

M4-H2,

M4-H7,

M5-H3,

M5-H6

and

M5-H8

were

dissolved

in

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Fraction M4-H2: Acquity UPLC BEH Amide, 2.1 x 150 mm, 1.7 µm (Waters);

194

gradient starting at 85% B, decreasing to 50% B in 4 min and to 10% B in 1 min,

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increasing to 85% B in 0.5 min and finally kept for 0.5 min.

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Fraction M4-H7: Acquity UPLC BEH C18, 2.1 x 150 mm, 1.7 µm (Waters); gradient

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starting with 5% B, increasing to 50% B in 4 min and to 95% in further 0.5 min,

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decreasing to 5% B in 0.5 min and held for 0.5 min.

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Fraction M5-H3: Acquity UPLC BEH C18, 2.1 x 150 mm, 1.7 µm (Waters); gradient

200

starting at 5% B, increased to 30% B in 4 min, further increased to 100% B in 0.5 min,

201

held for 0.2 min, then decreased to 5% B in 0.5 min and kept for 0.5 min.

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Fraction M5-H6 and M5-H8: Acquity UPLC BEH C18, 2.1 x 150 mm, 1.7 µm

203

(Waters); gradient starting at 5% B, increased to 50% B in 4 min, further increased to

204

100% B in 0.5 min, held for 0.2 min, then decreased to 5% B in 0.5 min and kept for

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0.5 min.


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Identification of Taste Modulators in Fractions M4-H2, M4-H7, M5-H3, M5-H6,

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M5-H8. Aliquots (1 mg/ml) were analyzed by means of MSn and let to the tentatively

208

identification of 13 peptides. Comparison of chromatographic and mass spectrometric

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data with commercial reference compounds unequivocally confirmed the identity of

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these peptides.

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pyro-Glutamyl-glutamyl-glutamic acid: UPLC-ToF-MS (ESI+): m/z 388.1354

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([M+H]+, measured); m/z 388.1356 ([M+H]+, calculated for C15H22N3O9); MS/MS (ESI+):

213

m/z 388.1,

214

UPLC-ToF-MS (ESI+): m/z 430.1933 ([M+H]+, measured); m/z 430.1938 ([M+H]+,

215

calculated for C17H28N5O8); MS/MS (ESI+): m/z 430.2, 412.1, 311.0. Aspartyl-tyrosyl-

216

arginine: UPLC-ToF-MS (ESI+): m/z 453.2101 ([M+H]+, measured); m/z 453.2098

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([M+H]+, calculated for C19H29N6O7); MS/MS (ESI+): m/z 453.2, 338.1, 321.1, 251.2,

218

175.1, 158,1. Aspartyl-alanyl-tryptophyl-proline: UPLC-ToF-MS (ESI+): m/z 488.2196

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([M+H]+, measured); m/z 488.2145 ([M+H]+, calculated for C23H30N5O7); MS/MS

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(ESI+): m/z 488.2, 470.1, 373.2, 355.1, 328.0, 187.9, 158.9. Arginyl-methionyl-proline:

221

UPLC-ToF-MS (ESI+): m/z 403.2300 ([M+H]+, measured); m/z 403.2127 ([M+H]+,

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calculated for C16H31N6O4S); MS/MS (ESI+): m/z 403.2, 385.0, 288.0. Valyl-prolyl-

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alanine: UPLC-ToF-MS (ESI+): m/z 286.1765 ([M+H]+, measured); m/z 286.1767

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([M+H]+, calculated for C13H24N3O4); MS/MS (ESI+): m/z 286.2, 268.1, 196.9, 186.9.

