Vegetable Broth

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Sensomics-based Molecularization of the Taste of Pot au Feu - a Traditional Meat/Vegetable Broth Maximilian Kranz, Florian Viton, Candice Smarrito-Menozzi, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05089 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 5, 2017

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Journal of Agricultural and Food Chemistry

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Sensomics-based Molecularization of the Taste of Pot

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au Feu - a Traditional Meat/Vegetable Broth

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Maximilian Kranz†, Florian Viton‡, Candice Smarrito-Menozzi‡, and

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Thomas Hofmann†,*

6 7

†

Chair of Food Chemistry and Molecular Sensory Science, Technical University of

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Munich, Lise-Meitner Str. 34, D-85354 Freising, Germany

9

‡

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Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, 1000 Lausanne 26,

Switzerland

11 12 13 14 15 16 17 18

*

To whom correspondence should be addressed

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PHONE

+49-8161-71-2902

20

FAX

+49-8161-71-2949

21

E-MAIL

[email protected]

22

1 Environment ACS Paragon Plus


1

ABSTRACT

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Targeted quantification of 49 basic taste-active molecules, followed by the calculation

3

of dose-over-threshold (DoT) factors, and taste re-engineering experiments revealed

4

minerals, nucleotides/nucleosides, amino acids, organic acids, and carbohydrates the

5

key compounds of Pot au Feu, a traditional broth preparation from beef cuts and

6

vegetables. Moreover, the dipeptide carnosine was identified to be the key inducer

7

for the white-meaty and thick-sour orosensation of the broth, next to anserine and 1-

8

deoxy-D-fructosyl-N-β-alanyl-L-histidine, the latter of which has been identified for the

9

first time by means of a sensory-guided fractionation of the broth. Sensory studies

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revealed the threshold concentration of carnosine in model broth to decrease by a

11

factor of 5 upon non-enzymatic glycosylation to reach 4.4 mmol/L for its Amadori

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product 1-deoxy-D-fructosyl-N-β-alanyl-L-histidine.

13 14 15

KEYWORDS: Sensomics, Maillard reaction, Amadori, carnosine, taste, taste modula-

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tion

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INTRODUCTION

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Pot-au-Feu is a popular dish, prepared from beef cuts, several vegetables and spic-

3

es, originally domiciled in the rural regions in northern France. The broth is highly

4

appreciated due to its full-bodied, complex and long-lasting savory taste and its typi-

5

cal aroma. By means of gas chromatography-olfactometry, the most odor-active vola-

6

tiles have been identified in beef-based broths and shown to be generated by Mail-

7

lard-type reactions, lipid autoxidation, as well as upon thiamin degradation.1-2 The

8

taste of meat preparations has been attributed to water-soluble, low-molecular weight

9

non-volatiles, such as, e.g. minerals, organic acids, amino acids, nucleotides, and

10

carbohydrates, respectively.3-5 Moreover, the dipeptides β-alanyl-L-histidine (carno-

11

sine, 1), β-alanyl-3-methyl-L-histidine (anserine, 2), and β-alanyl-L-glycine (3) were

12

reported to enhance the attractive white-meaty and long-lasting thick-sour orosensa-

13

tion of chicken broth when being present together with L-glutamic acid and sodium or

14

potassium ions.5-7 The carnosine homologue γ-aminobutyryl-L-histidine (homocarno-

15

sine, 4) (figure 1) was identified in several meat samples, albeit not so far evaluated

16

in its sensorial properties.8

17

Driven by the need to discover the key players imparting taste authenticity to

18

foods, the research area “sensomics” has made tremendous efforts in recent years in

19

mapping the comprehensive population of taste-active and taste-modulating com-

20

pounds in foods and relevant Maillard-type model systems by means of the taste dilu-

21

tion analysis (TDA) and the comparative taste dilution analysis (cTDA).9-12 For exam-

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ple, application of the cTDA to thermally treated alanine/glucose solutions and, later,

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to beef broth enabled the identification of the sweet-taste enhancing N-(1-

24

carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol inner salt, coined alapyridaine.13-14

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Further studies revealed only the (+)-(S)-enantiomer of alapyridaine to increase hu-

26

man sensitivity for sweetness, but also for umami and salt taste perception.13-16 Re-



1

cent application of the sensomics approach led to the identification of previously not

2

reported, taste modulating Maillard reaction products such as, e.g. N-(1-methyl-4-

3

oxoimidazolidin-2-ylidene) aminopropionic acid contributing to the typical thick-sour

4

and compley orosensation of the beef juice,5, 17 (S)-N2-(1-carboxyethyl)-guanosine 5’-

5

monophosphate as a key umami enhancer in yeast extracts,18-19 and 5-

6

acetoxymethyl-2-furaldehyde as sweet taste enhancer in traditional balsamic vine-

7

gar.20

8

The objective of the present study, therefore, was to quantitate the basic taste

9

compounds in Pot-au-Feu, to verify their sensory contribution by taste recombination

10

experiments and, then, screen for yet unknown taste modulators by means of activi-

11

ty-guided fractionation.

12 13 14

MATERIALS AND METHODS

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Chemicals. Ultrapure water for chromatographic separation was prepared with a

16

Milli-Q Gradient A 10 system (Millipore, Schwalbach, Germany), solvents were of

17

HPLC grade quality (Baker J.T., Deventer, Netherlands), and deuterated solvents

18

were purchased from Euriso-Top (Saarbruecken, Germany). The dipeptides L-

19

anserine and L-carnosine were obtained from Bachem (Bubendorf, Switzerland), L-

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homocarnosine from Aurora Fine Chemicals (Graz, Austria), L-Valine, D-galactose, D-

21

mannose, D-arabinose, sodium hydroxide, sucrose and calcium chloride from Merck

22

(Darmstadt, Germany). Stable isotope-labeled amino acids were supplied by Euriso-

23

Top (Saarbruecken, Germany). Yeast extract (Gistex XII LS) was obtained from FID

24

(Werne, Germany). All other chemicals were from Sigma Aldrich (Steinheim, Germa-

25

ny). For sensory analysis, bottled water (Evian) was adjusted to pH 5.9 with trace


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1

amounts of formic acid. A savory tasting model broth was prepared from monosodi-

2

um L-glutamate monohydrate (1.9 g), yeast extract (2.1 g), maltodextrin (6.375 g),

3

and sodium chloride (2.9 g), dissolved in bottled water (1 L) and adjusted to pH 5.9

4

with 1 % aqueous formic acid. Beef samples and vegetables were purchased from a

5

local food market.

6

Preparation of the Pot-au-Feu Broth (PaF). For the preparation of the Pot-au-

7

Feu, different parts of cattle were used: 624 g flat shoulder, 62 g round shoulder,

8

180 g bone marrow, 190 g knuckle of veal, 303 g oxtail, and 251 g flat rips were put

9

in a stainless steel cooking pot and, after adding cold water (5.0 L), were heated to

10

gentle boiling. Developing foam was removed constantly for the first 30 min, then,

11

sodium chloride (22.5 g) were added and, after boiling the broth for 2 h, 140 g leeks,

12

45 g onions, 108 g celery, 88 g may turnip, 0.35 g cloves, and 136 g carrots, each cut

13

into small pieces, were added to the broth. After boiling the broth for 3 h, the solids

14

were removed by decanting and filtration to afford the aqueous PaF-broth, which was

15

cooled to 4°C and separated from the fat layer by filtration. The broth was stored at -

16

24°C prior to further used.

