Expression of Multidisciplinary Flavour Science
BEHAVIOUR OF SELECTED FLAVOUR COMPOUNDS IN DAIRY
MATRICES: STABILITY AND RELEASE
K. BUHR1,3, B. Köhlnhofer1, A. Heilig2, J. Hinrichs2, and P. Schieberle1,3
1
2
3
Deutsche Forschungsanstalt für Lebensmittelchemie (DFA), Lichtenbergstraße 4,
D-85748 Garching, Germany
Institut für Lebensmittelwissenschaft und Biotechnologie (LTH), Garbenstraße 21,
D-70599 Stuttgart, Germany
Lehrstuhl für Lebensmittelchemie, Technische Universität München (TUM),
Lichtenbergstraße 4, D-85748 Garching, Germany
Abstract
Stability of ethyl hexanoate was found to be reduced in low fat dairy matrices.
Release of limonene, ethyl hexanoate, diacetyl and 2-methylbutanoic acid from
various dairy matrices and sunflower oil was measured by Proton Transfer Reaction Mass Spectrometry and compared with orthonasal detection thresholds.
Introduction
Despite increasing knowledge on interaction between flavour compounds and
various food matrices, it remains a challenge to adapt complex flavourings to dairy
matrices. This is mainly due to the complexity and variability of these matrices. In this
relation, numerous studies focused on individual aspects of flavour - dairy matrix
interaction. For example, it was shown that milk proteins, especially bovine serum
albumine and β-lactoglobuline, are able to specifically bind flavour compounds
resulting in a reduced availability for perception [1-3]. Furthermore it was shown that
higher fat contents lead to retention of lipophilic flavour compounds while the release
of more polar compounds like diacetyl is hardly affected [4-6]. Additional influencing
variables are different degrees of technological processing (heating, acidification or
fermentation) as well as addition of polysaccharides [4] or sugars [6].
While most of the studies mentioned above focus on individual aspects of flavour
- dairy matrix interaction and release, it is the objective of this study to perform a
systematic investigation on flavour - dairy matrix interaction and release of selected
aroma compounds by stepwise increasing the complexity of the matrix. The aroma
compounds diacetyl, 2-methylbutanoic acid, methoxyfuraneol, ethyl hexanoate,
limonene, δ-decalactone and vanillin were selected based on their industrial
relevance and in order to account for a wide range of functional groups, lipophilicity
and possible interaction mechanisms. Stepwise increase of the matrix complexity
was achieved by studying the release of the same aroma compounds from water,
sunflower oil as a replacement for milk fat, dispersions of whey protein, casein, milk
permeate as well as various dairy product matrices by Proton Transfer Reaction Mass Spectrometry (PTR-MS) [7]. Results were compared with orthonasal detection
thresholds of the same aroma compounds determined by the triangle test approach.
Additionally, the stability of the aroma compounds was investigated by Stable Isotope
Dilution Assays [8].
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Expression of Multidisciplinary Flavour Science
Experimental
Stability of aroma compounds in yogurt matrices. For investigation of stability of
aroma compounds in yogurt matrices, recovery of selected compounds from yogurt
matrices with varying fat contents was determined by Stable Isotope Dilution Analysis
according to [8].
Proton Transfer Reaction - Mass Spectrometry (PTR-MS). Aroma release from
model solutions as well as dairy product matrices was studied by Proton Transfer
Reaction - Mass Spectrometry (PTR-MS; Ionicon Analytik GmbH, Innsbruck, Austria)
according to the method described by Lindinger et al. [7].
For headspace sampling, 5 g of flavoured yogurt or 100 mL of model solution or
milk was placed in a 1 L Erlenmeyer flask, closed with a septum and left for 60 min at
ambient temperature for equilibration. The heated nose of the instrument was pierced
through the septum into the headspace of the flask. Additionally a disposable needle
was pierced through the septum in order to allow for replacement of the air
continuously sampled by the instrument at 170 mL/min. Analyses were performed at
120°C (inlet); 80°C (drift tube) and a drift voltage of 600 V.
The following mass fragments and dwell times were selected for monitoring the
release of the selected aroma compounds: diacetyl (m/z 87, 0.2 s), 2-methylbutanoic
acid (m/z 103, 0.2 s), ethyl hexanoate (m/z 117 and 145, 0.2 s), limonene (m/z 81
and 137, 0.2 s). Analyses of unflavoured matrices as well as fragmentation studies of
individual compounds prove no significant overlaps in mass fragments for the
selected aroma compounds. Due to their low volatilities, temporal release of vanillin,
δ-decalactone and methoxyfuraneol could not be monitored under these conditions.
Results
Stability of aroma compounds in dairy matrices. Aroma release is first of all
dependent on the quantities of aroma compounds present in the food matrix.
Therefore, recovery of limonene, vanillin, diacetyl, 2-methylbutanoic acid,
methoxyfuraneol, δ-decalactone, and ethyl hexanoate was determined by Stable
Isotope Dilution Analysis [8] before and after acidification of yogurt matrices with
varying fat contents. All aroma compounds except ethyl hexanoate showed excellent
recovery rates close to 100% [data not shown]. As shown in (Table 1), recovery rates
of ethyl hexanoate from yogurt matrices with max. 0.1% fat were 58% before and
45% after acidification.
