Influence of Free Amino Acids, Oligopeptides, and Polypeptides on the Formation of Pyrazines in Maillard Model Systems

Article abstract of DOI:10.1021/acs.jafc.5b01129

Pyrazines are specific Maillard reaction compounds known to contribute to the unique aroma of many products. Most studies concerning the generation of pyrazines in the Maillard reaction have focused on amino acids, while little information is available on the impact of peptides and proteins. The present study investigated the generation of pyrazines in model systems containing whey protein, hydrolyzed whey protein, amino acids, and glucose. The impact of thermal conditions, ratio of reagents, and water activity (a_w) on pyrazine formation was measured by headspace solid-phase microextraction with gas chromatography/mass spectrometry (HS-SPME-GC/MS. The presence of oligopeptides from hydrolyzed whey protein contributed significantly to an increased amount of pyrazines, while in contrast free amino acids generated during protein hydrolysis contributed to a lesser extent. The generation of pyrazines was enhanced at low a_w (0.33) and high temperatures (>120 °C). This study showed that the role of peptides in the generation of pyrazines in Maillard reaction systems has been dramatically underestimated.

Full text of DOI:10.1021/acs.jafc.5b01129

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Article
Influence of Free Amino Acids, Oligopeptides and Polypeptides
on the formation of Pyrazines in Maillard Model Systems
Gustavo Luis Leonardo Scalone, Tatiana Cucu, Norbert De Kimpe, and Bruno De Meulenaer
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01129 • Publication Date (Web): 14 May 2015
Downloaded from http://pubs.acs.org on May 20, 2015
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Journal of Agricultural and Food Chemistry
Influence of Free Amino Acids, Oligopeptides and Polypeptides on
the formation of Pyrazines in Maillard Model Systems
1,2
1
2
Gustavo Luis Leonardo Scalone
Meulenaer 1 *
Tatiana Cucu , Norbert De Kimpe and Bruno De
1 Department of Food Safety and Food Quality, nutriFOODchem group, member of
Food2Know.
Faculty of Bioscience Engineering, Ghent University, Coupure links 653, Bꢀ9000 Ghent,
Belgium
2 Department of Sustainable Organic Chemistry and Technology.
Faculty of Bioscience Engineering, Ghent University, Coupure links 653, Bꢀ9000 Ghent,
Belgium
* To whom correspondence should be addressed. Fax 32 9 2646215.
Eꢀmail: Bruno.DeMeulenaer@UGent.be
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ABSTRACT
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Pyrazines are specific Maillard reaction compounds known to contribute to the
unique aroma of many products. Most studies concerning the generation of pyrazines in
the Maillard reaction have focused on amino acids, while little information is available on
the impact of peptides and proteins. The present study investigated the generation of
pyrazines in model systems containing whey protein, hydrolyzed whey protein, amino
acids and glucose. The impact of thermal conditions, the ratio of the reagents and the
aw was measured on the pyrazine formation by HSꢀSPMEꢀGCꢀMS. The presence of
oligopeptides from hydrolyzed whey protein contributed significantly to an increased
amount of pyrazines, while in contrast free amino acids generated during protein
hydrolysis contributed to a lesser extent. The generation of pyrazines was enhanced at
low aw (0.33) and high temperatures (>120 ⁰C). This study showed that the role of
peptides in the generation of pyrazines in Maillard reaction systems has been
dramatically underestimated.
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KEYWORDS: Peptides; Maillard; fractions; pyrazines; flavor; model reactions; HSꢀ
SPMEꢀGCꢀMS.
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INTRODUCTION
The Maillard reaction comprises a set of complex chemical reactions, which are
initiated when a free amino group of an amino acid, a peptide, a protein or an amine
reacts with the carbonyl group of a reducing sugar. This nonꢀenzymatic browning
reaction gives rise to modifications in color, aroma, taste and nutritional value of
thermally treated foods and is influenced by many factors, such as temperature, time,
pH, type of buffer, water activity and reactant concentration.1 The reaction between free
amino acids and carbonyl compounds has been extensively studied1, whereas the
Maillard reaction between peptides and proteins with carbonyl compounds has been
less investigated.2ꢀ4 This in fact is quite surprising as the amount of free amino acids in
food is generally very low as compared to the amounts of peptides and especially
proteins.5
A limited number of studies have investigated the generation of volatiles in
protein – carbohydrate models. Nevertheless, these studies were performed under
unrealistically severe thermal conditions6ꢀ8.
Oligopeptides have been recognized as important flavor enhancers and
precursors of the Maillard reaction.2, 9ꢀ11 Moreover, peptides can be a flavor enhancer as
such (umami taste). In addition, the formation of flavor compounds due to the reaction
between peptides and carbohydrates has mainly been studied in model systems
containing glutathione12ꢀ15 and glycineꢀderived peptides such as diglycine, triglycine,
and tetraglycine.4 In these studies, pyrazines were indeed the most abundant volatiles.
Substituted and unsubstituted pyrazines are specific Maillard reaction products
which are known to contribute significantly to the unique roasted, nutty, meaty, earthy
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and popcornꢀlike aroma of many heated food products.16ꢀ17 Oh et al.4 found that the
amount of pyrazines formed from diglycine or tetraglycine was considerably lower
compared to that formed from either glycine or triglycine. Van Lancker et al.18ꢀ19 studied
the formation of pyrazines from di– and tripeptides with different amino acid sequences.
The results of these studies showed significant formation of pyrazines from dipeptides
compared to free amino acids and less pyrazine formation in tripeptides over dipeptides
and free amino acids.
The general mechanism of pyrazines formation via the Maillard reaction implies
in the first step the reaction of a dicarbonyl compound 1 produced by sugar degradation
and an amino acid 2, which leads to the formation of Strecker aldehydes 6 and αꢀ
aminoketones 7. A crucial step in this degradation reaction is the decarboxylation of the
intermediate 4 via a cyclic transition state (Figure 1 part I). The second step in the
pyrazine formation involves two possible pathways which start with the condensation
reaction of two αꢀaminocarbonyl compounds 7 and 7’ with the formation of a
dihydropyrazine 9. This dihydropyrazine can react in two distinct ways. The
dihydropyrazine can oxidize spontaneously with air to the corresponding pyrazine 11
(pathway A). Alternatively, after deprotonation the dihydropyrazine anion 10 can react
with a carbonyl compound 6 in an Aldolꢀtype reaction (pathway B). Whenever this
reacting carbonyl compound is a Strecker aldehyde 6, amino acid specific pyrazines 15
are formed (Figure 1 part II).