17

Preparation of a “Meat-only” and a “Vegetable-only” Broth. Following the

18

protocol described above for the PaF-preparation, a “vegetable-only” broth was pre-

19

pared by omitting all meat parts. In addition, a “meat-only” broth was prepared by

20

omitting the vegetables and spices from the recipe. The solutions were stored at -

21

24°C prior to sensory evaluation.

22

Preparation of a Meat Extract. The meat parts obtained after preparation of

23

“meat-only” broth were freed from fat tissue and bones, subsequently chopped into

24

small pieces and minced. Aliquots (500 g) of the meat material were extracted with

25

methanol/water (70/30, v/v; 3 × 1.5 L) and, following concentration, meat extract (ap-

26

prox. 1.3 L) and the corresponding amount of “meat-only” broth (5.0 L) were recom-



1

bined. Aliquots (500 g) of this recombined broth were extracted with n-pentane

2

(3×400 mL) and solvents separated in vacuum to give the meat extract as an amor-

3

phous solid (yield: 47.76 g). The meat extract was stored at -24°C prior to chemical

4

analysis.

5

Solvent Extraction of the Pot-au-Feu Broth. Aliquots (500 g) of the PaF broth

6

were extracted three times with n-pentane (400 mL each). The aqueous layer was

7

lyophilized to afford the water-soluble fraction A (yield: 6.87 g), while the combined

8

organic phases were separated from solvent in vacuum to yield the lipid fraction B

9

(yield: 0.13 g).

10

Ultrafiltration. Following a literature protocol,5, 7 an aliquot (10 g) of lyophilized

11

PaF-fraction A was dissolved in water (500 mL) and separated by means of tangen-

12

tial-flow ultrafiltration using a Vivaflow 200 filtration unit (Sartorius, Goettingen, Ger-

13

many), equipped with a polyethersulfone (PES) membrane (5 kDa cutoff filter), to

14

yield the permeate fraction A1 (MW 5 kDa, 10 % in yield) as amorphous powders after lyophilization. Fractions

16

A1 and A2 were kept at -24°C until further used.

17

Gel Permeation Chromatography (GPC). An aliquot (1.0 g) of the freeze-dried

18

meat extract was dissolved in water (10 mL) and placed on top of a XK 50 glass col-

19

umn (100 × 5 cm, GE Healthcare Biosciences AB, Uppsala, Sweden), filled with a

20

slurry of Sephadex G-15 gel (GE Healthcare Biosciences AB, Uppsala, Sweden) in

21

water and conditioned with water adjusted to pH 4.0 with formic acid (1 g/100 g) as

22

reported earlier.5, 7 Operating with a flow of 2 mL/min, the effluent was monitored at

23

λ=220 nm by means of an UV-detector (Jasco UV-2075 Plus) and collected in seven

24

subfractions (I-VII) using a LKB 2070 Ultrorac II fraction collector (LKB Produkter,

25

Bromma, Sweden), followed by lyophilization.


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Subfractionation of GPC-Fraction III. Aliquots of the lyophilized GPC frac-

2

tion III (0.5 g) were solubilized in water (10 mL) by ultrasonification for 10 min and,

3

after membrane filtration (0.45 µm), the solutions were fractionated by means of pen-

4

tafluorphenylpropyl-(PFPP)-HPLC on a 250 × 21.2 mm i.d., 5 µm, Monochrom MS

5

column (Varian, Darmstadt, Germany), equipped with a 50 × 21.2 mm i.d., 5 µm,

6

guard column of the same type (Varian, Darmstadt, Germany). Monitoring the efflu-

7

ent with an evaporative light scattering detector (ELSD), chromatography was per-

8

formed at a flow rate to 18 mL/min using aqueous formic acid (0.1 % in water) as sol-

9

vent A and methanol/acetonitrile (50/50, v/v) containing 1 % aqueous formic acid as

10

solvent B. Starting with 0 % B for 10 min, the gradient was successively increased to

11

100 % B within 5 min and, then, held for another 5 min. Thereafter, the gradient was

12

re-adjusted to 0 % B within 5 min and kept for another 5 min prior to the next injec-

13

tion. The HPLC-effluent was collected separately into seven fractions, namely III-1 to

14

III-7, lyophilized twice and stored at -24°C prior to sensory experiments and chemical

15

analysis.

16

Subfractionation of Fraction III-3. The lyophilized HPLC-fraction III-3

17

(200 mg) was taken up in water (5.0 mL), ultrasonicated for 10 min, membrane fil-

18

tered (0.45 µm) and, then, the solution was separated by means of semi-preparative

19

hydrophilic interaction liquid chromatography (HILIC-HPLC) on a 300 × 21.5 mm i.d.,

20

10 µm, TSKgel Amide-80 column (Tosoh Bioscience, Stuttgart, Germany), equipped

21

with a 75 × 21.5 mm i.d., 10 µm, guard column of the same type (Tosoh Bioscience).

22

Monitoring the effluent with an ELSD detector, chromatography was performed at a

23

flow rate to 8 mL/min using a mixture (95/5, v/v) of acetonitrile and aqueous ammoni-

24

um acetate (100 mmol/L, adjusted to pH 3.5 with formic acid) as solvent B and aque-

25

ous ammonium acetate (5 mmol/L, pH 3.5) as solvent A. Starting with 25 % A for

26

10 min, the gradient was increased to 32 % A within another 25 min. Afterwards, the



1

gradient was increased to 100 % A within 8 min. These conditions were held for an-

2

other 12 min and afterwards, solvent A was reduced to 25 % A within 5 min, followed

3

by another 5 min for equilibration. The effluent was separated into six fractions,

4

namely III-3/1 to III-3/6, which were collected separately, freed from solvent, and ly-

5

ophilized twice prior to sensory evaluation and chemical analysis. Sensory evaluation

6

of the six HILIC-fractions in model broth revealed exclusively fraction III-3/6 to impart

7

a long-lasting complex and white-meaty orosensation.

8

Identification of Taste Modulating Compounds in HILIC-Fraction III-3/6.

9

Aliquots of the HILIC-fraction III-3/6 were dissolved in acetonitrile/water (1/1, v/v) and,

10

after membrane filtration (0.45 µm), were fractionated by means of preparative

11

HILIC-HPLC on a 250 × 21.0 mm i.d., 5 µm, NUCLEODUR HILIC column (Macherey-

12

Nagel, Dueren, Germany). Monitoring the effluent with an ELSD detector, chromatog-

13

raphy was performed isocratically at a flow rate to 20 mL/min with a solvent mixture

14

of 30% aqueous ammonium acetate (5 mmol/L; pH 3.0) and 70% of a mixture (95/5,

15

v/v) of acetonitrile and aqueous ammonium acetate (100 mmol/L; adjusted to pH 3.0

16

with formic acid). Three compounds were isolated from subfractions III-3/6a to III-

17

3/6c, separated from solvent in vacuum, and lyophilized twice to afford amorphous

18

powders in a purity of above 98%. Sensory experiments, LC-MS/MS and NMR stud-

19

ies, followed by co-chromatography with synthetic reference compounds led to the

20

identification of β-alanyl-L-histidine (carnosine, 1) and β-alanyl-3-methyl-L-histidine

21

(anserine, 2) in fractions III-3/6a and III-3/6b, respectively, and the previously not re-

22

ported 1-deoxy-D-fructosyl-N-β-alanyl-L-histidine (5) in fraction III-3/6c.

