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Sensomics-Assisted Elucidation of the Tastant Code of Cooked Crustaceans and Taste Reconstruction Experiments Andreas Dunkel, Stefanie Meyer, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b06069 • Publication Date (Web): 21 Jan 2016 Downloaded from http://pubs.acs.org on January 27, 2016
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Journal of Agricultural and Food Chemistry
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Sensomics-Assisted Elucidation of the Tastant Code of
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Cooked
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Experiments
Crustaceans
and
Taste
Reconstruction
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Stefanie Meyer1, Andreas Dunkel1,2 and Thomas Hofmann1,2*
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1
Chair of Food Chemistry and Molecular and Sensory Science, Technische
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Universität München, Lise-Meitner-Str. 34, D-84354 Freising, Germany, and
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Bavarian Center for Biomolecular Mass Spectrometry, Gregor-Mendel-Straße 4, D-85354 Freising, Germany.
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13 14 15 16 17 18 19 20
*
To whom correspondence should be addressed
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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] 24 25
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ABSTRACT
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Sensory-guided fractionation by means of ultrafiltration and cation exchange
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chromatography, followed by MS/MS quantitation, and taste re-engineering
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experiments revealed the key taste molecules coining the characteristic taste profile
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of the cooked meat of king prawns. Furthermore, quantitative analysis demonstrated
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that the taste differences between crustaceans are due to quantitative differences in
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the combinatorial code of tastants, rather than to qualitative differences in the tastant
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composition. Besides the amino acids glycine, L-proline, and L-alanine, the
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characteristic seafood-like sweet profile was found to be due to the sweet modulatory
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action of quaternary ammonium compounds, amongst which betaine, homarine,
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stachydrin, and trimethylamine-N-oxide were found as the key contributors on the
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basis of dose-activity considerations. The knowledge of this combinatorial tastant
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code sets the ground for the development of more sophisticated crustacean flavors
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that are lacking any heavy metal ions and allergenic proteins present when using
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crustacean extracts for food flavoring.
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Key Words: taste, taste dilution analysis, taste enhancer, prawns, lobster, seafood
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INTRODUCTION
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Due to their alluring aroma profile and their attractive taste centering around a
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unique sweet-umami balance, cooked crustaceans such as, e.g. shrimps, lobster or
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king prawns, are intimately linked with a very delicious product in consumers’ minds.
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Whereas a vast number of studies have been focused in the past on the elucidation
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of the volatile chemical odor code of foods and beverages,1 the knowledge on the
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chemical structures and sensory properties of the non-volatile key taste (modulating)
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molecules in crustaceans is still rather limited.
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Among the non-volatile constituents, amino acids, nucleotides, organic acids, and
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minerals were reported as contributors to the taste of fresh as well as thermally
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processed meat of crustaceans.2-5 Inosine-5ʹ-monophosphate (IMP), adenosine-5ʹ-
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monophosphate (AMP), and L-glutamic acid have been reported as key molecules
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determining the characteristic umami taste of crustacean meat,6-7 whereas the typical
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sweet profile of crustaceans such as, e.g. boiled snow and Chinese mitten crab, has
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been proposed to be elicited by the amino acids glycine, L-proline, and L-alanine, as
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well
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trimethylethanolamine (choline, 2), N-methylpipecolinic acid (homarine, 3), and N-
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trimethylglycine (betaine, 4), Figure 1, were identified in seafood like crabs, scallop,
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and lobster,2,8-13 however, their sensory impact remains largely unclear. Only betaine
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has been reported to affect the sweet profile of seafood,2,14 and to enhance the
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glycine- and L-alanine-induced response of the amino acid taste receptor in fish.15-17
as
sodium
ions.2,6,8-9
Moreover,
trimethylamine-N-oxide
(1),
N-
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Driven by the need to discover the key players imparting the typical taste of foods,
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the research area “sensomics” has made tremendous efforts in recent years in
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mapping the comprehensive population of sensory active, low-molecular weight
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compounds, coined sensometabolome, and cataloging, quantifying, and evaluating
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the sensory activity of metabolites which are present in raw materials and/or are ACS Paragon Plus Environment
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produced upon food processing and storage, respectively.18-20 Aimed at decoding the
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typical taste signature of food products on a molecular level, the so-called taste
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dilution analysis (TDA) was developed as an efficient screening tool enabling the
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sensory-directed identification of the comprehensive population of taste-active
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sensometabolites in foods and beverages such as, e.g. black tea infusions,21 red
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wine,22 chicken broth,23 and Gouda cheese.24 Moreover, the sensomics approach
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accomplished the discovery of a series of taste-modulating molecules generated
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upon food processing by means of “kitchen-type” chemistry such as, e.g. (S)-
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alapyridaine in beef broth,25 N-(1-methyl-4-oxoimidazolidin-2-ylidene) α-amino acids
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in stewed beef juice,26 γ-glutamyl dipeptides in matured Gouda cheese,27 N2-lactoyl-
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guanosine
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carboxyethyl)guanosine 5’-phosphate in yeast extracts,29 and 5-acetoxymethyl-2-
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furaldehyde in traditional balsamic vinegar.30
5’-monophosphate
in
fermented
tuna
fish,28
(S)-N2-(1-
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As the entire nonvolatile sensometabolome of crustacean meat has not yet been
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fully investigated, the objective of the present investigation was to identify and
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quantify taste active and taste modulatory compounds in the cooked meat of king
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prawns (Litopenaeus vannamei), to rank them in their sensory impact based on
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dose-activity considerations, and to validate their sensory relevance by means of
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taste re-engineering experiments. Finally, quantitative monitoring of selected taste
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compounds in cooked lobster (Homarus americanus) and Norway lobster (Nephros
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norvegicus) should visualize species differences in the combinatorial code of taste
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molecules.