225

Seryl-prolyl-valine: UPLC-ToF-MS (ESI+): m/z 302.1718 ([M+H]+, measured); m/z

226

302.1716 ([M+H]+, calculated for C13H24N3O5); MS/MS (ESI+): m/z 302.2, 284.1, 255.9,

227

214.9, 184.9, 156.9, 117.9, 72.1. Tyrosyl-glycyl-aspartyl-glycine: UPLC-ToF-MS

228

(ESI+): m/z 411.1519 ([M+H]+, measured); m/z 411.1516 ([M+H]+, calculated for

229

C17H23N4O8); MS/MS (ESI+): m/z 411.1, 393.0, 335.9, 318.0, 135.9. Lysyl-aspartyl-

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glutaminyl-proline: UPLC-ToF-MS (ESI+): m/z 487.2523 ([M+H]+, measured); m/z

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487.2516 ([M+H]+, calculated for C20H35N6O8); MS/MS (ESI+): m/z 487.2, 469.1, 372.0,

370.0,

240.9,

147.9.

pyro-Glutamyl-glutaminyl-alanyl-threonine:



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243.9, 227.0. Phenyl-alanyl-glutamic acid: UPLC-ToF-MS (ESI+): m/z 295.1296

233

([M+H]+, measured); m/z 295.1294 ([M+H]+, calculated for C14H19N2O5); MS/MS (ESI+):

234

m/z 295.1, 277.2, 249.2, 148.1, 120.0. Tyrosyl-valine: UPLC-ToF-MS (ESI+): m/z

235

281.1506 ([M+H]+, measured); m/z 281.1501 ([M+H]+, calculated for C14H21N2O4);

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MS/MS (ESI+): m/z 281.0, 263.0, 235.1, 164.0, 135.8. Asparaginyl-asparaginyl-alanyl-

237

leucine: UPLC-ToF-MS (ESI+): m/z 431.2270 ([M+H]+, measured); m/z 431.2254

238

([M+H]+, calculated for C17H31N6O7); MS/MS (ESI+): m/z 431.2, 413.1, 385.1, 300.0,

239

272.0.

240

488.2487 ([M+H]+, measured); m/z 488.2469 ([M+H]+, calculated for C19H34N7O8);

241

MS/MS (ESI+): m/z 488.2, 470.1, 453.1, 342.1, 314.1, 228.9, 211.9.

Asparaginyl-glycyl-glycyl-leucyl-glutamine:

UPLC-ToF-MS

(ESI+):

m/z

242

UPLC-MS/MS Screening for Prolyl and pyro-Glutamyl Dipeptides. Peeled

243

deeply frozen cocoa beans (1 g) were ground to powder (Grindomix GM 200, Retsch,

244

Haan, Deutschland) and extracted with methanol/water (20 ml, 70:30, v/v, 3-fold,

245

15 min) in an ultrasonic bath, supernatants were combined, methanol evaporated and

246

the aqueous extract finally freeze-dried. An aliquot of the obtained dry extract (10 mg)

247

was dissolved in water (5 mL), membrane filtered (0.45 µm) and aliquots (2 µL) were

248

analyzed by means of UPLC-ToF-MS. For the identification of prolyl dipeptides the flow

249

rate was set to 0.4 ml/min and 5 mM NH4Ac-buffer in water (pH 2; A) and 5 mM NH4Ac-

250

buffer in acetonitrile/water (95:5; v/v; pH 2; B) were used as mobile phase.

251

Chromatographic separation was performed on an Acquity UPLC BEH Amide column,

252

2.1 x 150 mm, 1.7 µm (Waters) with the following gradient: starting at 100% B, held for

253

2 min, decreased to 80% B in 4 min, decreased to 65% B in 11 min, decreased to 0%

254

B in 0.5 min, held for 1 min, increased to 100% B in 0.5 min and held for 1 min. For the

255

identification of pyro-glutamyl dipeptides the same analytical column and flow rate

256

were used and 5 mM NH4Ac-buffer in water (pH 3.5; A) and 5 mM NH4Ac-buffer in

257

acetonitrile/water (95:5; v/v; pH 3.5; B) were used as mobile phase with the following 10 ACS Paragon Plus Environment

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gradient: starting at 95 % B, held for 1 min, decreased to 30% B in 4 min, held for

259

1 min, increased to 95% B in 0.5 min and held for 1.5 min.