23

1-Deoxy-D-fructosyl-N-β-alanyl-L-histidine, 5, Figure 2: LC-MS (ESI-): m/z

24

387.15 (100, [M-H]-); 1H NMR (400 MHz, 300 K, D2O, COSY) δ 2.77 [m, 2H, H-C(7)],

25

3.13 [dd, J = 15.4, 8.2 Hz, 1H, H-C(11)], 3.21 – 3.27 [m, 1H, H-C(11)], 3.28 – 3.32

26

[m, 2H, H-C(1)], 3.33 – 3.44 [m, 2H, H-C(8)] , 3.63 – 3.75 [m, 1H, H-C(3)] , 3.76 –


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3.85 [m, 2H, H-C(6), H-C(8)], 3.87 – 3.91 [m, 1H, H-C(5)], 3.99 – 4.03 [m, 2H, H-C(4),

2

H-C(6)], 4.53 [dd, J = 8.2, 5.2 Hz, 1H, H-C(10)], 7.28 [d, J = 1.0 Hz, 1H, H-C(13)],

3

8.61 [d, J = 1.4 Hz, 1H, H-C(14)].

4

26.98 [C(11)], 30.26 [C(8)], 44.28 [C(7)], 53.01 [C(1)], 53.92 [C(10)], 63.91 [C(6)],

5

68.80 [C(4)], 69.20 [C(5)], 69.76 [C(3)], 95.21 [C(2)], 116.65 [C(13)], 129.49 [C(12)],

6

133.15 [C(14)], 171.60 [C(9)], 176.13 [C(15)].

13

C NMR (100 MHz, 300 K, D2O, HSQC, HMBC) δ

7

Maillard Preparation of 1-Deoxy-D-fructosyl-N-β-alanyl-L-histidine (5) and

8

[13C6]-1-Deoxy-D-fructosyl-N-β-alanyl-L-histidine. Potassium hydroxide (4 mmol)

9

was added to a suspension of carnosine (4 mmol) in methanol (100 mL), followed by

10

heating under reflux for 2 h. After cooling and filtration, the filtrate was separated from

11

solvent in vacuum to afford the carnosine-potassium salt, which was taken up in

12

methanol (50 mL) and, after adding glucose or [13C6]-labeled glucose (4 mmol), re-

13

spectively, heated for 2 h at 80°C under reflux. After cooling, the solvent was sepa-

14

rated in vacuum, the residue was dissolved in acetonitrile/water (50/50, v/v; 10 mL)

15

and, after membrane filtration (0.45 µm), fractionated by means of preparative HILIC-

16

HPLC on a 250 × 21.0 mm i.d., 5 µm, NUCLEODUR HILIC column (Macherey-Nagel,

17

Dueren, Germany), using the chromatographic conditions described above. The ma-

18

jor peak was collected, separated from solvent in vacuum and, after adding water (5

19

mL), lyophilized twice. Comparison of chromatographic (HILIC) and spectroscopic

20

data (UV-Vis, LC-MS/MS, 1H-NMR) of the synthesized compound with that isolated

21

from Pot-au-Feu, followed by co-chromatography confirmed its chemical structure as

22

1-deoxy-D-fructosyl-N-β-alanyl-L-histidine (5).

23

Quantitative Analysis of Basic Taste Compounds by Means of High-

24

Performance Ion Chromatography (HPIC). Quantification of Cations. A defined

25

volume of the PaF-broth was membrane filtered (0.45 µm), 1/10 (v/v) diluted with wa-

26

ter, and aliquots (2 µL) used for cation quantitation using a ICS-2000 Ion Chromatog-



1

raphy System (Dionex, Idstein, Germany), consisting of a GS 50 gradient pump, an

2

AS 50 autosampler, an AS 50 thermal compartment, and an ED 50A electrochemical

3

detector operating in conductivity mode. System control and data acquisition were

4

accomplished using Chromeleon Version 6.80 software (Dionex). Quantification was

5

based on external standard calibration. Concentrations of standard solutions ranged

6

from 0.1 mg/L to 70 mg/L (six-point calibration). Cations were analysed on a 250 ×

7

2 mm Dionex IonPac CS 18 analytical column, equipped with a Dionex IonPac CG

8

18 (50 × 2 mm) guard column and a self-regenerating cation suppressor CSRS 300

9

(2 mm, suppressor current: 5 mA, Dionex). Isocratic elution using aqueous methane

10

sulfonic acid (5 mmol/L) at a temperature of 40°C and a flow rate of 0.3 mL/min was

11

applied.

12

Quantification of carbohydrates, polyols, anions and organic acids. A defined

13

volume of the PaF-broth was membrane-filtered (0.45 µm) and used directly and, in

14

addition, after diluting with water (1/10, v/v) for the quantitative analysis of carbohy-

15

drates, polyols, anions, and organic acids, respectively. Aliquots (2 - 25 µL) were in-

16

jected into an ICS-2500 Ion Chromatography System (Dionex, Idstein, Germany),

17

following the protocol reported recently.7

18

Quantitative Analysis of Basic Tastants by High Performance Liquid

19

Chromatography/Tandem Mass Spectroscopy (HPLC-MS/MS). Amino Acids and

20

Dipeptides. Free amino acids and the dipeptides carnosine (1), anserine (2) and ho-

21

mocarnosine (4) were quantified by means of LC-MS/MS analysis, using stable iso-

22

tope labeled internal standards. The PaF-broth was diluted with water (1/40 to 1/500;

23

v/v), membrane-filtered (0.45 µm), and aliquots (990 µL) were then spiked with an

24

aliquot (10 µL) of an internal standard solution, containing stable isotope labeled

25

amino acids (1 mg/L, each), followed by MS analysis using an API 4000 QTrap LC-

26

MS/MS mass spectrometer (AB Sciex, Darmstadt, Germany), equipped with a


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TSKgel Amide-80 column (150 × 2.00 mm, i.d., 3 µm, Tosoh Bioscience, Stuttgart,

2

Germany), using the protocol reported recently.20-21 Dipeptides were quantified with

3

isotope-labeled histidine as an internal standard, using multiple reaction monitoring

4

(MRM) with defined mass transitions in the ESI+-mode. Given in parentheses are the

5

used mass transitions, declustering potential (DP in V), collision energy (CE in V),

6

and cell exit potential (CXP in V): carnosine (m/z 227.0→110.0; +36/ +33/ +33), an-

7

serine (m/z 241.0→109.0; +76/ +33/ +6), homocarnosine (m/z 241.1→110.0; +56/

8

+17/ +14). Quantitative data were obtained by comparing the peak areas of the cor-

9

responding mass traces with those of defined standard solutions. Values are given as

10

the mean of triplicates.