Peptides cannot follow the typical Strecker degradation due to the absence of a
free carboxyl group at the αꢀcarbon with respect to the free amino group, making the
decarboxylation impossible. Van Lancker et al.18 has suggested a mechanism of
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pyrazine formation in model systems containing dipeptides and dicarbonyl compounds
as shown in Figure 2. In accordance to the reaction with free amino acids, the reaction
of the αꢀdicarbonyl compound 1 with the dipeptide starts with the formation of an αꢀ
ketoimine 17. Afterwards, deprotonation occurs at the αꢀposition of the amide moiety
resulting in a 1,5ꢀHꢀshift leading to enolization of the carbonyl group of intermediate 17.
Hydrolysis of the imino moiety of 4ꢀhydroxyꢀ2ꢀazadiene 18 produces the αꢀaminoketone
7 and instead of forming the Strecker aldehyde, a complex αꢀketoamide 19 is formed.2
Although it was shown before that peptides may generate pyrazines in the
Maillard reaction, their potential contribution to the overall pyrazine formation in food
has not been compared to the contribution of amino acids. Therefore, this study aimed
to evaluate the impact of trypsinogenic whey hydrolysis products on the formation of
pyrazines in a Maillard reaction model, thereby trying to discriminate the role of free
amino acids and peptides generated respectively. In addition the impact of other factors
such as heating conditions, the ratio of reagents and the water activity on the formation
of pyrazines were investigated.
MATERIALS AND METHODS
Chemicals.
DL–norvaline (99%), L–cysteine (99.5%), L–4ꢀhydroxyproline (99%), DL–valine
(99%), DL–alanine (99%), L–tryptophan (99.5%), L–citruline (99%), sarcosine (99%),
DL–histidine (99%), L–isoleucine (99.5%), DL–leucine (99%) and glutamine (99,5%)
were purchased from Fluka (Sigma–Aldrich, Bornem, Belgium). DL–lysine
monohydrochloride (98%), DL–methionine (99%), glycine (99.5%), L–arginine
hydrochloride (99%), L–phenylalanine (99%), L–glutamic acid (99%), Dꢀ(+)ꢀglucose
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(99.5%), trypsin from porcine pancreas, trypsinꢀchymotrypsin inhibitor from glycine max
(soybean), insulin from bovine pancreas, insulin chain B oxidized from bovine pancreas,
cytochrome C from equine heart, vitamine B12, ValꢀTyr, pyrazine (99%), 2ꢀ
methylpyrazine (99%) and sodium bromide (99%) were purchased from Sigma–Aldrich
(Bornem). L–lysine (97%), arginine (98%), proline (99%), asparagine monohydrate
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(99%),
2,5ꢀdimethylpyrazine
(99%),
2ꢀethylpyrazine
(98%),
2ꢀethylꢀ3,5(6)ꢀ
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dimethylpyrazine (99.5%) and trichloroacetic acid (99%) were purchased from Acros
organics, Thermo – Fisher Scientific (Erembodegem, Belgium). Aspartic acid (99%),
DL–threonine (99%), L–tyrosine (99%) were purchased from Merck (Darmstadt,
Germany). DL–serine, 2,3ꢀdimethylpyrazine (99%) and 2,6ꢀdimethylpyrazine (96%)
were purchased from Janssen Chimica (Geel, Belgium). Magnesium chloride
hexahydrate (99%), calcium carbonate (99%), potassium iodide (99.5%) and sodium
chloride (98%) were purchased from Chemlab Analytical (Zedelgem, Belgium). Whey
protein isolate LACPRODAN DIꢀ9224 was donated from Arla foods (Aarhus, Denmark).
Hydrolysis of whey protein.
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Whey protein isolate (84% protein) was dissolved in potassium phosphate buffer
0.1M pH 7.8 at a concentration of 5 mg/ml, heated at 95 ⁰C for 5 min and then cooled
down at room temperature. Trypsin was added at a ratio of 1:20 (w:w), and then
incubated at 37 ⁰C in a water bath during 20 h. The obtained solution was heated at 95
⁰C for 5 min to inactivate the enzyme and then frozen for posterior use. The free amino
acid content of the hydrolyzed protein was analyzed and reported (Table 1). The nonꢀ
hydrolyzed whey protein isolate did not contain free amino acids in detectable amounts.
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The protein hydrolyzate was furthermore characterized with size exclusion
chromatography as shown in Figure 1 and outlined below.
Size exclusion chromatography.
Samples of whey protein and hydrolyzed whey protein were analyzed with an
ÄKTA explorer LC coupled to a UV detector (GE Healthcare, Zaventem, Belgium)
equipped with a Superdex Peptide 10/300 GL column (GE Healthcare, Zaventem).
Working conditions were: mobile phase ꢀ ammonium hydrogen carbonate buffer 0.15M
pH 7.8; flow ꢀ 0.5 ml/min; UV detection ꢀ 214 nm.
Free amino acid analysis.
Samples of whey protein and hydrolyzed whey protein were treated with a
solution of 15% of trichloroacetic acid, further centrifugated, filtered and analyzed using
HPLC – analysis with fluorescence detection.20
Selection of the reaction conditions.
Thermal conditions:
Sterilization conditions: Samples were mixed in a volumetric flask and the
volume was adjusted to 5 ml with 0.1M potassium phosphate buffer pH 7.8, then
transferred to a SPME vial (Gerstel, Mülheim, Germany) and closed with a pressure cap
(silicon/PTFE 55⁰ shore A 1.5mm magnetic. Gerstel, Mülheim). The vial was heated in a
stirring oil bath at 130 ⁰C to obtain an inside temperature of 120 ⁰C, for 2 h, after which
the vials were immediately cooled down in an ice bath.
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Roasting conditions: Samples were mixed in a volumetric flask and the volume
was adjusted to 5 ml with 0.1M potassium phosphate buffer pH 7.8, then transferred to
a SPME vial containing 1.5 g of sand. Subsequently the samples were frozen and
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freeze dried in a VaCo5 freeze dryer (Zirbus Technology, Bad Grund, Germany) to
ensure sample homogeneity. Finally, the samples were closed with pressure caps and
heated in an oven (Memmert, Fisher scientific, Erembodegem) at 180 ⁰C for 90 min.
Preliminary experiments showed that a considerable improvement of the reproducibility
could be obtained dissolving, freezing and freezedrying instead of dry mixing the
various ingredients.
Reactant type.
Two groups of samples were analyzed to evaluate the yields of pyrazines
formation under roasting and sterilization conditions.
Samples containing hydrolyzed whey protein and glucose were prepared at 8 different
ratios (protein:glucose) (w:w) ranging from 1:1.33 to 1:0.08 as indicated in Table 2.
Samples containing lysine and glucose were prepared at 8 different ratios (lysine:
glucose) (w:w) ranging from 1:2.53 to 1:0.15 as indicated in Table 3. The samples were
analyzed using HS SPME – GC MS.