260

Sensory Analyses. Training of the Sensory Panel. 12 subjects (seven women and

261

five men with an age between 24 and 30) with no history of known taste disorders and

262

who had given informed consent to take part in the present sensory tests, were trained

263

weekly over at least two years to become familiar with the methodologies and language

264

of sensory experiments. The panelists were trained to differentiate taste qualities, to

265

evaluate taste intensities and to detect taste recognition thresholds. Therefore,

266

aqueous solutions (1 ml) of standard taste compounds dissolved in bottled water (pH

267

5.9, adjusted with 0.1% formic acid) were used: sucrose (50 mmol/L) for sweet taste,

268

lactic acid (20 mmol/L) for sour taste, monosodium L-glutamate (5 mmol/L) for umami

269

taste, caffeine (1 mmol/L) for bitter taste, sodium chloride (15 mmol/L) for salty taste

270

for the puckering astringency and the velvety astringent, mouth-drying oral sensation

271

gallotannic acid (0.05%) and quercetin-3-O-β-D-glucopyranoside (0.002 mmol/L),

272

respectively.11,16,26 Additional training was performed to familiarize panelists with a

273

savory taste matrix and the effects of taste modulators. Hence, an aqueous model

274

broth (pH 5.9, adjusted with 0.1% formic acid) containing monosodium L-glutamate

275

(10 mmol/L), sodium chloride (50 mmol/L), maltodextrin (6.4 g/L) and yeast extract (2.1

276

g/L) was prepared.27 Taste modulators were added to the model broth e.g. guanosine

277

monophosphate (0.14 mmol/L) or glutathione (2 mmol/L) and presented in

278

3-Alternative Forced Choice (AFC)-tests.

279

General conditions. Sensory analyses were performed in an air-conditioned

280

sensory panel room at 22-25 °C and panelists wore nose-clips to prevent cross-modal

281

interactions with olfactory inputs. Subjects used the sip-and-spit method, nevertheless

282

isolated fractions and commercially purchased reference compounds were analytically



283

checked to be free of impurities prior to sensory analyses by means of LC-ToF-MS and

284

1H-NMR.

285

(Comparative) Taste Profile Analysis. For taste profile analysis a solution

286

(0.1%; m/v) of the AE in bottled water was prepared and sensory panelists were asked

287

to rate the intensities of the qualities sweet, sour, bitter, salty, umami and astringent

288

on a scale between 0 (not detectable) and 5 (strongly detectable). For comparative

289

taste profile analysis panelists were requested to evaluate the intensities of saltiness,

290

umami taste and kokumi sensation of the model broth on the same scale. Hence, a

291

solution (0.1%; m/v) of the AE in model broth was presented to the panel and taste

292

intensities were rated in comparison to the unspiked model broth by the subjects.

293

Taste Dilution Analysis (TDA). Aliquots of MPLC fractions M1-M10 were dissolved

294

in either bottled water (20 mL) in “natural” concentration ratios for TDA or model broth

295

(mb) for modified taste dilution analysis (TDAmb). For TDA, aqueous solutions were

296

diluted serially 1+1 with water and were presented to the panel in order of increasing

297

concentrations. Using 3-AFC tests panelists were asked to determine the dilution step

298

at which a difference between sample and control (blank) could be detected. The

299

concentration of this dilution step was defined as taste dilution (TD) factor. Comparable

300

procedure was performed for TDAmb with the difference of using model broth instead

301

of water to dilute sample and as control sample (blank). TD(mb)-factors were determined

302

in two separate sessions, each, averaged and values between individuals and

303

sessions did not differ more than plus/minus one dilution step.3,19 After determination

304

of the TD(mb)-factors sensory study subjects were asked to described perceived taste

305

differences of spiked samples in comparison to blanks.

306

Taste Recognition Threshold Concentrations. The threshold concentration of a

307

compound where the taste quality was just detectable, was determined in bottled water

308

(pH 5.9 adjusted with 0.1% formic acid) for the intrinsic taste and in model broth (pH 12 ACS Paragon Plus Environment

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309

5.9 adjusted with 0.1% formic acid) for the taste modulating properties, respectively.

310

Test compounds were dissolved, serially diluted 1+1 and presented to the panel in

311

order of ascending concentrations using duo-trio-tests. The taste threshold of an

312

individual was calculated as the arithmetic mean of the last not correct recognized and

313

the first correct recognized concentration. For determination of the total threshold

314

concentration the geometric mean of individual thresholds was used and given as

315

µmol/L.19 Values between individuals and independent sessions differed by not more

316

than plus or minus one dilution step.