11

Creatine and Creatinine. Creatine and creatinine were quantified by means of

12

HPLC-MS/MS analysis using an API 3200 LC-MS/MS mass spectrometer (AB Sciex,

13

Darmstadt, Germany), equipped with a 150 × 2.0 mm i.d., 3 µm, Luna PFP column

14

(Phenomenex, Aschaffenburg, Germany). Prior to analysis, aliquots of the PaF-broth

15

were diluted with water (1/100; v/v) and membrane-filtered (0.45 µm). Diluted sam-

16

ples of the PaF-broth (990 µL) were spiked with an aliquot (10 µL) of a [2H3]-

17

creatinine solution (500 mg/L) and were injected into the HPLC-MS/MS system.

18

Chromatography was performed at a flow rate of 0.25 mL/min with isocratic elution,

19

using a mixture of water and acetonitrile (97/3, v/v), containing 0.1% formic acid.

20

Creatine and creatinine were analyzed using multiple reaction monitoring (MRM) with

21

defined mass transitions in the ESI+-mode. Given in parentheses are the used mass

22

transitions, declustering potential (DP in V), collision energy (CE in V), and cell exit

23

potential (CXP in V): creatine (m/z 132.0→90.2; +26/ +19/ +4), creatinine

24

(m/z 114.0→44.1; +31/ +29/ +5), [2H3]-creatinine (m/z 117.0→47.1; +31/ +29/ +5).

25

Quantitative data were obtained by comparing the peak areas of the corresponding



1

mass traces with those of defined standard solutions. Values are given as the mean

2

of triplicates.

3

Nucleotides and Nucleosides. Nucleotides and nucleosides were quantified by

4

means of HPLC-MS/MS analysis using an API 4000 QTrap LC-MS/MS mass spec-

5

trometer (AB Sciex, Darmstadt, Germany), equipped with a 150 × 4.6 mm, i.d., 5 µm,

6

polymeric ZIC-pHILIC column (Merck, Darmstadt, Germany), following the protocol

7

reported recently.21 Aliquots of the PaF-broth were diluted with water (1/10; v/v) and

8

membrane-filtered (0.45 µm) prior to analysis.

9

Carbohydrate-6-phosphates. Sugar-6-phosphates were quantified by means

10

of LC-MS/MS analysis using a Xevo TQ-S (Waters UK Ltd., Manchester, UK) mass

11

spectrometer, equipped with a 150 × 2.1 mm, 1.7 µm, Acquity UPLC BEH Amide col-

12

umn (Waters UK Ltd., Manchester, UK). Aliquots of the PaF-broth were diluted with

13

water (1/10; v/v) and membrane filtered (0.45 µm) prior to analysis. The sample solu-

14

tion (990 µL) was spiked with an internal standard solution of [13C6]-labeled glucose

15

(10 µL) and aliquots (2 µL) were injected into the LC-MS/MS system. Chromatog-

16

raphy was performed with a mixture (95/5, v/v) of acetonitrile and aqueous ammoni-

17

um acetate (100 mmol/L; adjusted to pH 3.0 with acetic acid) as solvent B and aque-

18

ous ammonium acetate (5 mmol/L; adjusted to pH 3.0 with acetic acid) as solvent A

19

(flow rate: 0.35 mL/min): Starting with a mixture of 5 % A and 95 % B for 1 min, the

20

content of solvent A was linearly increased to 50 % within 17 min. Thereafter, the

21

content of solvent A was increased to 100 % within 1 min and, then, held for another

22

2 min. The starting conditions were then re-adjusted within 1 min. Carbohydrate-6-

23

phosphates and the stable-isotope labeled internal standard [13C6]-glucose were ana-

24

lyzed in the negative electrospray ionization mode (ESI-), using optimized tuning pa-

25

rameters for each compound. The following mass transitions, cone voltages (CV in

26

V), and collision energies (CE in V) were used as given in parentheses: glucose-6-


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1

phosphate (m/z 259.1→79.0; -2/ -30), fructose-6-phosphate (m/z 259.0→79.0; -2/ -

2

34) and [13C6]-glucose-formate (m/z 231.0→92.0; -10/ -13). Quantitative data were

3

obtained via calibration curves, calculated by plotting peak area ratios of each ana-

4

lyte to the internal standard using linear regression. Values are given as the mean of

5

triplicates.

6 7

1-Deoxy-D-fructosyl-N-β-alanyl-L-histidine. Aliquots (50 mL) of the PaF-broth

8

(50 mL) were spiked with a solution of the internal standard [13C6]-1-deoxy-D-

9

fructosyl-N-β-alanyl-L-histidine (10 µL) in ACN (50%) (2 mg/mL) and, after homoge-

10

nization via ultrasonification (30 min), the samples were applied to a Strata C18-E

11

cartridge (1 g, 6 mL, Phenomenex, Aschaffenburg, Germany) preconditioned with

12

methanol and water (5 mL each). The cartridge was then eluted with water (100 mL)

13

and an aliquot (2 µL) of the effluent was directly injected into an API 4000 QTrap LC-

14

MS/MS mass spectrometer (AB Sciex, Darmstadt, Germany), equipped with a

15

150 × 2.00 mm, i.d., 5 µm, TSKgel Amide-80 column (Tosoh Bioscience, Stuttgart,

16

Germany). Chromatography (flow rate: 0.2 mL/min) was performed with a mixture

17

(95/5, v/v) of acetonitrile and aqueous ammonium acetate (100 mmol/L, adjusted to

18

pH 3.2 with acetic acid) as solvent B and aqueous ammonium acetate (5 mmol/L;

19

adjusted to pH 3.2 with acetic acid) as solvent A. Starting with a mixture of 80 % A

20

and 20 % B for 5 min, solvent B was increased to 100 % within another 12 min. The

21

target compound and the corresponding stable isotope labeled internal standard

22

were analyzed in the positive electrospray ionization mode (ESI+), using optimized

23

tuning parameters with for each compound with mass transitions, declustering poten-

24

tial (DP in V), collision energy (CE in V), and cell exit potential (CXP in V) given in

25

parentheses: 1-deoxy-D-fructosyl-N-β-alanyl-L-histidine (m/z 389.2→305.0; +71/ +25/

26

+4), [13C6]-1-deoxy-D-fructosyl-N-β-alanyl-L-histidine (m/z 395.3→310.1; +81/ +25/



1

+10). Quantitative data are given as the mean of triplicates, assessed by using cali-

2

bration curves that are calculated by plotting peak area ratios of each analyte to the

3

internal standard versus the concentration ratios followed by linear regression.

4

Gelatin. An aliquot (25 mL) of the PaF-broth was heated under reflux with hy-

5

drochloric acid (36 %; 25 mL) for 6 h. After hydrolysis, the release of L-4-

6

hydroxyproline was quantified in comparison to an untreated sample of the PaF-

7

broth, using the amino acid quantitation method described above.22

8

Sensory Analyses. General Conditions and Panel Training. Sensory evalua-

9

tion of single compounds and fractions was performed by a trained panel of 15 volun-

10

teers, who gave informed consent to participate the sensory tests of the present in-

11

vestigation and had no history of known taste disorders, while using nose clips to

12

avoid any retro-nasal aroma impressions. To remove solvent traces and buffer com-

13

pounds from fractions and isolated compounds, the individual samples were sus-

14

pended in water and were freeze-dried twice after removing the volatiles in high vac-

15

uum. Analytical techniques (1H-NMR, HPLC-MS) revealed that food fractions treated

16

by that procedure were essentially free of solvents and buffer compounds. The panel

17

was trained to recognize and distinguish the taste of aqueous solutions of standard

18

taste compounds by using the sip-and-spit method as described earlier.23-24 For train-

19

ing the thick-sour and white-meaty orosensation, a model broth solution was evaluat-

20

ed before and after spiking carnosine (40 mmol/L).

