The effect of high pressure on the formation of volatile products in a model Maillard reaction

Article abstract of DOI:10.1039/a901186b

Reaction progress in the formation and subsequent decay of several of the volatile products from a model Maillard reaction between lysine and xylose has been followed at pH 7 and 10 and at elevated pressures. At low pH, the buildup and decay of 5-methyl-4-hydroxy-3(2H)-furanone and several minor products were observed. The application of high pressure results in a much diminished maximum concentration of each although the time to the maximum is unaffected. At pH 10, products contain nitrogen heterocycles with 2-methylpyrazine being the principal one which builds up and only slowly decays with time. Again, the yield is greatly reduced by pressure. The results are interpreted in terms of the inhibition by pressure of the formation of the precursor the Amadori rearrangement product which affects subsequent products. In some instances rates of formation are also found to be slightly inhibited while degradation of these products is accelerated. The corresponding mechanisms are examined in the light of these results.

Full text of DOI:10.1039/a901186b

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The effect of high pressure on the formation of volatile products in
a model Maillard reaction
Mark Bristow and Neil S. Isaacs
Department of Chemistry, University of Reading, Reading, UK RG6 6AD
Received (in Cambridge, UK) 11th February 1999, Accepted 30th July 1999
Reaction progress in the formation and subsequent decay of several of the volatile products from a model Maillard
reaction between lysine and xylose has been followed at pH 7 and 10 and at elevated pressures. At low pH, the
buildup and decay of 5-methyl-4-hydroxy-3(2H)-furanone and several minor products were observed. The
application of high pressure results in a much diminished maximum concentration of each although the time to
the maximum is unaffected. At pH 10, products contain nitrogen heterocycles with 2-methylpyrazine being the
principal one which builds up and only slowly decays with time. Again, the yield is greatly reduced by pressure.
The results are interpreted in terms of the inhibition by pressure of the formation of the precursor, the Amadori
rearrangement product which affects subsequent products. In some instances rates of formation are also found to
be slightly inhibited while degradation of these products is accelerated. The corresponding mechanisms are examined
in the light of these results.
Introduction
In recent years, the application of high pressures in processing
1–3
of food materials has been the subject of intense scrutiny.
Benefits associated with this method of treatment include steril-
ization from moulds, yeasts and bacteria while retaining fresh
flavour, consequences which have an immediate application in
the preservation and marketing of fruit-based foods in par-
ticular. It has been our aim to elucidate the chemical changes
which occur on pressure treatment of foods especially those
changes which differ from those which are products of thermal
treatment. In this paper, further studies of a model Maillard
reaction are discussed.
The Maillard reaction is the name given to the complex
sequence of events which occur when protein and carbohydrate
4,5
are heated together at temperatures in excess of 80 ЊC,
Scheme 1. The generation of a multitude of flavour compounds
and brown colouration is the basis of the familiar changes which
occur during cooking. It would be surprising if the application
of high pressure during such a complex sequence of reactions
produced no changes in the products and their consequent
flavours and colours, information which would be needed
before exploitation of this technology could proceed.
The Maillard reaction occurs in two or three distinct stages.
Initially condensation of a carbonyl function from carbo-
hydrate with an amino function in the protein leads to an imine
which rearranges to the amino form (Amadori rearrangement),
6
an isolable intermediate, 2, Scheme 2. This subsequently is
transformed into a variety of products, both volatiles and
brown polymer (‘melanoidin’). We have previously shown that
7
4
Scheme 1 Stages in the Maillard reaction (after Hodge ).
the first stage, the formation of the Amadori rearrangement
product (ARP) is accelerated by pressure while the second, its
degradation, is retarded.
8
examined by Ames and Apriyantono who identified a range of
9
products at varying pH and by Hayashi who studied the effect
The ARP therefore is formed more rapidly but decomposes
more slowly under high pressure conditions. The present work
examines the subsequent reactions which give rise to a large
number of volatile products, mainly oxygen and nitrogen
heterocycles, the flavour constituents. For this purpose, lysine
and xylose were chosen as the model protein and carbohydrate
since reactions between these two take place more rapidly and
at lower temperatures than those between other analogous
ingredients. The effect of pressure on this system has been
of pressure on products. In this work, the yields, both as a
function of time and pressure, of six principal products are
reported together with their rates of formation and decay
and associated volumes of activation. It must be said, however,
that systems of this kind are immensely complex and produce
a remarkable array of products many of which are transient
and evolve with time towards polymeric materials. The
products obtained vary with the particular reagents as well as
with conditions of pH, temperature and time.