Elucidation of the role of protein hydrolysis products on the formation of
pyrazines:
Four groups of model systems were prepared as follows:
(1) 15 mg of hydrolyzed whey protein and 15 mg of glucose, (2) 15 mg native whey
protein and 15 mg glucose, (3) 15 mg native whey protein, 15 mg glucose and the
addition of a mixture of free amino acids corresponding to the free amino acid content of
the tryptic digestion of whey protein as reported in Table 1, (4) a mixture of free amino
acids corresponding to the free amino acid content of the tryptic digestion of whey
protein as reported in Table 1 and 15 mg glucose.
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The reaction mixtures were dissolved in 5ml 0.1M potassium phosphate buffer
pH 7.8 and then transferred to SPME vials containing 1.5 g of sand, freeze dried, closed
with pressure caps and thermally treated under previously described roasting
conditions. The samples were analyzed using HS SPME – GC MS.
Impact of water activity on pyrazine generation from hydrolyzed whey protein.
Samples containing 15 mg of hydrolyzed whey protein and 7.5 mg of glucose
were dissolved in 5ml 0.1M potassium phosphate buffer pH 7.8 and transferred to
SPME vials containing 1.5 g of sand. Subsequently the vials were freezedried and
immediately capped. Afterwards, the aw value of the samples was determined using a
4TEV dew point water activity meter (Aqualab, Decagon Devices, Pullman, USA)
obtaining an initial value of 0.16.
Subsequently freezedried samples were transferred to different hermetic plastic
recipients containing saturated solutions of: magnesium chloride (32% RH), potassium
carbonate (43% RH), sodium bromide (56% RH), potassium chloride (67% RH), sodium
chloride (75% RH). All the samples were incubated in triplicates during 7 days at 30 ⁰C
inside an incubator (Memmert, Fisher scientific, Erembodegem) in order to reach the
desired water activity in each sample. After reaching equilibrium, all samples were
thermally treated under previously described roasting conditions and analyzed by
means of HS SPME – GC MS.
HS SPME – GCMS analysis.
The volatiles produced during the different experimental conditions were
extracted by means of headspace solid – phase microextraction (HS – SPME) for 30
minutes at 35 ⁰C with a DVD/Car/PDMS fiber (Supelco, Bornem) with a multipurpose
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sampler (MPS – 2) (Gerstel). GC – MS analyses of the SPME extract were performed
with an Agilent 6890 GC Plus apparatus coupled to a quadrupole mass spectrometer
5973 MSD (Agilent Technologies, Diegem, Belgium) and equipped with an DBꢀ5
capillary column (30 m length x 0.25 m i.d; 0.25 ꢁm film thickness) (Agilent
Technologies, Diegem). Working conditions were: transfer line to MSD 250 ⁰C, carrier
gas (He) 1.0 ml/min; ionization: EI 70eV; acquisition parameters: scanned m/z: 40ꢀ200
(2ꢀ10 min), 40ꢀ300 (10ꢀ20 min), 40ꢀ400 (>20 min); oven temperature started at 35 ⁰C,
held 5 min, programmed from 35 – 80 ⁰C at 2 ⁰C/min, held 2 min. Pyrazines were
identified by comparison of the mass spectrum with mass spectral libraries (Nist 98,
Wiley 6th and HPCH2205) and by comparison of the calculated linear retention indices
with literature values.
The generation of pyrazines was followed in a semiquantitative way by
considering the absolute peak area of each individual pyrazine18, 21ꢀ22. Although this
approach does not allow absolute quantitation of each individual pyrazine, it is generally
accepted as suitable to evaluate the pyrazine formation in a reliable way.
Statistical analysis.
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All analyses were performed with SPSS Statistics version 22 at a significance
level of 95% (p = 0.050). Data were normally distributed (KolmogorovꢀSmirnov test: p <
0.050 for all standardized residuals). Therefore, one way ANOVA was selected for
statistical analysis. The Games – Howell correction was applied to control the familyꢀ
wise error rate at 5% for all multiple pairwise comparisons.
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RESULTS AND DISCUSSION
Selection of the reaction conditions and ratio of the model systems.
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In a first series of experiments the impact of the reactant ratio (glucose and
hydrolyed whey protein) on the formation of pyrazines was evaluated for both of thermal
treatments applied.
The results shown in Table 2 indicate that during the roasting conditions (180
°C/90 min) significant quantities or the following pyrazines were produced in model
systems containing hydrolyzed whey protein and glucose: 2ꢀmethylpyrazine, 2,5(6)ꢀ
dimethylpyrazine,
2,3ꢀdimethylpyrazine,
2ꢀethylꢀ3ꢀmethylpyrazine,
3ꢀethylꢀ2,5ꢀ
dimethylpyrazine, 2ꢀethylꢀ3,5ꢀdimethylpyrazine and 5ꢀethylꢀ2,3ꢀdimethypyrazine.
The same reactants under sterilization conditions (120 °C/120 min) yielded
mainly 2ꢀmethylpyrazine and 2,5(6)ꢀdimethylpyrazine. However, the peak areas of the
produced pyrazines were considerably lower compared to those observed during
roasting conditions. These observations can be due to the fact that indeed a lower
amount of pyrazines was produced, which can be explained by the different reaction
conditions: temperature and presence of water in the systems. It should be noted as
well, however, that by addition of 2 ꢁg of 2ꢀethylꢀ3ꢀmethylpyrazine to similar amounts of
nonꢀthermally treated samples, a much higher peak area was observed for the dry
(107.89 x 106) vs the wet sample (6.68 x 106). This indicated that the partitioning of the
pyrazines between the samples and its headspace is totally different for a dry or
aqueous sample.
The ratio between glucose and hydrolyzed whey protein played a crucial role in
the formation of pyrazines. The generation of pyrazines under dry roasting conditions
was low in model systems where glucose was present in low amounts (ratio 1:0.08 and
1:0.16). This effect can likely be explained since the amount of dicarbonyl compounds
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generated from glucose degradation was low, which had an impact on the formation of
pyrazines and resulted in lower peak areas. Higher peak areas were detected for ratios
up to 1:0.5 and 1:0.67. At higher protein/glucose ratios, a decrease in the formation of
pyrazines was again observed. This may be due to the excessive formation of different
carboxylic acids such as acetic acid from glucose23 which might decrease the pH of the
model systems. It is generally known that the pH has an impact on the generation of
pyrazines1 hindering their formation at low pH due to the protonation of the amino
groups of the amino acids and peptides. Also under sterilization conditions the peak
area of the formed pyrazines was affected by the protein/glucose ratio although to a
lesser extent.
It is broadly known that in Maillard reaction models free amino acids have a high
reactivity. In order to compare the reactivity of the glucose/hydrolyzed whey protein with
a glucose/amino acid mixture, lysine was selected. Pyrazine formation at different
lysine/glucose ratios was monitored for both heating conditions see (Table 3).