21

Taste Dilution Analysis (TDA) and Comparative Taste Dilution Analysis

22

(cTDA). According to a literature protocol,25 a taste dilution analysis (TDA) was per-

23

formed with the GPC-fractions in order to determine the intrinsic taste quality of sin-

24

gle fractions, while taste modulating activities were evaluated by means of the com-

25

parative taste dilution analysis (cTDA).14 To achieve this, the corresponding effluent

26

fractions of three GPC were dissolved in natural concentration ratios in water, adjust-


Page 14 of 37

Page 15 of 37


1

ed to pH 5.9 with trace amounts of aqueous 1% formic acid (for TDA), or in model

2

broth (for cTDA), respectively. Each fraction was diluted stepwise one-to-one with

3

water (for TDA) or model broth (for cTDA) and, then, presented in order of increasing

4

concentrations to trained sensory panellists, who were asked to evaluate the taste

5

quality and to determine the detection threshold in a duo test. The dilution step show-

6

ing a difference in taste between the blank water (for TDA) or the blank model broth

7

(cTDA), respectively, and the diluted GPC fraction was defined as the taste dilution

8

factor (TD-factor).

9

Taste Recognition Thresholds Concentrations. Prior to sensory analysis, puri-

10

ties of the tested compounds were checked by means of 1H NMR spectroscopy and

11

LC-MS analysis. Taste recognition thresholds were determined either in water

12

(pH 5.9) for intrinsic taste or in model broth for taste modulatory or enhancement ef-

13

fects by twelve trained panellists using a triangle test with ascending concentrations

14

of a particular compound.21, 26 Taste threshold concentrations were approximated by

15

averaging the threshold values of the individual panellists in three independent ses-

16

sions.

17

High-Performance Liquid Chromatography - Mass Spectrometry (HPLC-

18

MS/MS). LC-MS/MS analysis was performed using an Dionex Ultimate 3000 HPLC-

19

system connected to either an API 3200 MS/MS, or an API 4000 QTrap MS/MS de-

20

vice (AB Sciex, Darmstadt, Germany) running in the positive or negative electrospray

21

ionization (ESI+, ESI-) mode, respectively. Zero grade air served as nebulizer gas

22

(45 psi), and as turbo gas for solvent drying (55 psi, 425°C). Nitrogen served both as

23

curtain gas (35 psi) and collision gas (8.7x10–7 psi). Both quadrupoles were set at

24

unit resolution. ESI+ and ESI- mass and product ion spectra were acquired with direct

25

flow infusion. For ESI+, the ion spray voltage was set at + 5500 V and - 4500 V for

26

ESI- . Energies for declustering potential (DP) entrance potential (EP), collision ener-



Page 16 of 37

1

gy (CE), and cell exit potential (CXP) as well as MS/MS parameters for measuring in

2

the MRM mode were optimized for each compound individually, detecting the frag-

3

mentation of molecular ions into specific product ions after collision with nitrogen. For

4

instrumental control and data acquisition, Sciex Analyst software v1.5 (Applied Bio-

5

science) was used.

6

Carbohydrate-6-phosphates were analyzed on a Waters Xevo TQ-S tandem

7

mass spectrometer (Waters UK Ltd., Manchester, UK) in the negative electrospray

8

ionization (ESI-) mode, coupled with an Acquity UPLC I-class system (waters, Milford,

9

MA, USA). The ion source parameters were set as follows: capillary voltage (-2.8 kV),

10

sampling cone voltage (20 V), source offset (50 V), source temperature (150°C),

11

desolvation temperature (500°C), cone gas (150 L/h), collision gas (0.15 mL/min),

12

and nebulizer gas (7 bar). The Xevo TQ-S mass spectrometer was operated with

13

MassLynx 4.1 software (Waters UK Ltd., Manchester, UK) and data processing was

14

performed using TargetLynx 4.1 SCN 813 software (Waters UK Ltd., Manchester,

15

UK). The MS/MS parameters were individually tuned for each compound after colli-

16

sion with argon gas.

17

Preparative High-Performance Liquid Chromatography (HPLC). For pre-

18

parative chromatography, the HPLC system (Jasco, Groß-Umstadt, Germany) con-

19

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

20

valve with a 1000 µL loop (Rheodyne, Bensheim, Germany), a MD-2010 Plus multi-

21

wavelenth detector, and a Sedex 85 LT-ELSD detector (Sedere, Alfortville, France). Nuclear Magnetic Resonance Spectroscopy (NMR). 1H,

22 23

13

C, homonuclear

1

H-1H correlation spectroscopy (1H-1H-COSY), heteronuclear single quantum coher-

24

ence spectroscopy (HSQC) and heteronuclear multiple bond correlation spectrosco-

25

py (HMBC) NMR measurements were performed on an Avance III 500 MHz

26

equipped with a CTCI probe and an Advance III 400 MHz spectrometer with a BBO


Page 17 of 37


1

probe (Bruker, Rheinstetten, Germany), respectively. Chemical shifts were refer-

2

enced to the solvent signal. Data processing was performed by using Topspin Ver-

3

sion 3.2 (Bruker, Rheinstetten) and MestReNova version 6.2.1 software (Mestrelab

4

Research, Santiago de Compostela, Spain).

5 6 7

RESULTS AND DISCUSSION

8 9

Aiming at the identification of compounds contributing to the typical white-meaty and

10

long-lasting thick-sour orosensation induced of Pot-au-Feu (PaF), the freshly pre-

11

pared broth was presented to trained sensory panellists, who were asked to rate the

12

individual intensities of the taste qualities sweet, sour, salty, bitter, astringent, umami,

13

and white-meaty on a linear scale between 0 (not detectable) and 5 (strongly detect-

14

able). The PaF-broth exhibited a pronounced umami taste judged with a score of 2.8,

15

followed by saltiness (2.3) and the white-meaty and thick-sour orosensation (2.3),

16

respectively (Table 1). Sweetness and sourness were rated with somewhat lower

17

scores of 1.0 and 0.9, respectively, while astringency and bitterness were only mar-

18

ginally perceived (scores: 0.4 and 0.2).

19

Solvent Extraction and Molecular Weight Fractionation of PaF-Broth. As

20

the taste-active compounds of aqueous preparations from beef were expected to be

21

polar and water-soluble,5 a defined volume of the PaF-broth was de-fatted by extrac-

22

tion with n-pentane. Aqueous and organic phases were freed from solvent in vacuum

23

to yield the water-soluble fraction A (98.1 wt% of dry mass) and hydrophobic fraction

24

B (1.9 wt% of dry mass). After solvent separation, sensory evaluation showed that

25

the taste profile of fraction A, dissolved in water, matched well with that of the whole



1

PaF-broth (data not shown), thus demonstrating the water-soluble compounds as the

2

key taste contributors.

