E. T. Contis et al.

(Editors)
Food Flavors: Formation, Analysis and Packaging Influences
© 1998 Elsevier Science B.V. All rights reserved 529

Effect of antioxidants on the formation of volatilesfromthe Maillard

Reaction.
A. Amoldi, M. Negroni, and A. D'Agostina,

Dipartimento di Scienze Molecolari Agroalimentari, Universita di Milano, Via Celoria 2,

20133 Milano, Italy

Abstract
The aim of this report was to study the involvement of free radicals on the
formation of volatiles from the Maillard reaction. An aqueous model system of
glucose/lysine was heated in the presence of different amounts of antioxidants, such as a-
tocopherol, 2,6-di-^er^butyl-4-methylphenol (BHT), and rosemary extract, or of a,a'-
azobis-isobutyronitrile (ADBN) which is a pro-oxidant (free radical initiator). The
differences in volatile formation between the standard model system and those with
additives are relatively small. BHT, at pH 4, decreases pyrazine, while it increases slightly
2-methylpyrazine and 2,5-dimethylpyrazine and much more 2-acetylpyrrole; at pH 6 it
produces a decrease in furaneol. a-Tocopherol, at pH 4, decreases pyrazine,fiiraneol,2,3-
dihydroxy-6-methyl-4H-pyran-4-one, and 2-acetylpyrrole, while at pH 6 it decreases 2-
furanmethanol, and furaneol. Rosemary extract, at pH 4, decreases all the pyrazines and 2-
fliranmethanol and 2-acetylpyrrole; at pH 6 it produces a decrease of furaneol and 2,3-
dihydroxy-6-methyl-4H-pyran-4-one. AIBN at both pH's decreases the pyrazines and
increases 2-acetylpyrrole.

1. UNTRODUCTION

The reaction between amino acids and sugars (the Maillard reaction) produces
many volatile heterocyclic compounds whose structures, odor thresholds, and
concentrations affect the aroma of a food [1]. As part of an investigation on the effects of
lipids in the Maillard reaction we studied the formation of volatile compounds from an
aqueous glucose/lysine model system heated in the presence of either an antioxidant or a
pro-oxidant (free radical initiator). a-Tocopherol, 2,6-di-?^r^butyl-4-methylphenol (BHT),
and rosemary extract [2] were used as antioxidants and a,a'-azobis-isobutyronitrile
(AIBN) as a free radical initiator.
A previous paper [3] from our laboratory has presented some data on this same
subject. However, recently the experimental methodology was changed because the
Maillard reaction is very sensitive to pH. Previous model systems were heated at 120 and
100 °C in closed tubes after having set the pH to the desired value. Under these
conditions, the pH slowly decreases to reach values below pH 4. To prevent the drop in
pH, model systems were heated at 100 °C in a flask equipped with an autoclavable
electrode; pH was monitored during all heating steps and maintained constant by the
addition of dilute base using a procedure proposed by Ames and coworker [4].
530

2. EXPERIMENTAL

Mixtures containing equimolar amounts of xylose and lysine (70 mL of 0.5 M

water solution) and 60 mg of additive were heated under reflux for 2 h in a flask equipped
Avith an autoclavable electrode (ATI Russel). During this time the pH was monitored and
kept constant at the value of 4.0 and 6.0 by the addition of diluted sodium hydroxide
(NaOH). At the end of the heating time, the pH was adjusted to 8.0. Tetradecane was
added as a first internal standard, and the volatile compounds were recovered by
continuous extraction with dichloromethane (CH2CI2). After careful concentration of the
solvent to 1 mL, pentadecane was added as a second internal standard and the samples
were quantified by gas chromatography-flame ionization detection (GC-FID) on a DANI
86.10 gaschromatographer. Two internal standards, tetradecane and pentadecane, were
used. Compound concentration was calculated after determination of the correction
factors and were expressed in "mg / model system". Peaks were tentatively identified by
GC-mass spectrometry (GC-MS) on a Shimadzu QP-5000 by comparison with the
NIST62 spectra library and commercial standards as far as possible. Ions were generated
by EI at 70 eV. A capillary column SPB-1701 (30 m x 0.2 mm, film 1 ^m) was used.
Temperature program: 37 °C x 10 min, 4 °C to 200 °C, then isothermal. Each experiment
was repeated at least three times. Drawings from the same model systems (one every 15
min) were analyzed with an UV-visible spectrometer at 420 nm in order to detect the color
development.