Considerably higher amounts of pyrazines were detected in the lysine containing
reaction systems under both heating conditions when compared to hydrolyzed whey. In
addition, the detected pyrazines were more diverse, particulary for the roasted
samples. Under roasting conditions, the detected amounts of 2ꢀmethylpyrazine and
2,5(6)ꢀdimethylpyrazine were considerably less influenced by the lysine/glucose ratio.
However the lysine/glucose ratio did have a considerable effect on the amount of
pyrazines detected. At the lower lysine/glucose ratios, formation of pyrazines in roasting
conditions decreased considerably, while this tendency was not observed under
sterilization conditions. These experiments showed that depending upon the amino
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compounds participating in the reactions such as free amino acids or peptides involved
in the pyrazine formation, the impact of the amino compounds/glucose ratio on the
pyrazine formation will be different.
As it was noticed that considerable more pyrazines were detected under roasting
conditions (180°C/90 min) either for glucose/hydrolyzed whey protein and
glucose/lysine models, this heat treatment was further on used.
Elucidation of the role of protein hydrolysis products on the formation of
pyrazines:
From the previous experiments it was obvious that the hydrolyzed whey proteins
were able to generate pyrazines in Maillard reaction systems. However, it was not clear
if the compounds responsible for the pyrazine generation are the free amino acids
produced during hydrolysis (Table 1), or the peptidic fraction, or both. Therefore the
formation of pyrazines by the hydrolyzed whey protein was compared with the pyrazine
generation in a mixture of nonꢀhydrolyzed proteins to which the free amino acids formed
upon trypsinogenic hydrolysis were added according to the amounts reported in (Table
1). Nonꢀhydrolyzed whey protein was included as a control. In addition, glucose
together with the free amino acids generated upon trypsinogenic hydrolysis of whey was
considered as well. Experiments were conducted under dry heating conditions
considering two protein/glucose ratios.
In model systems containing free amino acids as described in Table 1 and
glucose, no pyrazines were detected (data not shown). Most likely the amount of
reagents was not sufficient to generate sufficient Maillard products. Model systems
containing hydrolyzed whey protein and glucose led to the formation of the highest
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amounts of pyrazines (Table 4). In model systems containing native whey protein and
glucose, the generation of pyrazines was significantly lower than in samples of
hydrolyzed whey. The native whey protein isolate did not contain any free amino acids,
suggesting that the limited generation of pyrazines was due to thermal degradation of
the protein in the selected reaction conditions, releasing amino compounds which can
react with glucose and form pyrazines. Model systems, containing native whey with the
addition of the mixture of free amino acids (Table 1) and glucose, produced comparable
and statistically similar amounts of pyrazines as the model systems containing the
native whey protein isolate and glucose.
These results allowed to conclude that the amount of free amino acids present in
the whey hydrolyzate was not sufficient to produce pyrazines in measurable amounts
using the heating conditions earlier described. Compared to the results from the
experiments with the hydrolyzed whey, it is obvious that the peptides generated upon
hydrolysis played an important role in the generation of pyrazines.
Regarding the pathways that involve the peptidic fraction into the generation of αꢀ
aminoketones and their posterior condensation into pyrazines, Oh et al.4 studied model
systems with glycine containing diꢀ, triꢀ and tetrapeptides. The authors suggested that
tetraglycine could be degraded to diglycine and further degraded into glycine to produce
pyrazines. Simultaneously, triglycine was suggested to be degraded to diglycine and
glycine. However, these results are limited to glycineꢀcontaining peptides. Yan et al.24
studied the peptide bond cleavages during the Maillard reaction and their results show
that some peptide bonds can be more resistant or labile depending on the amino acid
sequence in the peptide chain. Therefore, the use of diꢀ, triꢀ and tetraglycine can only
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represent some peptides. The mechanism previously depicted in Figure 2 implies the
formation of pyrazines in model systems containing dipeptides and dicarbonyl
compounds. The alternative mechanism was suggested since dipeptides cannot follow
the typical Strecker degradation due the absence of a free carboxyl group to form αꢀ
aminoketones.
It is obvious that a similar mechanism can be responsible for the formation of
pyrazine from oligopeptides as well.
Impact of water activity on pyrazine generation from hydrolyzed whey protein.
Lu et al.25 studied the effects of the water content on volatile generation and
peptide degradation in the Maillard reaction using glycerol to limit the water content of
the samples. However, SmarritoꢀMenozzi.26 reported that glycerol is an underestimated
flavor precursor in the Maillard reaction. Therefore, the water content of the samples
was controlled by equilibrating them in recipients filled with saturated salt solutions,
obtaining atmospheres with fixed relative humidities as described by Greenspan et al.27.
Thus, after equilibration, samples with a particular water activity were obtained.
The results presented in Table 5 show that the model systems incubated with a
water activity of 0.33 are the ones which produced the highest amounts and variety of
pyrazines. This may be explained by the potential effect of water activity on the peptide
degradation. Oliyai et al.28 studied the chemical stability of a hexapeptide under several
factors such as pH, temperature and moisture content finding that the moisture content
degraded significantly the hexapeptide, leading to an increased reactivity. In addition
water activity has been pointed out to have an impact on reaction rates29 since water in
food often acts as a plasticizer, leading to enhanced mobility and chemical reactivity as
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315
316
317
318
319
320
321
322
323
324
325
326
327
328
well.30 However, increasing the water activity to values higher than 0.33 lead
progressively to a lower generation of pyrazines, which can be explained by the fact that
water is a product of several condensation steps in the Maillard reaction.31.
This is the first paper systematically evaluating the impact of peptides generated
by trypsinogenic hydrolysis on the formation of pyrazines via the Maillard reaction. The
results suggest that the contribution of peptides to the generation of pyrazines is
considerably high, while the role of free amino acids is only minor and more than likely
less important.
The role of peptides in food on the generation of pyrazines has probably been
underestimated and requires more attention.
Aknowledgements
The authors thank the Erasmus Mundus program for the financial support of this
investigation. Siele Ceuppens, Monica Ovando Chaves, Laurens De Coen and Luke
Sebastian Scalone are acknowledged for their valuable advice.
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REFERENCES
(1) Nursten, H. E., The Maillard reaction chemistry, biochemistry, and implications. Royal Society of
Chemistry: 2005.
(2) Van Lancker, F.; Adams, A.; De Kimpe, N., Chemical modifications of peptides and their impact on
food properties. Chemical Reviews 2011, 111, 7876-7903.
(3) Ho, C. T.; Oh, Y. C.; Zhang, Y. G.; Shu, C. K., Peptides as flavor precursors in model Maillard reactions
Acs Symposium Series 1992, 490, 193-202.
(4) Oh, Y. C.; Shu, C. K.; Ho, C. T., Some volatile compounds formed from thermal interaction of glucose
with glycine, diglycine, triglycine, and tetraglycine. J. Agric. Food Chem. 1991, 39, 1553-1554.