3

To gain insight into the molecular weight of the taste compounds, fraction A was

4

separated using tangential-flow ultrafiltration with a 5 kDa cut-off filter, yielding frac-

5

tions A1 (< 5 kDa) and A2 (≥ 5 kDa). After lyophilisation, fractions A1 and A2 were

6

taken up in water in their native concentrations, the pH was adjusted to pH 6.9, and

7

then used for taste profile analysis (Table 1). The high molecular weight fraction A2

8

was perceived as nearly tasteless with only a slight bitter and astringent taste,

9

whereas the low-molecular weight fraction A1 showed almost the identical taste pro-

10

file as the whole PaF-broth. In conclusion, the key molecules evoking the typical taste

11

of the PaF were shown to be water-soluble and exhibit low molecular weight.

12

Influence of the Condiments on the Taste Profile of the PaF Broth. To nar-

13

row down the enormous complexity of the PaF broth and to gain a first insight into the

14

source of the key taste compounds, a “vegetable only” and a “meat only” broth, re-

15

spectively, was prepared and compared to the taste profile of the PaF broth. The

16

“vegetable only” broth showed only weak taste intensity, e.g. none of the taste de-

17

scriptors rated with scores above 1.0 and the white-meaty and thick-sour orosensa-

18

tion was not detectable al all (Table 2). In comparison, the taste profile of the “meat

19

only” broth matched well with that of the whole PaF broth with the exception of a

20

somewhat lower intensities for the white-meaty and thick-sour orosensation (2.3 vs.

21

2.0) and umami taste (2.8 vs. 2.5). These data clearly demonstrated that the key

22

taste molecules of the PaF broth origin from the meat parts used for PaF preparation

23

and that in particular the white-meaty and thick-sour orosensation may be slightly

24

enhanced when the meat was thermally treated in the presence of vegetables.

25

Quantitation of Basic Tastants in PaF Broth and Taste Re-engineering Ex-

26

periments. In order to evaluate the impact of literature-known basic taste com-


Page 18 of 37

Page 19 of 37


1

pounds on the taste profile of the PaF broth, cations, anions, polyols, carbohydrates

2

and organic acids were quantitated by means of high-performance ion chromatog-

3

raphy (HPIC), while amino acids, dipeptides, creatine/creatinine, carbohydrate-6-

4

phosphates, nucleotides and nucleosides were quantitatively determined by means

5

of UHPLC-MS/MS and stable isotope dilution analysis (SIDA). In addition, a dose-

6

over-threshold (DoT) factor was calculated for each compound as the ratio of the

7

concentration of a target compound in the PaF broth and its taste threshold concen-

8

tration.5, 20 Thereafter, the taste compounds were grouped into six classes, namely I

9

to VI, depending on their taste quality (Table 3).

10

The bitter tasting group I contained creatine and creatinine, taurine, hypoxan-

11

thine, xanthine and inosine, as well as the bitter tasting amino acids L-tyrosine, L-

12

leucine, L-isoleucine, L-lysine, L-valine, L-arginine and L-histidine. With a calculated

13

DoT factor of 1.0, only creatinine reached its bitter taste threshold concentration.

14

Group II comprised the umami-like compounds L-glutamine, L-glutamic acid, L-

15

asparagine, L-aspartic acid, 5’-AMP, 5’-IMP, and succinic acid. According to its DoT

16

factor, only L-glutamic acid (1.7) exceeded its taste threshold and, therefore, is ex-

17

pected to show a direct impact on the taste of the PaF, while the other compounds

18

may contribute by taste enhancement. Salty tasting compounds were in group III,

19

comprising the anions chloride and phosphate and the cations of sodium, magnesi-

20

um, potassium and calcium. The highest DoT factors were found for sodium (16.4)

21

and chloride (9.7), followed by potassium with a DoT factor of 1.0. Group IV com-

22

prised sweet tasting compounds like carbohydrates and carbohydrate-6-phosphates,

23

polyols, and the amino acids L-alanine, L-ornithine, L-methionine, L-4-hyroxyproline,

24

L-proline, L-serine

25

L-alanine

26

centrations. Among the sour tasting compounds pyroglutamic acid, malic acid, citric

and L-threonine. The highest DoT-factor of 1.7 was determined for

and L-ornithine, while other compounds did not reach their threshold con-



1

acid, formic acid and lactic acid, summarized in group V, pyroglutamic acid (1.3) and

2

lactic acid (1.0) showed the highest taste contribution with DoT-factors of 1.3 and 1.0,

3

respectively. Group VI represented the dipeptides carnosine, anserine, and homo-

4

carnosine that were reported to induce a white-meaty orosensation in the presence of

5

L-glutamic

6

was found for carnosine.

acid and minerals.5-7 Among these peptides, the highest DoT-factor of 0.4

7

To study whether the compounds already identified (Table 3) can already ex-

8

plain the typical taste profile of the PaF broth, a basic taste recombinant was pre-

9

pared by dissolving all 49 compounds from tastant groups I to V plus compounds 1 ,2

10

and 4 from group VI in their natural concentrations in water. In addition, gelatin was

11

added in its levels as found in PaF broth and the pH adjusting to 6.9 prior to sensory

12

analysis. Comparative taste dilution analysis of the authentic broth and the recombi-

13

nant revealed that the intensity scores for the acidic taste (0.9), bitterness (0.2) and

14

astringency (0.4) matched perfectly. Moreover, saltiness (2.3 vs. 2.4 ± 0.2) and the

15

umami-like taste quality (2.8 vs. 2.9 ± 0.3) were evaluated almost identical (Table 2).

16

Whereas the sweet note was somewhat higher (1.0 vs. 1.3) in the recombinant, the

17

white-meaty and thick-sour orosensation was perceived as slightly lower (2.3 vs. 2.1)

18

when compared to the PaF broth (Table 2). In conclusion, the taste re-engineering

19

experiment demonstrated the basic taste compounds, summarized in Table 3, as the

20

key molecules coining the typical taste profile of the PaF.

21

In order to investigate as to whether the small difference in the white-meaty

22

and thick-sour orosensation is due to additional, yet unknown compounds, an activi-

23

ty-guided fractionation was performed. As the meat was found as the major precursor

24

of the taste compounds in PaF (Table 2), the “meat only” broth was used for further

25

analysis to somewhat reduce the complexity of the PaF broth.


Page 20 of 37

Page 21 of 37


1

Activity-Guided Fractionation of a “Beef-only” Broth. Aiming at the identi-

2

fication of unknown taste modulating compounds originating from beef meat, the

3

“meat only” broth was combined with a methanol/water extract prepared from the

4

cooked meat parts, de-fatted with n-pentane, followed by gel permeation chromatog-

5

raphy (GPC) on a Sephadex G-15 resin to received seven GPC fractions (I-VII),

6

which were individually lyophilized twice and used for comparative taste dilution as-

7

say (cTDA)14 (Figure 3). Aliquots of the GPC fractions I-VII were solubilized in model

8

broth in their native concentrations in PaF and the sensory panel was asked to com-

9

pare the gustatory impact of the samples in comparison to the blank model broth so-

10

lution. Whereas fractions I and VII did not show any sensory impact, GPC fraction III

11

increased the thick-sour and white-meaty oral impression with a TD factor of 16 (Fig-

12

ure 3). The other fractions showed lower TD-factors for bitterness (fractions II, IV, VI)

13

and umami taste (fraction II and IV).