317

Sensory Evaluation of HPLC Sub-Fractions. Lyophilized HPLC fractions were

318

dissolved in model broth in three different concentrations: a three-fold “natural”

319

concentration level, a 1:10 dilution and a 1:100 dilution. Panelists were asked to

320

determine the sample which showed taste modulating activity in duplicate duo-trio-

321

tests for each concentration level of each HPLC fraction. After selecting the different

322

sample, the taste modulating effect has to be described. Samples with significance

323

level of α ≤ 0.1 were rated as distinguishable.28

324

High Performance Liquid Chromatography (HPLC). Preparative separation of

325

MPLC fractions M4 and M5 was accomplished on a HPLC system (Jasco) consisting

326

of two PU-2087 Plus pumps, a DG-2080-53 degasser, a Rh 7725i Rheodyne injection

327

valve (Rheodyne, Bensheim, Germany) and an ELSD (Sedex Model 85). The

328

evaporative light scattering detector was equipped with a low flow nebulizer, operated

329

at 40 °C, used air as operating gas (3.5 bar) and the split ratio was set to 1 ml/min for

330

the

331

Chromatography Data System, Version 1.9 (Jasco). Preparative chromatography was

332

performed on a preparative Hyperclone ODS C-18 column, 250 x 21.2 mm, 5 µm

333

(Phenomenex) operated with a flow rate of 18.0 ml/min.

detector.

Data

acquisition

was

performed


by

means

of

Chrompass


334

UPLC-ToF-MS. Analytical chromatographic separation and acquisition of mass

335

spectra of HPLC sub-fractions were performed on an Acquity UPLC core system

336

(Waters) connected to a Synapt G2-Si HDMS mass spectrometer (Waters) operating

337

in positive electrospray ionization (ESI+) mode and the following parameters: scan time

338

(MSe, centroid) 0.1 s, collision energy ramp 20-40 eV, capillary voltage (+2.5 kV),

339

sampling cone (20 V), source temperature (120 °C), desolvation temperature (400 °C),

340

cone gas (30 L/h) and desolvation gas (850 L/h). The MS system was calibrated over

341

a mass range from m/z 100 to m/z 1200 using a solution of sodium formate (0.5 mmol/l)

342

in 2-propanol/water (9:1; v/v). All data were lock mass corrected on leucine

343

enkephaline (m/z 556.2771 [M+H]+). Chromatographic separations were performed on

344

an Acquity UPLC BEH C18 column, 2.1 x 150 mm, 1.7 µm (Waters) or an Acquity

345

UPLC BEH Amide column, 2.1 x 150 mm, 1.7 µm (Waters), respectively. Data

346

acquisition and processing was done by using MassLynx software, Version 4.1

347

(Waters).

348

(LC)-MS/MS Systems. Different systems were used for mass spectral analyses,

349

but all were operated in electrospay ionization (ESI) mode. LC-MS/MS system 1

350

consisted of an API QTrap 5500 (Sciex, Darmstadt, Germany) connected to a Nexera

351

X2 UHPLC system (Shimadzu Europa GmbH, Duisburg, Germany) consisting of two

352

LC pump systems 30AD, a DGU-20A5 degasser, a SIL-30AC autosampler, a

353

CTO-30A column oven and a CBM-20A controller. The following ion source

354

parameters were used for LC-MS/MS system 1: ion spray voltage (+5500 V), curtain

355

gas (2.4 bar), gas 1 (3.1 bar), gas 2 (3.8 bar) and source temperature (425 °C). This

356

system was used for fullscan and fragmentation experiments. Data acquisition and

357

instrumental control was performed using Analyst 1.6.2 software. This system was

358

used in MRM mode for screening of known taste modulators as described above.


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Further MSn fragmentation experiments were performed on the MS system 2: High

360

Capacity Ion Trap Ultra system (HCT Ultra, PTM Discovery; Bruker, Billerica, USA)

361

with manual syringe infusion was used with following parameters: ion spray voltage

362

(-4000 V), evaporation gas (1.4 bar), dry gas (7 ml/min), dry gas temperature (325 °C).