J. Chem. Soc., Perkin Trans. 2, 1999, 2213–2218
This journal is © The Royal Society of Chemistry 1999
2213

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Fig. 1 Liquid chromatogram of products formed at pH 7.
Fig. 2 Liquid chromatogram of products formed at pH 10.
Reactions carried out to a maximum of 2 kbar were con-
ducted in a high pressure sampling cell from which a series of
aliquots of the reaction solution could be obtained while main-
taining pressure.
Scheme 2 Formation of the Amadori rearrangement product (ARP, 2).
Analysis of reaction solutions
Samples (20 mL) were extracted by ether (3 × 100 mL), the
Experimental
Buffer solutions
combined extracts dried (MgSO ) and carefully rotary evapor-
4
ated at <30 ЊC. Final adjustment to 0.1 mL in a graduated vial
was made by evaporation in a stream of nitrogen and 5-methyl-
hexan-3-one (0.2 µL, 10% in ether) was added as a GLC
standard.
Acetate. Sodium acetate (10 g) was dissolved in water (100
mL) and the pH adjusted to 7.00 by the addition of sodium
hydroxide. The solution was degassed by ultrasound and stored
over nitrogen.
Finally the solution was analysed by GLC using a 60 m capil-
lary column (DB-3, J and W Scientific) with the following tem-
perature programme; 30 ЊC isothermal, 15 min; 30 ЊC to 225 ЊC
Ϫ1
Bicarbonate. Potassium bicarbonate (0.2 M) was adjusted to
pH 10.00 by the addition of sodium hydroxide. The solution
was degassed by ultrasound and stored over nitrogen.
ramped at 4 ЊC min ; 225 ЊC isothermal for 10 min giving a
total analysis time of 74 min. Typical chromatograms are
shown in Figs. 1 and 2. Area counts for the major peaks relative
to the standard were then recorded. The reproducibility of
these measurements was about ±5%. For each of the aqueous
samples before extraction, the absorbance at 400 nm for a 1 cm
pathlength sample was also measured, Fig 3.
Reactions at 1 bar
Xylose (30 g, 0.2 mol) and lysine monohydrochloride (36 g, 0.2
mol) were dissolved in an appropriate buffer solution, either
acetate or bicarbonate (200 mL) and the pH further adjusted to
Identification of products
7
.00 or 10.00 by the addition of alkali. A nitrogen atmosphere
The gas chromatograms, Figs. 1, 2 obtained showed more than
fifty components, mostly base-line separated. About twenty
of these were conclusively identified and others somewhat ten-
tatively, see Tables 1 and 2 for comparison with authentic speci-
was introduced and the solution stirred magnetically and
heated maintaining the temperature at 100 ± 0.5 ЊC. Aliquots
(
20 mL) were removed periodically and analysed.
11
mens and for their linear retention indexes (LRIs), defined by
eqn. (1), in which LRI = LRI for unknown compound x, t =
Reactions at high pressure
x
x
A sample (approximately 25 mL) of each solution made up as
described above was placed in a cylindrical poly(tetrafluoro-
ethylene) (PTFE) vessel closed by a piston. This was placed in
the high pressure vessel described previously set at 100 ЊC.
The desired pressure was applied and the reaction allowed to
proceed for a specified time after which the vessel was depres-
surized, the sample removed and analysed. Kinetic plots were
built up point by point, each being duplicated.
LRI = 100 [(t Ϫ t )/(t
Ϫ t ) ϩ n]
(1)
x
x
n
n ϩ 1
n
10
retention time for compound x, t = retention time for n-alkane
n
of n carbon atoms eluting before x, tn ϩ 1 = retention time for
n-alkane of (n ϩ 1) carbon atoms eluting after x, by which
comparisons between these values and literature values may be
compared.
2
214
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Table 1 Identification of products in Fig. 1
ϩ
M
ϩ
M
No.
Name
No.
Name
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
pentane-2,3-dione
ethyl propanoate
pyrazine
3-methylenepentan-2-one
butyl acetate
methylpyrazine
2-furylmethanol
1-acetoxypropan-2-one
2-acetylfuran
2,5-dimethylpyrazine
2(3H)-furanone
100
102
80
98
116
94
16
17
18
19
2-furoic acid
112
142
154
178
163
124
112
140
102
152
135
126
194
162
2-methyl-3,5-dihydroxy-4H-pyran-4-one, 5
2,5,5-trimethylcyclohexane-1,3-dione
bifuran, C H O
1
0
10
3
20
5-acetyl-7-methyl-2,3-dihydro-1H-pyrrolizine
U1
U2
U3
U4
U5
U6
U7
U8
U9
unidentified
unidentified
unidentified
unidentified
unidentified
unidentified
unidentified
unidentified
unidentified
98
116
110
108
84
124
112
114
128
1
1
1
1
1
1
2-propanoylfuran
3-methylcyclopentane-1,2-dione
5-methyl-4-hydroxy-3(2H)-furanone, 1
2,5-dimethyl-4-hydroxy-3(2H)-furanone
Table 2 Identification of products in Fig. 2
ϩ
M
ϩ
M
No.