3. RESULTS AND DISCUSSION

The overall effect of the anti or pro-oxidants on the Maillard reaction can be drawn
from color development seen at 420 nm. Figure 1 shows the increase in the absorbance at
420 nm in model systems at pH 4 and 6. BHT at the concentration used in these
experiments appears to favor browning at both pH values while a-tocopherol and
rosemary extract seem to have no effect at pH 4 and are weak inhibitors at pH 6.
AIBN inhibits browning at pH 4 and has no effect at pH 6 by 120 min.
The discussion on the volatile compounds will be limited to selected compounds.
Figures 2 and 3 compare the amount of these volatiles extracted from the systems at pH 6
and 4, respectively. The differences in volatile formation between the standard model
systems and those with additives are relatively small especially when considering the
experimental error that is rather large. While these data will be treated with suitable
statistical methods in the fixture, here the discussion will be limited to volatiles that are
affected at a greater degree.
BHT, at pH 4, decreases pyrazine, while it increases slightly 2-methylpyrazine (2-
MP) and 2,5-dimethylpyrazine (2,5-MP) and much more 2-acetylpyrrole (2-AP). At pH 6,
BHT produces a decrease in furaneol. a-Tocopherol decreases pyrazine, furaneol, 2,3-
dihydroxy-6-methyl-4H-pyran-4-one (DHP), and 2-acetylpyrrole (2-AP) at pH 4, while at
pH 6 it decreases 2-furanmethanol (FM), and fiiraneol. Rosemary extract, at pH 4,
decreases all the pyrazines and 2-furanmethanol (FM) and 2-acetylpyrrole (2-AP); at pH 6
it produces a decrease of furaneol and 2,3-dihydroxy-6-methyl-4H-pyran-4-one (DHP).
AIBN at both pH's decreases the pyrazines and increases 2-acetylpyrrole (2-AP).
 531

- reference
-pH6+BHT
-pHd+tocoph.
-pH6+rosm.
-PH6+AIBN

45 60 75 120
Time (min)

0,6 ~ pH4

0,5-

0,4- —•— reference

X J7^ --^pH4+BHT
S0,3- -o—pH4+tocoph.
< —A—pH4+rosm.
--0-PH6+AIBN
0,2 1

0.1 -

0<
(\ 15 30 45 60 75 90 105 120
Time (min)

Fig.1: Colour formation (absorbance at 420 nm) as a function of time

532

4.5 n

pyrazine
• reference B 6 H T Dtocoph. Drosm. BAIBN

0,C35n

0,04

0,03
m!^

1
0,02-

0,01
m
^^
2,3-DMP

| S reference DBHT Dtocoph. E3rosm. BAIBN |

®1
5

?3- T
WK^
2

0^
W/. Y- ^ ^ —
furaned
01 reference BBHT Dtocoph. Qrosm. QAIBN |Dreference DBHT Dtocoph. Drosm. HAIBN [

DHP
nreference eaBHT Dtocoph. Drosm. BAIBN

Fig. 2: Comparison of volatlles In model system at pH 6 with the indication of the confidence Interval (P=0.1)
2-MP=2-methylpyrazlne, 2,5-DMP=2,5-dimethylpyrazine, 2,3-DMP=2,3-dlmethylpyrazlne,
DHP=2,3-dlhydroxy-6-methyl-4H-pyran-4-one, FM=2-furanmethanol, 2-AP=2-acetylpyrrole
 533