363

Data acquisition was performed by Esquire Control 6.1 and data analysis by MZmine

364

2.29 software.

365

LC-MS/MS system 3 for pyro-glutamyl and prolyl dipeptides: Acquity UPLC core

366

system (Waters) coupled to a Xevo TQ-S system (Waters). ESI+ source parameters

367

were set as follows: capillary voltage +3.8 kV, source temperature 150 °C, desolvation

368

temperature 400 °C, cone gas 150 L/h and desolvation gas 850 L/h. Data acquisition

369

and processing was done by using MassLynx software, Version 4.1 (Waters).

370

Quantitative Magnetic Resonance Spectroscopy (qNMR). qNMR experiments

371

were performed on a Bruker DRX 400 MHz spectrometer (Bruker, Rheinstetten,

372

Germany) with a Broadband BBFOplus probe (BB, 1H). D2O was used as solvent and

373

quantitation was done using L-tyrosine as external calibration standard (5.21 mM) with

374

its specific resonance signal (7.10 ppm). Spectra were recorded at 298 K and data

375

analysis was performed by using the ERETIC 2 methodology of TopSpin 3.2 software

376

(Bruker) as reported earlier.29

377 378

RESULTS AND DISCUSSION

379

Recently, the aqueous extract of overfermented cocoa beans has been reported as

380

a novel source of previously unknown taste modulating substances.12 In order to

381

identify further umami, kokumi and salt taste modulating components overfermented

382

(14 d) unroasted cocoa beans were defatted with n-pentane, extracted with

383

acetone/water (70:30, v/v), the solvent removed under vacuum and to remove bitter 15 ACS Paragon Plus Environment


384

and astringent compounds like catechins and procyanidines the remaining aqueous

385

extract successively extracted with dichloromethane, methyl acetate, ethyl acetate and

386

n-butyl acetate.20 Evaporation of residual solvents and lyophilisation provided a dry

387

powder of the aqueous extract.

388

To get a first insight into the taste modulating effects, a solution of the freeze-dried

389

aqueous extract in model broth (0.1 %, m/v) was prepared and presented to a trained

390

sensory panel. Panelists were asked to rate the intensities of umami and salty taste,

391

as well as the kokumi effect on a scale between 0 (not detectable) and 5 (strongly

392

detectable) in comparison to a model broth without any additive. Addition of the

393

aqueous extract to the model broth led to the increase of the umami taste from 2.5 to

394

3.2 (±0.26) and of the kokumi effect from 1.0 to 1.5 (±0.38). No difference was

395

described for saltiness. To exclude that the effects were caused by the intrinsic taste

396

of the aqueous extract a taste profile analysis in bottled water (pH 5.9, 0.1 %, m/v) was

397

conducted which did not show any umami, salty or kokumi intensities, but bitterness

398

(2.2), astringency (1.8) and sourness (0.7).

399

Activity-Guided Identification of Taste Modulating Compounds in the

400

Aqueous Extract. In order to locate the compounds which elicited the taste

401

modulating effects, the aqueous extract was separated in 10 fractions (M1 to M10) by

402

means of MPLC-ELSD (Figure 1A). These fractions were freed from solvent,

403

lyophilized twice and used for the modified taste dilution analysis (TDAmb).3 Four

404

fractions, namely M1, M2, M4 and M5, showed umami enhancing or kokumi effects.