(5) Arnoldi, A., Thermal processing and nutritional quality. The Nutrition Handbook for Food Processing
2002, 265-286.
(6) Jing, H.; Kitts, D. D., Chemical and biochemical properties of casein-sugar Maillard reaction products.
Food Chem. Toxicol. 2002, 40, 1007-1015.
(7) Ferretti, A.; Flanagan, V. P.; Ruth, J. M., Nonenzymatic browning in a lactose-casein model system. J.
Agric. Food Chem. 1970, 18, 13-18.
(8) Ferretti, A.; Flanagan, V. P., Lactose-casein (Maillard) browning system - volatile components. J.
Agric. Food Chem. 1971, 19, 245-249.
(9) Schlichtherle-Cerny, H.; Amado, R., Analysis of taste-active compounds in an enzymatic hydrolysate
of deamidated wheat gluten. J. Agric. Food Chem. 2002, 50, 1515-1522.
(10) Hashiba, H., Glucose-diglycine condensation product participating in oxygen-dependent browning.
J. Agric. Food Chem. 1975, 23, 539-542.
(11) Jiang, Z. M.; Brodkorb, A., Structure and antioxidant activity of Maillard reaction products from
alpha-lactalbumin and beta-lactoglobulin with ribose in an aqueous model system. Food Chem. 2012,
133, 960-968.
(12) Zhang, Y. G.; Chien, M. J.; Ho, C. T., Comparison of the volatile compounds obtained from thermal-
degradation of cysteine and glutathione in water. J. Agric. Food Chem. 1988, 36, 992-996.
(13) Zhang, Y. G.; Ho, C. T., Volatile compounds formed from thermal interaction of 2,4-decadienal with
cysteine and glutathione. J. Agric. Food Chem. 1989, 37, 1016-1020.
(14) Zhang, Y. G.; Ho, C. T., Comparison of the volatile compounds formed from the thermal-reaction of
glucose with cysteine and glutathione. J. Agric. Food Chem. 1991, 39, 760-763.
(15) Zhang, Y. G.; Ho, C. T., Formation of meatlike aroma compounds from thermal-reaction of inosine
5'-monophosphate with cysteine and glutathione. J. Agric. Food Chem. 1991, 39, 1145-1148.
(16) Maga, J. A., Pyrazine update Food Reviews International 1992, 8, 479-558.
(17) Adams, A.; De Kimpe, N., Chemistry of 2-acetyl-1-pyrroline, 6-acetyl-1,2,3,4-tetrahydropyridine, 2-
acetyl-2-thiazoline, and 5-acetyl-2,3-dihydro-4H-thiazine: Extraordinary Maillard flavor compounds.
Chemical Reviews 2006, 106, 2299-2319.
(18) Van Lancker, F.; Adams, A.; De Kimpe, N., Formation of pyrazines in Maillard model systems of
lysine-containing dipeptides. J. Agric. Food Chem. 2010, 58, 2470-2478.
(19) Van Lancker, F.; Adams, A.; De Kimpe, N., Impact of the N-terminal amino acid on the formation of
pyrazines from peptides in maillard model systems. J. Agric. Food Chem. 2012, 60, 4697-4708.
(20) Mestdagh, F.; Kerkaert, B.; Cucu, T.; De Meulenaer, B., Interaction between whey proteins and
lipids during light-induced oxidation. Food Chem. 2011, 126, 1190-1197.
(21) Adams, A.; Borrelli, R. C.; Fogliano, V.; De Kimpe, N., Thermal degradation studies of food
melanoidins. J. Agric. Food Chem. 2005, 53, 4136-4142.
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(22) Yu, A. N.; Zhang, A. D., The effect of pH on the formation of aroma compounds produced by
heating a model system containing L-ascorbic acid with L-threonine/L-serine. Food Chem. 2010, 119,
214-219.
(23) Davidek, T.; Robert, F.; Devaud, S.; Vera, F. A.; Blank, I., Sugar fragmentation in the Maillard
reaction cascade: Formation of short-chain carboxylic acids by a new oxidative alpha-dicarbonyl
cleavage pathway. J. Agric. Food Chem. 2006, 54, 6677-6684.
(24) Yang, C.; Wang, R.; Song, H. L., The mechanism of peptide bonds cleavage and volatile compounds
generated from pentapeptide to heptapeptide via Maillard reaction. Food Chem. 2012, 133, 373-382.
(25) Lu, C. Y.; Hao, Z. G.; Payne, R.; Ho, C. T., Effects of water content on volatile generation and peptide
degradation in the Maillard reaction of glycine, diglycine, and triglycine. J. Agric. Food Chem. 2005, 53,
6443-6447.
(26) Smarrito-Menozzi, C.; Matthey-Doret, W.; Devaud-Goumoens, S.; Viton, F., Glycerol, an
underestimated flavor precursor in the Maillard reaction. J. Agric. Food Chem. 2013, 61, 10225-10230.
(27) Greenspan, L., Humidity fixed-points of binary saturated aqueous-solutions. J Res Nbs a Phys Ch
1977, 81, 89-96.
(28) Oliyai, C.; Borchardt, R. T., Chemical pathways of peptide degradation. VI. Effect of the primary
sequence on the pathways of degradation of aspartyl residues in model hexapeptides. Pharm. Res. 1994,
11, 751-8.
(29) Labuza, T. P., The effect of water activity on reaction-kinetics of food deterioration. Food Technol-
Chicago 1980, 34, 36-40.
(30) Roos, Y. H., Glass transition-related physicochemical changes in foods. Food Technol-Chicago 1995,
49, 97-102.
(31) Eichner, K.; Karel, M., Influence of water-content and water activity on sugar amino browning
reaction in model systems under various conditions. J. Agric. Food Chem. 1972, 20, 218-223.