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MATERIALS AND METHODS
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Chemicals and Materials. The following chemicals were purchased from the
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sources given in parentheses: L-amino acids, organic acids, inorganic salts,
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nucleosides, nucleotide phosphates, trigonelline hydrochloride, trimethyllysine
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hydrochloride,
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iodomethane-d3, picolinic acid (Sigma-Aldrich, Steinheim, Germany); γ-L-glutamyl
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dipeptides (Bachem, Weil am Rhein, Germany); hydrochloric acid, petrol ether, silver
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oxide, formic acid (Merck, Darmstadt, Germany); betaine anhydrous, choline
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chloride, trimethylamine N-oxide dihydrate (TMAO), ammonium hydroxide solution
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(25%) (Fluka, Neu-Ulm, Germany). Stable isotope labeled compounds such as amino
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acids, nucleotides, betaine-d11, and choline-d9 used for stable isotope dilution assays
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(SIDA) were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA,
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USA). Solvents were of high-performance liquid chromatography (HPLC) grade (J.T.
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Baker, Deventer, Netherland), and deuterated solvents were supplied by Euriso-Top
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(Saarbrücken, Germany). Deionized water used for chromatography was prepared
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by means of a Milli-Q water gradient A 10 system (Millipore, Schwalbach, Germany).
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For sensory analysis, bottled water (Evian) was adjusted to pH 6.8 with trace
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amounts of formic acid. Deep-frozen samples of king prawns (Penaeus vannamei)
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from aquacultures and Norway lobster (Nephros norvegicus) from the North Atlantic,
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both containing head and shell, as well as lobster tails (Homarus americanus) from
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the Northwest Atlantik were obtained commercially from a local vendor.
trifluoroacetic
acid,
chloramine-T
trihydrate,
iodomethane,
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Kitchen-type Preparation of Cooked King Prawns, Lobster, and Norway
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Lobster. Deep-frozen crustaceans were thawed by maintaining them for 1.5 h at
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room temperature. Four king prawns and four Norway lobsters, respectively, were
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blanched in hot water (4 L, 90°C) for 1 min, while the lobster tail was cooked in water
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(4 L) for 3 min at 100°C. After cooling to room temperature, intestines and the
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carcasses were removed. ACS Paragon Plus Environment
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Preparation of Crustacean Extracts. Portions (100 g) of crustacean meat were
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homogenized with water/methanol (80/20, v/v; 200 mL) for 3 min by means of an
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Ultra-Turrax T 25 basic (Ika Labortechnik, Stauffen, Germany) and, after
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centrifugation at 9000 rpm for 20 min at 7 °C (Avanti J-E, Beckmann-Coulter, Krefeld,
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Germany), the residues were re-extracted with water/methanol (80/20, v/v; 100 mL),
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centrifuged as described above, and lyophilized using a Christ Delta 1-24 LSC
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freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany) to
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give the non-soluble fraction of king prawns (yield: ~22% of cooked fresh weight),
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lobster (yield: ~20%), and Norway lobster (yield: ~18%). The pooled liquid
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supernatants were separated from solvent in vacuum and freeze-dried to give the
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extracts of king prawns (yield: ~8% of cooked fresh weight), lobster (yield: ~7%), and
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Norway lobster (yield: ~8%). All fractions were stored at -20 °C until further analysis.
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Stirred-Cell Ultrafiltration. An aliquot (2 g) of the king prawn soluble extract was
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dissolved in water (200 mL) and separated into a low molecular (KPLMW; < 1kDa;
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80% yield) and a high molecular weight fraction (KPHMW; ≥ 1kDa; 20% yield) using an
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Amicon 8400-type ultrafiltration cell (Amicon, Witten, Germany) equipped with a
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YM1-type cellulose filter (1 kDa cutoff; Millipore, Bedford, MA, USA) at a nitrogen
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pressure of 0.2 MPa. Both fractions collected from various separations were pooled
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accordingly and kept at -20 °C until further analysis.
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Quantitation of Candidate Basic Taste Compounds. Organic acids, Cations,
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and Anions. Aliquots (100 mg) of the soluble crustacean extracts were dissolved in
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water (3 mL), membrane filtered (0.45 µm) and without any further dilution (for
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organic acids) or after 1:10 dilution (anions, cations) analyzed by high-performance
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ion chromatography using a Dionex IC 2500 system (Dionex, Idstein, Germany)
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consisting of a GS50 gradient pump, an AS50 autosampler, an AS50 thermal
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compartment, and an ED 50 electrochemical detector following the protocol reported ACS Paragon Plus Environment
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recently.24 For cation analysis, a Dionex ICS-2000 apparatus was used with a digital
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conductivity detector, a CSRS 300 suppressor cell, an AS autosampler, and an
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eluent generator equipped with a RFIC EluGen cartridge EGC II MSA (Dionex,
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Idstein, Germany). Data analysis was performed using the Chromeleon software
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6.80. The quantitative data are given as the mean of four replicates (RSD for each
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data point