405

Fraction M6, M7 and M8 revealed a strong bitter taste (Figure 1B). Furthermore, a

406

taste dilution analysis (TDA) in bottled water was performed to ensure that the

407

observed taste modulating effects are not caused by the intrinsic taste of present

408

compounds.19 The sensory panel described an umami taste for fraction M1, M2 and

409

M3, as well as a bitter taste or astringency for fractions M4 to M9. Fractions M1 and 16 ACS Paragon Plus Environment

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410

M2 did not modulate the taste of the model broth but highlighted an intrinsic umami

411

taste and fractions M4 and M5, which showed taste modulating effects in model broth,

412

only revealed a bitter or astringent sensation in water, respectively. Consequently,

413

these four fractions were screened by means of LC-MS/MSMRM on the following known

414

taste modulators, L-amino acids, nucleotides and γ-glutamyl peptides using previously

415

described methods.15,19,2017,21,22 Umami tasting amino acids L-glutamic acid,

416

L-glutamine, L-aspartic

417

M2, M4 and M5. Fractions M1 and M2 exhibited the presence of the umami tasting

418

(enhancer)

419

monophosphate (5’-CMP).3,27 Furthermore, kokumi active compounds γ-glutamyl-

420

alanine, γ-glutamyl-lysine and γ-glutamyl-glutamine were identified via LC-MS/MSMRM.

421

Because of the identification of the above mentioned umami and taste modulating

422

compounds in fractions M1 and M2 further investigations on taste modulators were

423

focused on fractions M4 and M5.

acid and L-asparagine were identified in fraction M1 but not in

nucleotides

uridine-5’-monophosphate

(5’-UMP)

and

cytidine-5’-

424

Therefore, fractions M4 and M5 were further separated by means of preparative

425

RP-HPLC-ELSD and the taste modulating potential of the single HPLC fractions was

426

evaluated in sensory experiments. Fraction M4 was separated into seven fractions

427

(M4-H1 to M4-H7), evaporated, freeze-dried and three solutions with different

428

concentrations of each fraction were prepared in model broth and presented to a

429

trained sensory panel in duo-trio-tests in comparison to an unspiked model broth

430

(Figure 2). Sub-fraction M4-H2 induced a kokumi effect at all concentration levels and

431

M4-H7 showed umami enhancing potential. Separation of M5 revealed eight fractions

432

(M5-H1 to M5-H8) which were sensory evaluated as described above. M5-H3 and M5-

433

H8 revealed umami enhancing effects and M5-H6 was described as kokumi active.

434

To identify the taste modulating compounds in sub-fractions of M4 and M5 they

435

were analyzed via UPLC-ToF-MS and various MSn experiments. As several short 17 ACS Paragon Plus Environment


436

peptides are known as taste modulaters,8,9,13,17 generally, the strategic focus was set

437

on peptides. UPLC-ToF-MS analysis in the positive ionization mode (ESI) of M4-H7

438

highlighted several signals and revealed a m/z 302.1718 as one of the most intense

439

pseudomolecular ions. Suggesting a molecular mass of 301.1716 Da the empirical

440

formula of C13H23N3O5 was calculated.

441

A typical fragmentation pattern for peptides of the protonated molecule m/z 302.2

442

was observed by means of MS/MS analysis on a triple quadrupole mass spectrometer

443

(Figure 3). Compared to the pseudomolecular ion the mass difference of 18 Da

444

matches to the cleavage of a H2O molecule giving the b3 fragment ion m/z 284.1. The

445

a3 fragment ion (m/z 255.9) indicated the loss of a carbonyl group. Besides, the

446

characteristic mass loss of 99 Da of the b3 fragment ion was in line with the cleavage

447

of valine, fitting perfectly to the observed b2 fragment ion (m/z 184.9). Moreover, the

448

ion at m/z 214.9 (y2) showed in comparison to the y1 fragment ion (m/z 117.9) a mass

449

difference of 97 Da which could be assigned to a proline moiety. Using the y2 fragment

450

ion it was possible to calculate the mass difference of 87 Da compared to the

451

pseuomolecular ion, and consequently, could be assigned to serine. Taking all these

452

data in consideration, the tripeptide seryl-prolyl-valine (SPV) could be proposed.

453

In sub-fraction M4-H7 an intense signal at m/z 403.2300 ([M+H]+) was detected by

454

means of UPLC-ESI-ToF-MS. For the molecular mass of 402.2127 Da the empirical

455

formula C16H30N6O4S was calculated. The MS2 spectrum showed a signal at m/z 385.0,

456

which could be assigned to the cleavage of a molecule H2O of the pseudomolecular

457

ion and another intense signal at m/z 288.0 (Figure 4). This mass difference of 115 Da

458

could be referred to the cleavage of proline generating the b2 fragment ion.