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TABLES AND ARTWORK
O
Z
HO R2
Z
R2
R1
N
O
Z
R1
OH
R1
OH
+
+ H2O
R2
H2N
N
H
O
O
O
O
O
O
H
1
2
3
4
I
R2
R1
N
Z
O
H2O
O
6
NH2
+
+ CO2
R1
H
Z
R2
OH
5
7
:B
R1
R1
H
H2N
H
N
R1
N
R4
R4
O
R4
+
HO
R2
R2
O
OH
R3
R3
7'
N
H
R2
N
R3
NH2
7
8
9
ꢀH+
OH
R1
N
N
R1
R2
N
R1
R2
N
N
R4
R3
H+
O
6
Pathway B
R4 = H
Z
II
H
+
R2
R3
H
Z
N
R3
B:
13
ꢀH2O
12
10
Pathway A
R1
R2
N
R1
R2
N
R1
R2
N
R4
R3
Z
Z
R3
N
R3
N
N
H
B:
14
15
11
Figure 1. Strecker degradation (part I) and pyrazine formation (part II)
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O
Z2
Z1
O
O
H
R2
R1
N
O
NRH
R2
N
+
R1
N
Z1
NH2
NRH
H
O
Z2
H
O
O
1
16
17
O
Z2
O
O
O
R3
R4
N
R1
R2
H
H2O
R2
R1
N
NRH
R2
NH2
N
R1
NRH
+
N
Z1
H
N
Z1
OH
O
O
Z2
11
7
19
18
Figure 2. Hypothetical formation mechanism of αꢀaminoketones from the reaction of
peptides with an αꢀdicarbonyl compound. Adapted from Van Lancker et al. 18
Figure 3. Gel permeation chromatogram of hydrolyzed whey protein after tryptic
hydrolysis. Nonꢀhydrolyzed whey elutes as one unique peak at 16.96 minutes. Peak
position for weight markers are shown on top.
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Table 1. Free amino acid composition of native whey protein isolate under tryptic
hydrolysis
mg/100 mg
Amino acid
hydrolyzed whey
Lysine
Leucine
0.9916
0.1340
0.0960
0.0728
0.0546
0.0421
0.0389
0.0359
0.0297
0.0218
0.0104
Valine
Isoleucine
Arginine
Tyrosine
Threonine
Tryptophan
Phenylalanine
Methionine
Alanine
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Table 2. Pyrazines (GCꢀMS peak Area x 10 ⁶) detected in model reactions of dry glucose and hydrolyzed whey mixtures under roasting conditions (180 ⁰C/90 min.), and aqueous
glucose and hydrolyzed whey solutions under sterilization conditions (120 ⁰C/120 min.).
Hydrolyzed
whey 15 mg
Glucose
1.25 mg
4.15 ± 0.12 a
Hydrolyzed
whey 15 mg
Glucose
2.5 mg
9.46 ± 0.23 b
Hydrolyzed
whey 15 mg
Glucose
Hydrolyzed
whey 15 mg
Glucose
Hydrolyzed
whey 15 mg
Glucose
Hydrolyzed
whey 15 mg
Glucose
Hydrolyzed
whey 15 mg
Glucose
Hydrolyzed
Experimental
conditions
whey 15 mg
Glucose
20 mg
Compund
5 mg
7.5 mg
10 mg
15 mg
17.5 mg
Roasting
Sterilization
Roasting
16.21 ± 0.47 c
0.94 ± 0.04 a
37.81 ± 1.23 bd
6.75 ± 0.66 ac
1.13 ± 0.03 a
N/D
23.10 ± 0.36 d
1.75 ± 0.01 b
42.39 ± 3.04 d
9.64 ± 0.41 be
1.80 ± 0.07 b
N/D
22.54 ± 1.95 cd
3.46 ± 0.24 c
35.25 ± 2.01 b
7.49 ± 0.19 c
2.10 ± 0.15 c
N/D
20.97 ± 1.20 cd
5.45 ± 0.08 de
25.41 ± 1.83 a
11.16 ± 0.04 d
2.16 ± 0.01 c
N/D
18.86 ± 1.47 cd
5.35 ± 0.29 de
22.75 ± 2.22 a
8.32 ± 0.20 e
2.54 ± 0.14 d
N/D
20.87 ± 0.65 d
6.32 ± 0.39 e
20.22 ± 1.26 a
9.66 ± 0.14 b
2.11 ± 0.03 c
N/D
2ꢀMethylpyrazine α
2,5(6)ꢀDimethylpyrazine α
2,3ꢀDimethylpyrazine α
N/D
N/D
24.45 ± 1.35 a
N/D
35.22 ± 1.88 b
5.65 ± 0.52 a
N/D
Sterilization
Roasting
N/D
Sterilization
Roasting
N/D
N/D
N/D
N/D
3.45 ± 0.04 a
4.24 ± 0.19 b
4.29 ± 0.16 b
4.73 ± 0.06 c
4.96 ± 0.22 c
4.92 ± 0.12 c
2ꢀEthylpyrazine α
Sterilization
Roasting
N/D
3.79 ± 0.20 a
N/D
8.22 ± 0.44 bd
N/D
N/D
N/D
N/D
N/D
N/D
10.84 ± 0.21 c
N/D
6.78 ± 0.33 b
11.00 ± 0.51 c
N/D
5.14 ± 0.13 c
9.38 ± 0.48 d
N/D
3.59 ± 0.33 a
8.80 ± 0.05 bd
N/D
3.37 ± 0.18 a
9.32 ± 0.50 d
N/D
3.43 ± 0.21 a
7.92 ± 0.37 b
N/D
2.42 ± 0.14 da
2ꢀEthylꢀ5ꢀmethylpyrazine α
3ꢀEthylꢀ2,5ꢀdimethylpyrazine α
2ꢀEthylꢀ3,5ꢀdimethylpyrazine β
5ꢀEthylꢀ2,3ꢀdimethylpyrazine β
Sterilization
Roasting
N/D
N/D
3.77 ± 0.18 a
N/D
6.70 ± 0.39 b
N/D
Sterilization
Roasting
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
2.07 ± 0.04 a
N/D
1.22 ± 0.05 a
2.28 ± 0.14 a
N/D
1.06 ± 0.06 a
2.04 ± 0.16 a
N/D
1.03 ± 0.08 a
1.54 ± 0.08 ba
N/D
1.16 ± 0.08 a
1.26 ± 0.56 b
N/D
1.23 ± 0.11 a
1.27 ± 0.09 b
N/D
1.12 ± 0.03 a
Sterilization
Roasting
N/D
N/D
N/D
N/D
Sterilization
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D (not detected). α Identification confirmed by GC retention index and mass spectra of authentic compounds. β Tentatively identified by matching mass spectra library.
Data points represent mean values of 3 independent determinations. Values in the same line followed by different superscript letters are significantly different (p < 0.05).
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Table 3. Pyrazines (GCꢀMS peak Area x 10 ⁶) detected in model reactions of dry glucose and lysine mixtures under roasting conditions (180 ⁰C/90 min.), and aqueous glucose and
lysine solutions under sterilization conditions (120 ⁰C/120 min.).