0,9 n 0,09
0,8 0,08
0,7 0,07
0,6 T-
0.0,5
'0.4
r
0,3
0,2 .I
0,1 -
0-1 '••••••• — • - - - " • •-------• 1
Dvrazine 2-MP
= reference HBHT Dtocoph. Drosm. fiSAIBN | a reference HBHT Dtocoph Grosm. HAIBN \

0.035 ^ n
003
0,025
a>0,02^
^0,015 -
m
0,01 -
1^M
0,005 S
^^^ 2,5-DMP
B reference E9BHT Itocoph Drosm. BAIBN 1

0,03

furaneol
nreference SBHT Dtocoph Drosm. DAIBN

1,4 n
1,2
1 -
o. 0,8
e
0.6
^
0,4
0,2- [|[[||[
2-AP
DHP
1 Preference DBHT Dtocoph. Drosm. SAIBN [ Ireferer^ce DBHT Dtocoph Drosm. BAIBN

: impossible to quantify

Fig. 3: Comparison of volatiles in model system at pH 4 with the Indication of the confidence interval (P=0.1).
2-MP=2-methylpyrazine;2,5-DMP=2,5-dimethylpyrazine;2-FM=2-furanmethanol;
2-AP=2-acetylpyrrole; DHP=2,3-dihydroxy-6-methyl-4H-pyran-4-one;
534

There are some relevant diflferences between the results of this work and our
previous research [3]. The methodology was changed in an important way in that now the
pH is kept constant during all thermal treatments. This produces a depletion of the
compounds formed because while the pH varies, the mechanism of the Maillard reaction
changes and different compounds are more or less favored [5]; for example, the formation
of 2-furancarboxyaldehyde takes place only at low pH. Therefore, the observed
discrepancies can be explained by the fact that the dependence on pH is so relevant that
small differences in its value during heating can conceal or be confused for the additive
effect. This underlines how carefiilly experiments must be planned in this field to obtain
consistent results.
The free radical initiator AIBN is thermally decomposed to give gaseous nitrogen
(N^) and two alkyl radicals that can initiate a free radical chain. The presence of radicals in
a reaction mixture has an inhibitory effect on the Maillard reaction (MR) as demonstrated
by the slower formation of color at 420 nm and by reduction of pyrazines. The other
compounds are less sensitive.
BHT at the concentration used in the model system (0.27 mmol/model system)
seems to be favorable for browning while this does not happen with the other two
antioxidants. The effects on aroma are less clear and depend on the pH.
In conclusion free radicals may be involved in some way in the Maillard reaction,
but the effects are moderate in the model systems and difficult to discuss. Antioxidants
may play a role, but more experimental work is necessary for a practical exploitation of
these information's.

These researches were financed by the European Union: project FAIR, contract €796-1080.

4. REFERENCES
1. S. Fors, in "The Maillard Reaction in Foods and Nutrition", G.R. Waller and M.S. Feather
(eds), ACS Symposium Series 215, American Chemical Society, Washington DC, 1983, p.
185.
2. S.S. Chang, B. Ostric-Matijasevic, O.A. Hsieh, C.-L. Huang, J. Food. Sci. 42 (1977),
1102
3. A. Amoldi, E. Corain, in "Flavour Science. Recent Developments". A.J. Taylor, D.S.
Mottram (eds). The Royal Society of Chemistry, Cambridge, 1996,217.
4. J. M. Ames, A. Apriyantono, in "Thermally Generated Flavors. Maillard, Microwave and
Extrusion Processes". T.H. Parliment, M.J. Morello, R.J. McGorrin (eds), ACS
Symposium Series 543, American Chemical Society, Washington, DC, 1994,228.
5. F. Ledl, E. Schleicher, Angew. Chem. Int. Ed. 29 (1990), 565