459

Fragmentation of the b2 fragment by means of MS3 resulted in only a few signals. One

460

signal at m/z 271.0 generated by the cleavage of an ammonia-group and another one

461

at m/z 156.9 which exhibited a difference of 131 Da from the b2 fragment. This could 18 ACS Paragon Plus Environment

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462

be assigned to the cleavage of methionine. The residual b1 fragment ion (m/z 156.9)

463

was assigned to arginine and hence, the tripeptide arginyl-methionyl-proline (RMP)

464

was proposed. The same strategy was applied to the other taste-modifying fractions

465

(M5-H3, -H6, -H8) resulting in the summarized data in Table 1. The presence of the

466

identified peptides was confirmed by means of UPLC-MS/MS using purchased

467

reference compounds for data alignment and co-chromatography.

468

Identification of Proline Containing Dipeptides. As the UPLC-ToF-MSe

469

screening of the taste modulating sub-fractions highlighted first hints for the presence

470

of proline containing dipeptides and such peptides were previously described in cocoa

471

beans,25 an UPLC-MS/MS method was developed to screen for 39 proline containing

472

dipeptides. MS/MS parameters were tuned software-based for each analyte in ESI+

473

mode. The pairs of dipeptides PL/PI and LP/IP could neither be separated by

474

hydrophilic liquid interaction chromatography (HILIC) nor distinguished via MRM-

475

transitions. With exception of PN, PC, EP, WP and CP all further dipeptides could be

476

identified (Supporting Information).

477

Identification of pyro-Glutamic Acid Moiety Containing Dipeptides. After the

478

identification of two short pyro-glutamyl containing peptides in sub-fraction M4-H2 and

479

the knowledge of taste modulating properties of pyro-glutamyl dipeptides,8,9 an UPLC-

480

MS/MS method to screen for 20 dipeptides containing one of the proteinogenic amino

481

acid next to the pyro-glutamyl moiety was developed (Figure 5), which enabled with

482

the exception of pEC the identification of all pyro-glutamyl dipeptides (Supporting

483

Information).

484

Sensory Evaluation of the Identified Peptides. Identified peptides by means of

485

activity guided fractionation (Table 1), MS experiments as well as co-chromatography

486

with reference compounds were evaluated via different sensory tests. Prior to sensory

487

analyses the purity of the peptides was checked by means of 19 ACS Paragon Plus Environment

1H-qNMR


488

spectroscopy.29 The analyzed purity was between 40 and 71 % due to impurities

489

(water) which did not influence the sensory analyses. First, the intrinsic taste in water

490

(pH 5.9) of these peptides was evaluated by determination of human taste threshold

491

concentrations. Two-alternative forced-choice tests with ascending concentrations of

492

the respective peptide were presented to a trained sensory panel. Six of these peptides

493

did not show any intrinsic taste up to concentrations of 2 mmol/L (4, 5, 7, 8, 9, 13),

494

while some were described as sour (1, 2, 11, 12), two to be astringent (3, 6) and one

495

as bitter (10). Corresponding threshold concentrations ranged from 190 – 1000 µmol/L

496

(Table 2).

497

Furthermore, the taste modulating activity was evaluated by determination of the

498

threshold concentrations in model broth (pH 5.9). Peptides were dissolved in model

499

broth, presented to a trained sensory panel in ascending concentrations and evaluated

500

by duo-trio-tests. As given in Table 2, except from 10 and 11 all peptides showed

501

significant lower threshold concentrations (TC) in model broth in comparison to their

502

intrinsic taste in water. Taste modulating effects were described as salt taste

503

enhancing (1, 2, 5, 6, 12), umami enhancing (4) and kokumi active (3, 7, 8, 9, 13).

504

Lowest TC were observed for the salt enhancing compound 1 (55 µmol/L) and kokumi

505

active component 3 (90 µmol/L), while their threshold values in an aqueous solution

506

were at 560 (1) and 700 µmol/L (3), respectively. All other TC ranged between 160 -

507

440 µmol/L but showed far lower values than in aqueous solution. To the best of our

508

knowledge, for the peptides (1-9, 12, 13) no taste modulating effect was described so

509

far.