Lysine
14.61 mg
Glucose
2.5 mg
Lysine
14.61 mg
Glucose
4.5 mg
Lysine
14.61 mg
Glucose
9 mg
Lysine
14.61 mg
Glucose
13.5 mg
42.74 ± 0.01 c
15.11 ± 0.39 bc
450.48 ± 1.28 c
Lysine
14.61 mg
Glucose
18 mg
Lysine
14.61 mg
Glucose
22.5 mg
Lysine
14.61 mg
Glucose
27 mg
Lysine
14.61 mg
Glucose
31.5 mg
Lysine
14.61 mg
Glucose
37 mg
Experimental
conditions
Compund
Roasting
Sterilization
Roasting
49.05 ± 0.11 a
36.80 ± 0.15 b
6.75 ± 0.47 a
35.96 ± 0.83 b
13.28 ± 0.19 b
402.27 ± 1.03 b
19.07 ± 1.37 b
10.35 ± 0.22 c
2.39 ± 0.06 b
8.37 ± 0.12 a
N/D
48.49 ± 0.87 a
16.97 ± 0.40 c
448.55 ± 0.51 c
20.95 ± 1.74 b
15.11 ± 0.13 e
2.42 ± 0.03 b
15.37 ± 0.08 c
N/D
45.16 ± 0.01 d
25.45 ± 1.15 d
40.69 ± 1.13 e
30.33 ± 1.46 e
43.36 ± 0.18 cd
34.82 ± 2.04 f
40.57 ± 0.08 e
38.87 ± 1.14 g
2ꢀMethylpyrazine α
2,5(6)ꢀDimethylpyrazine α
2,3ꢀDimethylpyrazine α
2ꢀEthylpyrazine α
N/D
430.58 ± 0.01 a 426.55 ± 3.67 a
433.84 ± 3.74 a 392.45 ± 9.19 b 293.06 ± 0.63 d 251.48 ± 1.27 e
dc
Sterilization
Roasting
N/D
2.23 ± 0.03 a
N/D
8.92 ± 0.77 a
4.12 ± 0.02 b
1.65 ± 0.16 a
N/D
25.24 ± 0.59
27.52 ± 0.57 c
14.62 ± 0.07 f
3.17 ± 0.12 c
15.31 ± 0.33 c
N/D
263.29 ± 1.66 f
2.00 ± 0.05 c
135.47 ± 1.47 f
N/D
29.69 ± 1.33 c
12.62 ± 0.27 g
3.34 ± 0.29 c
11.81 ± 0.06 d
N/D
25.57 ± 0.45 c
10.62 ± 0.03 c
3.41 ± 0.29 c
10.58 ± 0.06 e
N/D
24.37 ± 1.14 d
8.07 ± 0.05 h
3.20 ± 0.23 c
8.88 ± 0.02 f
N/D
75.22 ± 0.04 i
2.84 ± 0.08 d
26.95 ± 0.57 g
N/D
13.81 ± 0.11 d
2.86 ± 0.17 bc
13.44 ± 0.16 b
N/D
239.29 ± 0.69 d 278.17 ± 0.20 e
1.10 ± 0.05 a 1.56 ± 0.08 b
115.96 ± 2.02 d 121.61 ± 0.80 e
Sterilization
Roasting
N/D
Sterilization
Roasting
N/D
45.50 ± 0.50 a
N/D
68.30 ± 0.16 b
211.13 ± 2.29 c
182.96 ± 0.53 g 117.95 ± 2.19 h
2ꢀEthylꢀ5ꢀmethylpyrazine α
Sterilization
Roasting
N/D
N/D
N/D
2.53 ± 0.05 d
91.24 ± 0.07 c
N/D
2.75 ± 0.25 d
48.87 ± 0.56 a
N/D
47.89 ± 0.02 a
N/D
4.80 ± 0.10 a
67.30 ± 0.57 b
N/D
10.58 ± 0.20 b
89.62 ± 0.92 c
N/D
32.99 ± 0.17 cd
3ꢀEthylꢀ2,5ꢀ
dimethylpyrazine α
Sterilization
Roasting
N/D
31.58 ± 0.51 d
N/D
N/D
31.68 ± 0.32 d
N/D
32.41 ± 2.70 cd
35.29 ± 0.55 c
17.83 ± 0.43 e
9.96 ± 0.11 b
2ꢀEthylꢀ3,5ꢀ
dimethylpyrazine β
Sterilization
Roasting
N/D
N/D
N/D
N/D
N/D
N/D
N/D
7.86 ± 0.09 a
N/D
22.07 ± 0.20 b
N/D
9.69 ± 0.09 a
101.59 ± 3.64 c
N/D
56.94 ± 0.90 b
191.70 ± 5.51 d 222.19 ± 0.69 e 219.87 ± 6.20 e
85.99 ± 0.39 f
N/D
23.77 ± 0.10 e
32.70 ± 0.01 b
N/D
8.07 ± 0.58 a
13.72 ± 0.79 ab
N/D
2.70 ± 0.17 f
5ꢀEthylꢀ2,3ꢀ
dimethylpyrazine β
Sterilization
Roasting
N/D
89.30 ± 1.28 c
N/D
N/D
94.04 ± 0.60 c
N/D
N/D
80.42 ± 4.16 d
N/D
N/D
2,5ꢀDiethylpyrazine β
Sterilization
Roasting
N/D
N/D
N/D
N/D
N/D
N/D
N/D
3.11 ± 0.07 a
N/D
2.95 ± 0.09 a
7.44 ± 0.07 b
N/D
10.30 ± 0.16 b
11.18 ± 0.54 c
14.75 ± 0.01 d
16.65 ± 0.54 e
11.59 ± 0.29 c
N/D
8.40 ± 0.15 e
5.59 ± 0.02 f
N/D
3.36 ± 0.13 a
2.75 ± 0.07 a
N/D
1.37 ± 0.06 f
2,3ꢀDiethylꢀ5ꢀ
methylpyrazine β
Sterilization
Roasting
N/D
N/D
N/D
N/D
N/D
14.60 ± 0.32 c
N/D
13.05 ± 0.70 c
18.20 ± 0.03 d
N/D
19.25 ± 0.11 d
15.09 ± 0.75 c
N/D
16.99 ± 0.43 e
3,5ꢀDiethylꢀ2ꢀ
methylpyrazine β
Sterilization
Roasting
N/D
N/D
N/D
N/D
N/D
N/D
N/D
3.29 ± 0.06 a
N/D
11.11 ± 0.10 a
8.43 ± 0.16 b
N/D
8.86 ± 0.06 b
9.88 ± 0.14 f
N/D
9.38 ± 0.11 c
4.50 ± 0.02 g
N/D
6.59 ± 0.09 f
2.38 ± 0.23 a
N/D
2.78 ± 0.11 g
2ꢀEthylꢀ3,5,6ꢀ
trimethylpyrazine β
Sterilization
Roasting
N/D
N/D
N/D
N/D
N/D
9.38 ± 0.10 c
N/D
10.72 ± 0.19 d
N/D
12.41 ± 0.08 e
N/D
2ꢀIsopropylꢀ3,5ꢀ
dimethylpyrazine β
Sterilization
N/D
N/D
N/D
N/D
N/D
N/D
N/D (not detected). α Identification confirmed by GC retention index and mass spectra of authentic compounds. β Tentatively identified by matching mass spectra library.