Table 1.
Recovery of 8 mg/kg ethyl hexanoate in various dairy matrices 48 h after
sample preparation.
Fat content
[g/100 g]
0.1
4
12
20
Recovery in non-acidified
yogurt matrices [mg/kg]
4.85 ± 0.59 (58.7%)
6.79 ± 0.05 (87.1%)
6.61 ± 0.37 (84.7%)
8.54 ± 0.12 (109.5%)
Recovery in acidified
yogurt matrices [mg/kg]
3.53 ± 0.05 (45.3%)
7.69 ± 0.03 (98.6%)
7.75 ± 0.30 (99.4%)
8.09 ± 0.13 (103.7%)
Low recovery rates were independent of microbial fermentation or acidification by
glucono-δ-lactone. However, low stability of ethyl hexanoate may be compensated by
higher release rates from low-fat dairy matrices as shown in the next section.
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Expression of Multidisciplinary Flavour Science
Aroma release from dairy matrices as measured by PTR-MS. Comparison with
orthonasal detection thresholds. As shown in (Figure 1), fat has the most pronounced
retention effect on the more lipophilic aroma compounds limonene and ethyl
hexanoate. Although not as pronounced as measured by PTR-MS, this is mirrored by
increased orthonasal threshold values. As shown in (Table 2), odour threshold values
above solutions in sunflower oil increase by a factor of 70 in the case of limonene
and a factor of 8 in the case of ethyl hexanoate compared to threshold values above
aqueous solutions.
While limonene release from milk permeate, casein and whey protein dispersions
shows reduced release rates as compared to water, the same differences are not
present in the case of ethyl hexanoate. Interestingly, for both compounds retention
from whole milk is much more pronounced than would be expected by addition of the
retention effects from casein dispersion, whey protein dispersion, milk permeate, and
sunflower oil as a semblance for milk fat.
Ethyl Hexanoate
1000000
Normailsed Signal Intensity [cps]
Normalised Signal Intensity [cps]
Limonene
1000000
100000
10000
1000
100
10
0.0001 0.001
0.01
0.1
1
10
100
1000
100000
10000
1000
100
10
0.001
10000
Concentration in matrix [mg/L]
0.01
1000000
100000
10000
1000
100
10
0.01
0.1
1
10
Concentration in matrix [mg/L]
1000
10000
2-Methyl Butanoic Acid
Normalised Signal Intensity [cps]
N o rm a lis e d S ig n a l In te n s ity [c p s ]
Diacetyl
0.1
1
10
100
Concentration in matrix [mg/L]
100
1000000
100000
10000
1000
100
10
1
0.1
1000
1
10
100
1000
Concentration in matrix [mg/L]
10000
Figure 1. Release of selected aroma compounds from water ( ), sunflower oil (‹),
milk permeate solution („), casein dispersion ( ), whey protein
dispersion (S) and whole milk (X) as measured by Proton Transfer
Reaction - Mass Spectrometry (PTR-MS).
When comparing orthonasal detection thresholds as shown in (Table 2), the
limonene threshold in whole milk (6.5 mg/L) corresponds with the PTR-MS
measurements and is found to be between the thresholds above water (0.2 mg/L)
and sunflower oil (14.7 mg/L). Although release rates of ethyl hexanoate from whole
milk are significantly higher as compared to sunflower oil, the orthonasal detection
thresholds of ethyl hexanoate in whole milk (0.3 mg/L) was found to be even higher
than in sunflower oil (0.04 mg/L). This is indicating possible perceptual interactions
with other volatile compounds from the milk matrix leading to a reduced sensitivity for
the respective aroma compound.
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Expression of Multidisciplinary Flavour Science
Table 2.
Orthonasal detection thresholds and LogP-values [9] of limonene, ethyl
hexanoate, diacetyl and 2-methylbutanoic acid above solutions in water,
whole milk and sunflower oil.
Log P
Limonene
Ethyl hexanoate
2-Methylbutanoic acid
Diacetyl
3.61
2.69
1.18
-0.60
Water
[mg/L]
0.2
0.005
0.5
0.015
Whole milk
[mg/L]
6.5
0.3
10.3
0.050
Sunflower oil
[mg/L]
14.7
0.04
0.1
0.010
2-Methylbutanoic acid does not show any significant differences in release from
water, milk permeate, casein or whey protein dispersions while aroma retention is
more pronounced in whole milk than in sunflower oil, which is mirrored by an
increased orthonasal detection threshold above solutions of 2-methylbutanoic acid in
whole milk.
Due to its ability to form dimers as well as hydrogen bonds in aqueous media,
diacetyl release is enhanced in an aprotic medium like sunflower oil. However, as an
original aroma compound in whole milk, its orthonasal detection threshold is
increased by a factor of 3.3 compared to water.
Acknowledgement
This work is supported by the German Federal Ministry of Economy and Technology
(BMWi/AIF) via Research Association of the German Food Industry (FEI), Project No.
AIF-FV 15158 N.
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