Name
No.
Name
1
2
3
4
5
6
7
8
2-methylpent-1-ene
propan-1-ol-2-one
propanoic acid
butan-3-ol-2-one
butan-1-ol-2-one
methylpyrazine
4-methylpentan-4-ol-2-one
2,5-dimethylpyrazine
84
74
74
88
88
94
116
108
9
10
11
12
13
14
15
U1
2(5H)-furanone
N,N-diethylformamide
84
101
112
114
128
126
144
147
3-methylcyclopentane-1,2-dione
5-methyl-4-hydroxy-3(2H)-furanone
2,5-dimethyl-4-hydroxy-3(2H)-furanone
3-hydroxy-2-methylpyran-4-one
2,3-dihydro-3,5-dihydroxy-6-methylpyran-4-one
unidentified
Fig. 4
used for fitting experimental to calculated concentration curves
by inspection.
Discussion
Products formed at pH 7
5
-Methyl-4-hydroxy-3(2H)-furanone 1. This is the principal
volatile constituent from xylose and lysine and imparts a char-
acteristic caramel odour to the product. This compound is not
stable under reaction conditions but its concentration rises to a
maximum and then decays away completely with time. Under
our conditions the maximum occurred at a time of 60 min. The
effect of high pressure, however, was mainly on the amount
formed. As pressure was increased the time to reach maximum
concentration and the rate profile in general were unaltered but
the amount of this product diminished until, at 6 kbar, hardly
any was seen to form, Fig. 4.
Rates of formation of 1 were obtained from concentration vs.
time data, Fig. 4, on the assumption of two consecutive reac-
tions. Rates of formation were of first order but rates of decay
best fitted a zero order plot, and were most reliably obtained
from experiments using authentic 1 as starting material heated
Fig. 3
In addition, identification was assisted by GC/MS analysis.
The difference between the two chromatograms emphasises the
sensitivity of the product ratios to the pH in which they are
formed.
Rate measurements
Where appropriate, data were fitted by computer to a linear, or
exponential function in order to obtain relative rate constants.
For the analysis of rates from buildup and decay curves such as
in Fig. 4, a programme analysing the sequential reaction (2) was
k
a
k
b
A
B
C
(2)
J. Chem. Soc., Perkin Trans. 2, 1999, 2213–2218
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Table 3 Rates of formation and decay of 5-methyl-4-hydroxy-3(2H)-
furanone as a function of pressure, pH 7, 100 ЊC
enolizations are unlikely to be contributing significantly to the
volume of activation.
That decomposition of the furanone occurs is evident from
its disappearance from the gas chromatogram with time but the
product was not identified. When the reaction was followed by
Ϫ1
Ϫ1
Pressure/bar
1
Ao
ka/min
kb/min
235
76.5
38.5
24.9
13.3
0.0297
0.0211
0.0205
0.0140
0.0133
0.0297
0.0213
0.0205
0.0223
0.0201
NMR spectroscopy carried out in D O, no spectral changes
2
1
2
4
6
000
000
000
000
were apparent. This suggests the product is chemically very
similar and a ring-opened form, 4, is a possible contender,
Scheme 4. The mass spectra of both starting material and
in the same buffer solution and under the same conditions as in
the Maillard reaction, Table 3.
The rate of the formation step falls slightly with pressure
‡
corresponding to an apparent activation volume, ∆V , calcu-
3
Ϫ1
lated from eqn. (3) as ϩ4 ± 1 cm mol . The rate of decay of
‡
Ϫd(ln k)/dp = ∆V /RT
(3)
Scheme 4 Possible degradation route of furanone, 1.
the furanone was not reliably obtained from these data, but
from separate experiments on the pure compound heated in the
same buffer solution at the same temperature. Reactions fol-
lowed zero-order kinetics suggesting enolization as the slow
step followed by rapid scavenging of the enol. Pressure slightly
accelerated the decomposition, the volume of activation was
product are identical, m/z = 114 being the highest peak in each
ϩ
case corresponding to M for 1 and (M Ϫ 18) for the assumed
product, 4, but no peaks at higher mass corresponding to
self-aldolization products were observed.