Data points represent mean values of 3 independent determinations. Values in the same line followed by different superscript letters are significantly different (p < 0.05).
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Table 4. Pyrazines (GCꢀMS peak Area x 10 ⁶) detected in model reactions of dry mixtures of glucose and hydrolyzed whey, whey and whey with the addition of an amount of free
amino acids as described in Table 1, under roasting conditions (180 ⁰C/90 min)
Hydrolyzed whey 15 mg
Glucose 7.5 mg
23.10 ± 0.36 a
42.39 ± 3.04 a
1.80 ± 0.07 a
Hydrolyzed whey 15 mg
Glucose 15 mg
20.97 ± 1.20 b
25.41 ± 1.83 b
2.17 ± 0.01 a
Whey 15 mg
Glucose 7.5 mg
8.99 ± 0.62 c
9.02 ± 0.69 c
N/D
Whey 15 mg
Glucose 15 mg
8.53 ± 0.26 c
9.29 ± 0.82 c
N/D
Whey 15 mg + AA (*)
Glucose 7.5 mg
9.66 ± 1.05 d
10.99 ± 1.17 d
N/D
Whey 15 mg + AA (*)
Glucose 15 mg
8.06 ± 0.66 e
9.37 ± 0.84 c
N/D
Compound
2ꢀMethylpyrazine α
2,5(6)ꢀDimethylpyrazine α
2,3ꢀDimethylpyrazine α
2ꢀEthylpyrazine α
4.23 ± 0.19 a
4.73 ± 0.06 a
N/D
N/D
N/D
N/D
2ꢀEthylꢀ5ꢀmethylpyrazine α
3ꢀEthylꢀ2,5ꢀdimethylpyrazine α
2ꢀEthylꢀ3,5ꢀdimethylpyrazine β
5ꢀEthylꢀ2,3ꢀdimethylpyrazine β
10.70 ± 0.11 a
5.14 ± 0.13 a
2.28 ± 0.14 a
8.81 ± 0.05 b
3.38 ± 0.18 b
1.54 ± 0.08 b
1.16 ± 0.08 a
1.64 ± 0.05 c
3.50 ± 0.13 d
3.53 ± 0.12 d
3.44 ± 0.42 d
N/D
N/D
N/D
N/D
N/D
N/D
N/D
N/D
1.03 ± 0.01 a
N/D
N/D
N/D
N/D
N/D (not detected). (*) concentration of amino acids as described in Table 1. α Identification confirmed by GC retention index and mass spectra of authentic compounds. β Tentatively
identified by matching mass spectra library.
Data points represent mean values of 3 independent determinations. Values in the same line followed by different superscript letters are significantly different (p < 0.05).
Table 5. Pyrazines (GCꢀMS peak Area x 10 ⁶) detected in model reactions of dry glucose and hydrolyzed whey mixtures incubated at different relative humidities under roasting
conditions (180 ⁰C/90 min)
Compound
0.16 aw
0.33 aw
0.38 aw
0.53 aw
0.62 aw
0.75 aw
2ꢀMethylpyrazine α
2,5(6)ꢀDimethylpyrazine α
2,3ꢀDimethylpyrazine α
2ꢀEthylpyrazine α
2ꢀEthylꢀ5ꢀmethylpyrazine α
3ꢀEthylꢀ2,5ꢀdimethylpyrazine α
2ꢀEthylꢀ3,5ꢀdimethylpyrazine β
5ꢀEthylꢀ2,3ꢀdimethylpyrazine β
2,5ꢀDiethylpyrazine β
23.10 ± 0.36 a
42.39 ± 3.04 a
1.8 ± 0.07 a
4.24 ± 0.19 a
11.00 ± 0.51 a
5.14 ± 0.13 a
2.28 ± 0.14 a
1.06 ± 0.06 a
N/D
54.45 ± 4.70 b
419.99 ± 13.07 b
5.43 ± 0.26 b
37.99 ± 1.23 c
343.52 ± 16.20 c
5.73 ± 0.54 b
30.40 ± 1.25 d
327.52 ± 14.63 c
5.02 ± 0.48 b
4.84 ± 0.43 ca
97.00 ± 6.95 cd
247.43 ± 12.75 b
22.04 ± 2.05 cd
22.68 ± 1.15 c
5.09 ± 0.45 a
15.20 ± 0.46 b
19.14 ± 1.28 a
10.60 ± 0.72 ab
43.83 ± 1.14 a
0.10 ± 0.01 b
22.39 ± 0.86 a
287.13 ± 5.82 d
4.78 ± 0.37 b
5.07 ± 0.28 ca
107.68 ± 6.33 bc
254.57 ± 4.56 b
22.26 ± 1.37 c
30.82 ± 2.75 b
9.92 ± 0.77 b
18.59 ± 1.21 a
26.53 ± 1.77 b
12.83 ± 1.02 a
42.22 ± 4.01 a
0.07 ± 0.01 b
19.71 ± 0.89 a
211.5 ± 12.18 e
4.59 ± 0.33 b
4.98 ± 0.32 ca
85.2 ± 8.00 d
163.00 ± 11.38 c
16.61 ± 1.39 d
22.60 ± 1.67 c
7.43 ± 0.70 ca
13.49 ± 1.27 b
20.04 ± 1.93 a
8.81 ± 0.65 b
22.55 ± 1.39 b
N/D
6.69 ± 0.46 b
5.62 ± 0.40 b
122.23 ± 8.24 b
262.84 ± 12.30 b
30.34 ± 2.08 b
28.34 ± 2.46 b
6.78 ± 0.33 a
18.72 ± 0.49 a
20.44 ± 0.37 a
13.03 ± 0.93 a
47.25 ± 2.70 a
11.80 ± 0.95 a
101.74 ± 6.33 b
249.39 ± 13.11 b
24.82 ± 1.93 c
19.73 ± 1.92 c
5.10 ± 0.43 a
16.19 ± 1.05 ab
18.07 ± 1.33 a
11.46 ± 0.84 a
46.88 ± 1.58 a
0.39 ± 0.03 b
2,3ꢀDiethylꢀ5ꢀmethylpyrazine β
3,5ꢀDiethylꢀ2ꢀmethylpyrazine β
2ꢀEthylꢀ3,5,6ꢀtrimethylpyrazine β
2,5ꢀDimethylꢀ3ꢀ(3ꢀmethylꢀbutyl)pyrazine β
3ꢀFuranꢀ2ꢀylmethylꢀ2,5ꢀdimethylpyrazine β
N/D
N/D
N/D
N/D
N/D
N/D (not detected). a Identification confirmed by GC retention index and mass spectra of authentic compounds. β Tentatively identified by matching mass spectra library.
Data points represent mean values of 3 independent determinations. Values in the same line followed by different superscript letters are significantly different (p < 0.05).
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