14

Due to the intense thick-sour and white-meaty orosensation detected in frac-

15

tion III, this GPC fraction was further separated by means of HPLC using a pen-

16

tafluorphenylpropyl-phase (PFPP-HPLC). Monitoring the effluent with an ELSD de-

17

tector, the effluent was separated into seven fractions, namely III-1 to III-7 (Figure

18

4A), which were solubilized in model broth and then sensorially compared to the

19

blank model broth. For HPLC-fractions III-2, III-3 and III-4 the panel observed an in-

20

crease of the thick-sour quality and white-meaty oral impression. Fraction III-5 in-

21

duced a sour taste and fraction III-6 enhanced the umami taste of the model broth,

22

whereas fractions III-1 and III-7 did not show any sensory activity (data not shown).

23

Aiming at the identification of molecules being responsible for the typical

24

white-meaty orosensation, fraction III-3 was fractionated by means of semiprepara-

25

tive hydrophilic interaction liquid chromatography (HILIC) to give six fractions, namely

26

III-3/1 to III-3/6 (Figure 4B). Sensory evaluation of the individual fractions revealed



1

fraction III-3/6 to evoke the targeted white-meaty and thick-sour impact when tested

2

in model broth. However, as NMR and LC-MS experiments demonstrated this frac-

3

tion to be still a complex mixture of compounds, the subfraction III-3/6 was further

4

separated by preparative HILIC-HPLC to yield fractions III-3/6a to III-3/6c (Figure

5

4C). Sensory experiments demonstrated that all three fractions enhanced the white-

6

meaty orosensation of the model broth.

7

LC-MS analysis of fractions III-3/6a and III-3/6b revealed the pseudomolecular

8

ions (M+H)+ m/z 241 and 227 in the ESI+-mode. By comparison of spectroscopic data

9

(LC-MS, NMR) and chromatographic data (retention times), followed by co-

10

chromatography with reference substances, III-3/6a and III-3/6b could be unequivo-

11

cally identified as the literature-known dipeptides β-alanyl-3-methyl-L-histidine (an-

12

serine, 2) and β-alanyl-L-histidine (carnosine, 1), respectively. However, the com-

13

pound in fraction III-3/c could not be assigned by comparison with any reference sub-

14

stance.

15

Identification of the White-Meaty and Thick-Sour Taste Compound in

16

Fraction III-3/6c. MS analysis of the target compound in fraction III-3/6c showed a

17

pseudomolecular ion ([M-H]+) of m/z 389. In ESI- the fragments m/z 387, 297, 225,

18

154 and 110 were observed in collision induced dissociation. The fragment ions

19

m/z 225,154 and 110 were identical to those of carnosine and the mass shift of the

20

pseudo-molecular ion m/z 387 to m/z 225 (carnosine, [M-H]-) was a first hint for the

21

cleavage of a glucose moiety. To unequivocally assign the chemical structure of the

22

target compound, 1D/2D-NMR experiments were applied. Comparing the 1H-NMR

23

spectrum of the target compound with that of carnosine revealed large similarity be-

24

tween both molecules and demonstrated the intact carnosine moiety within the target

25

molecule. Protons resonating between 3.63 and 4.03 ppm were assigned as the pro-

26

tons H-C(3), H-C(4), H-C(5) and H-C(6) of a glucose moiety and showed the ex-


Page 22 of 37

Page 23 of 37


1

pected homonuclear connectivity in a COSY experiment. A HSQC experiment

2

demonstrated C(1) and C(6) to be methylene groups and C(2) to be a quaternary

3

carbon showing a heteronuclear coupling with carbon C(6) in an HMBC experiment,

4

thus indicating a C(2)→C(6) ring closure. Moreover HMBC experiments revealed a

5

heteronuclear coupling between carbon C(7) and proton H-C(1), thus indicating the

6

linkage via the nitrogen atom of the β-alanyl moiety (Figure 2). Taking all these data

7

into consideration, the target compound in fraction III-3/6c was proposed to be 1-

8

deoxy-D-fructosyl-N-β-alanyl-L-histidine (5, Figure 2). To verify this proposal, a bina-

9

ry mixture of carnosine and glucose was reacted in methanol, the major reaction

10

product isolated by HILIC-HPLC in a purity of >98 %, followed by NMR and LC-

11

MS/MS analysis. Comparison of spectroscopic and chromatographic data demon-

12

strated the identity of the synthesized Amadori product with the compound isolated

13

from the beef broth, thus confirming 1-deoxy-D-fructosyl-N-β-alanyl-L-histidine (5),

14

the Amadori product from carnosine and glucose, as the taste enhancing molecule.

15

To the best of our knowledge, this taste molecule has not yet been previously report-

16

ed.

17

Sensory Analysis and Quantitation of 1-Deoxy-D-fructosyl-N-β-alanyl-L-

18

histidine (5). To investigate the taste activity of 1-deoxy-D-fructosyl-N-β-alanyl-L-

19

histidine (5), the purified Amadori product was checked for purity and identity by

20

means of 1H NMR and LC-MS prior to sensory analysis. Using a three-alternative

21

forced-choice (3-AFC) procedure, compound 5 did not show any intrinsic taste in wa-

22

ter (pH 5.9) up to a concentration of 30 mmol/L, but induced a white-meaty and thick-

23

sour orosensation in model broth (pH 5.9) above a taste threshold concentration of

24

4.4 mmol/L (Table 3). The quality of 5 in model broth was similar to that of the dipep-

25

tide carnosine, but 5 showed a five times lower threshold concentration when com-

26

pared to carnosine (22.7 mmol/L).



1

1-Deoxy-D-fructosyl-N-β-alanyl-L-histidine was then quantitated in PaF broth

2

by means of a stable isotope dilution analysis using the 13C6-labeled twin molecule as

3

the internal standard. HILIC-MS/MSMRM analysis revealed a concentration of

4

10 µmol/L of 5 in the PaF broth (Table 3). Although the threshold of 5 was five times

5

lower than that of carnosine, the concentration of the later peptide exceeded the lev-

6

els of the Amadori product by a factor of 1000, thus demonstrating carnosine as the

7

most important inducer of the white-meaty and thick sour orosensation in PaF broth.

8

Further studies in the future will, therefore, focus on developing high-yield, kitchen

9

chemistry-type approaches to increase the yield of generating the taste modulating 1-

10

deoxy-D-fructosyl-N-β-alanyl-L-histidine from natural precursors and to make this

11

compound available for convenience products.

12 13

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14

(1)

Guentert, M.; Bruening, J.; Emberger, R.; Koepsel, M.; Kuhn, W.; Thielmann,

15

T.; Werkhoff, P., Identification and formation of some selected sulfur-

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containing flavor compounds in various meat model systems. J. Agric. Food

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Chem. 1990, 38 (11), 2027-2041.