510

Moreover, the sensory activity of selected prolyl containing dipeptides was

511

exemplarily examined. In a pre-test, a series of these dipeptides was dissolved in two

512

different concentrations in model broth and evaluated for their taste modulating

513

potential by means of triangle tests. Some of the tested dipeptides, e.g. VP, SP, PS 20 ACS Paragon Plus Environment

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514

were found to modulate the saltiness und umami taste of the model broth.

515

Consequently, the following 11 prolyl dipeptides AP/PA, SP/PS, VP/PV, RP/PR and

516

PP were chosen to be precisely characterized by determination of their TC in water for

517

their intrinsic taste and the TC in model broth for the taste modulating activity. Different

518

effects of taste modulating activity, e.g. umami and salt enhancing properties, could be

519

observed for the single compounds in model broth and TC values ranged between 500

520

(RP) to 3500 µmol/L (PA). Interestingly, each dipeptide with the amino acid proline at

521

its C-terminus showed a lower TC in model broth than the corresponding isomeric

522

dipeptide, e.g. TC for VP was 600 µmol/L and for PV 900 µmol/L (Figure 6). In aqueous

523

solutions some of the dipeptides, e.g. PA, SP, RP, PP and KP exhibited an intrinsic

524

bitter taste which was in accordance to literature,30 nevertheless threshold values were

525

higher than in model broth and no intrinsic taste was observable for the other taste

526

modulating dipeptides.

527

To get a closer look on the taste modulating potential of the pyro-glutamyl

528

dipeptides, TC of 10 out of 19 identified dipeptides were exemplarily determined in

529

water and in model broth. A taste modulating effect in model broth for nine dipeptides

530

in a range from 300 µmol/L to 2700 µmol/L (Table 3) could be determined and the most

531

potent umami enhancers were pEP (300 µmol/L), pEE (320 µmol/L) and pEQ

532

(350 µmol/L).

533

Sensory Comparison of Structurally Related Dipeptides. After discovering the

534

taste modulating effects of several peptides containing either a proline or a pyro-

535

glutamyl moiety, it was interesting to compare the taste modulating potential of these

536

similar substances. As a third structure related group, γ-glutamyl peptides, which were

537

described in literature to show taste modulating effects as well,16,17 were compared to

538

the corresponding prolyl and pyro-glutamyl peptides (Figure 7). Three groups of

539

compounds were chosen, one with a C-terminal glutamic acid, a second with a C21 ACS Paragon Plus Environment


540

terminal glutamine and a third comprising a C-terminal valyl-leucine moiety. Threshold

541

concentrations for taste modulating effects were determined in model broth for each

542

analyte as previously described. Respective γ-glutamyl peptides exhibited the lowest

543

TC while corresponding prolyl peptides showed the highest concentration values

544

(Figure 8). For example, the TC for kokumi activity of γEE was at 250 µmol/L, while the

545

TC values for an umami enhancing effect of pEE and PE were at 320 µmol/L and

546

800 µmol/L, respectively.

547

Sensory Properties of a Mixture of pyro-Glutamyl Peptides. To evaluate a

548

potential additive or synergistic effect of the pyro-glutamyl dipeptides, a mixture of five

549

umami enhancing pyro-glutamyl dipeptides showing low threshold concentrations,

550

namely pER, pEE, pES, pEQ and pEF, was prepared in model broth. The final

551

concentration of each dipeptide in its lowest dilution level was 300 µmol/L,

552

consequently in total 1500 µmol/L for the mixture. The threshold concentration of the

553

mixture was determined as previously described for the single compounds.

554

Interestingly, an umami enhancing effect of the mixture with a TC of 125 µmol/L was

555

determined. Hence, this value was about 2.5 fold below the lowest TC of the most

556

potent single dipeptide pEE. These values indicated a combinatory effect which was

557

further examined based on the isobologram method,31 which is commonly used for the

558

evaluation of pharmacologic and toxic effects. A value M could be calculated and if M

559

Taste Modulating Peptides from Overfermented Cocoa Beans

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