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(2)

ruminant meat. Molecules 2013, 18 (6), 6748-81.

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(3)

Schlichtherle-Cerny, H.; Grosch, W., Evaluation of taste compounds of stewed beef juice. Z Lebensm Unters For 1998, 207 (5), 369-376.

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Resconi, V. C.; Escudero, A.; Campo, M. M., The development of aromas in

(4)

Warendorf, T.; Belitz, H. D.; Gasser, U.; Grosch, W., The Flavor of Bouillon. 2.

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Sensory Analysis of Nonvolatiles and Imitation of a Bouillon. Z Lebensm

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Unters For 1992, 195 (3), 215-223.


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identification of N-(1-methyl-4-oxoimidazolidin-2-ylidene)-alpha-amino acids as

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contributors to the thick-sour and mouth-drying orosensation of stewed beef

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juice. J. Agric. Food Chem. 2010, 58 (10), 6341-50.

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(6)

Methylated Compounds. Bull. Jpn. Soc. Sci. Fish. 1982, 48 (1), 89-95.

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Suyama, M.; Shimizu, T., Buffering Capacity and Taste of Carnosine and Its

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Dunkel, A.; Hofmann, T., Sensory-directed identification of beta-alanyl

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9

induced by chicken broth. J. Agric. Food Chem. 2009, 57 (21), 9867-77.

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Peiretti, P. G.; Medana, C.; Visentin, S.; Giancotti, V.; Zunino, V.; Meineri, G.,

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Determination of carnosine, anserine, homocarnosine, pentosidine and

12

thiobarbituric acid reactive substances contents in meat from different animal

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species. Food Chem. 2011, 126 (4), 1939-47.

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Toelstede, S.; Dunkel, A.; Hofmann, T., A Series of Kokumi Peptides Impart

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the Long-Lasting Mouthfulness of Matured Gouda Cheese. J. Agric. Food

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Chem. 2009, 57 (4), 1440-1448.

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Toelstede, S.; Hofmann, T., Quantitative studies and taste re-engineering

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experiments toward the decoding of the nonvolatile sensometabolome of

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Gouda cheese. J. Agric. Food Chem. 2008, 56 (13), 5299-5307.

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Toelstede, S.; Hofmann, T., Sensomics mapping and identification of the key

21

bitter metabolites in Gouda cheese. J. Agric. Food Chem. 2008, 56 (8), 2795-

22

2804.

23

(12)

Hufnagel, J. C.; Hofmann, T., Quantitative reconstruction of the nonvolatile

24

sensometabolome of a red wine. J. Agric. Food Chem. 2008, 56 (19), 9190-

25

9199.



1

(13)

Ottinger, H.; Hofmann, T., Identification of the taste enhancer alapyridaine in

2

beef broth and evaluation of its sensory impact by taste reconstitution

3

experiments. J. Agric. Food Chem. 2003, 51 (23), 6791-6796.

4

(14)

Ottinger, H.; Soldo, T.; Hofmann, T., Discovery and structure determination of

5

a novel Maillard-derived sweetness enhancer by application of the

6

comparative taste dilution analysis (cTDA). J. Agric. Food Chem. 2003, 51 (4),

7

1035-1041.

8

(15)

enhancer? Chem. Senses 2003, 28 (5), 371-379.

9 10

Soldo, T.; Blank, I.; Hofmann, T., (+)-(S)-alapyridaine - A general taste

(16)

Villard, R.; Robert, F.; Blank, I.; Bernardinelli, G.; Soldo, T.; Hofmann, T.,

11

Racemic and enantiopure synthesis and physicochemical characterization of

12

the novel taste enhancer N-(1-carboxyethyl)-6-(hydroxymethyl)pyridinium-3-ol

13

inner salt. J. Agric. Food Chem. 2003, 51 (14), 4040-4045.

14

(17)

Kunert, C.; Walker, A.; Hofmann, T., Taste modulating N-(1-methyl-4-

15

oxoimidazolidin-2-ylidene) alpha-amino acids formed from creatinine and

16

reducing carbohydrates. J. Agric. Food Chem. 2011, 59 (15), 8366-74.

17

(18)

Festring, D.; Hofmann, T., Discovery of N(2)-(1-carboxyethyl)guanosine 5'-

18

monophosphate as an umami-enhancing maillard-modified nucleotide in yeast

19

extracts. J. Agric. Food Chem. 2010, 58 (19), 10614-22.

20

(19)

Festring, D.; Hofmann, T., Systematic studies on the chemical structure and

21

umami enhancing activity of Maillard-modified guanosine 5'-monophosphates.

22

J. Agric. Food Chem. 2011, 59 (2), 665-76.

23

(20)

Hillmann, H.; Mattes, J.; Brockhoff, A.; Dunkel, A.; Meyerhof, W.; Hofmann, T.,

24

Sensomics analysis of taste compounds in balsamic vinegar and discovery of

25

5-acetoxymethyl-2-furaldehyde as a novel sweet taste modulator. J. Agric.

26

Food Chem. 2012, 60 (40), 9974-90.


Page 26 of 37

Page 27 of 37


1

(21)

Meyer, S.; Dunkel, A.; Hofmann, T., Sensomics-Assisted Elucidation of the

2

Tastant Code of Cooked Crustaceans and Taste Reconstruction Experiments.

3

J. Agric. Food Chem. 2016, 64 (5), 1164-75.

4

(22)

Amtliche Sammlung von Untersuchungsverfahren nach § 64 LFGB (2010)

5

Bestimmung des Hydroxyprolingehaltes in Fleisch und Fleischerzeugnissen;

6

Photometrisches Verfahren nach saurem Aufschluss L 06.00–8, Hrsg.: BVL,

7

Beuth Verlag GmbH, Berlin.

8

(23)

Rotzoll, N.; Dunkel, A.; Hofmann, T., Activity-guided identification of (S)-malic acid 1-O-d-glucopyranoside (morelid) and γ-aminobutyric acid as contributors

9 10

to umami taste and mouth-drying oral sensation of morel mushrooms

11

(Morchella deliciosa Fr.). J. Agric. Food Chem. 2005, 53 (10), 4149-4156.

12

(24)

Scharbert, S.; Hofmann, T., Molecular definition of black tea taste by means of

13

quantitative studies, taste reconstitution, and omission experiments. J. Agric.

14

Food Chem. 2005, 53 (13), 5377-5384.

15

(25)

Frank, O.; Ottinger, H.; Hofmann, T., Characterization of an intense bitter-

16

tasting 1 H, 4 H-quinolizinium-7-olate by application of the taste dilution

17

analysis, a novel bioassay for the screening and identification of taste-active

18

compounds in foods. J. Agric. Food Chem. 2001, 49 (1), 231-238.

19 20

(26)

Hillmann, H.; Hofmann, T., Quantitation of Key Tastants and Re-engineering the Taste of Parmesan Cheese. J. Agric. Food Chem. 2016, 64 (8), 1794-805.

21

22



Page 28 of 37

1

Table 1. Taste Profile Analysis of Pot au Feu (PaF) and the Ultrafiltration Fractions

2

A1 (5 kDa). intensities for individual taste qualitiesa taste descriptor

PaF

A2 (>5 kDa)

A1 (

Vegetable Broth

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