The reduction in the peak quantity of furanone produced
under high pressure is therefore due to the retardation in its rate
of formation together with the acceleration of its decompos-
ition. The decomposition of furanone was also studied in the
presence of xylose and was found to be accelerated four-
fold. Furthermore, pressure was now found to have a very large
3
Ϫ1
found to be Ϫ5 ± 1 cm mol .
The simulation also reproduces initial quantities of the
precursor to the furanone, A , whose values decrease
o
markedly with pressure, consistent with the reduced rate of
decay of the ARP or whatever other intermediate is controlling
this reaction. The mechanism of formation of 1 from the
ARP, 2, is plausibly via the deoxyoxopentose 3 as shown in
‡
3
Ϫ1
rate-accelerating effect, ∆V = Ϫ35 cm mol . NMR of the
final product showed a sugar residue to be present. These data
are consistent with a condensation reaction between furanone
and carbohydrate supplementing the route to disappearance of
the furanone, Scheme 5 and must further complicate interpret-
ation of rates measured under conditions of the Maillard
reaction.
12
Scheme 3.
Scheme 5 Reaction between furanone, 1, and xylose.
2
-Methyl-3,5-dihydroxy-(4H)-pyran-4-one, 5. A further major
product in the reaction between xylose and lysine is 2-methyl-
,5-dihydroxy-(4H)-pyran-4-one (5-hydroxymaltol) 5. This six-
carbon compound requires reaction of xylose with a C1 unit,
3
14
methanal or an equivalent in its formation, Scheme 6.
Methylations are a feature of the reaction as for example in the
formation of various methylated pyrazines at higher pH.
The degradation of 5 can lead to acyclic carbonyl com-
15
pounds and has been shown to be the precursor of 2-acetyl-
furan, a minor product detected in this study. Other routes
could also involve condensations with enolates building up large
and eventually coloured molecules. It was noted that, when a
pure sample of 5-hydroxymaltol was heated in water, some 50
products were obtained, furans, furanones, pyranones and
carboxylic acids. This intermediate is evidently able to act as a
second source of some of the other products of the reaction
and to create reactive species which later are incorporated in
melanoidins.
Scheme 3 Formation of 5-methyl-4-hydroxy-3(2H)-furanone, 1.
In such a complex sequence and with incomplete knowledge
of the charge states of the intermediates, it is difficult to predict
the volume of activation but the observed positive value sug-
gests that step b, the expulsion of the nitrogen residue, a dis-
sociative reaction, is rate-determining. This step is formally
similar to the decomposition of the Amadori rearrangement
product which has been previously shown also to have a
7
positive volume of activation. Retardation of the rate of
The rate of formation and the maximum yield of 5-hydroxy-
maltol diminish with pressure similar to the behaviour of 1,
Table 4; at 6 kbar, it becomes undetectable. Analysis of the
decomposition of the ARP in forming melanoidins was further
confirmed in the present work although it now appears that
this only applies at neutral or acidic pH while at high pH pres-
‡
3
Ϫ1
kinetic plots gave ∆V for formation = ϩ5 ± 1 cm mol
12
sure actually accelerates browning. Neutral proton transfers
are usually accompanied by no volume change so the various
although decay rates obtained from the sequential reaction
analysis were not of great reliability. It seems that as before,
2
216
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Table 4 Rates of formation of 5-hydroxymaltol
Ϫ3
Ϫ1
p/bar
k/10 min
1
58.5
57.3
41.7
31.7
1
2
4
000
000
000
Fig. 5
cycles make their appearance. In the present study, methyl-
pyrazine is the one formed in largest quantity and whose
formation has been studied. Other workers under similar con-
13
ditions have observed 2,5-dimethylpyrazine as predominant.
Unlike the oxygen heterocycles, this compound is reasonably
stable and builds up to a static concentration at 1 bar then
diminishes with a half life of about 20 h. However, the rates of
‡
app
formation are retarded quite strongly by pressure, ∆V
=
3
Ϫ1
ϩ12 cm mol and the maximum concentrations are greatly
reduced, Fig. 5. At high pressure furthermore, degradation
begins to become apparent and is accelerated. However, rates of
formation even at the highest pressures are hardly affected by
decomposition so that the reduction of the yield must be due to
the effect of pressure on the precursor.
Scheme 6 Formation and decay of hydroxymaltol, 5.
reduction of the rate of formation due to retardation of the
fission of the Amadori product is followed by more rapid
decomposition by associative steps.
Pyrazines can result from the condensation between two
α-aminoketone moieties. Methylpyrazine is plausibly formed
17
from the precursors shown in Scheme 8 but frequently numer-
ous methylated pyrazines are formed in similar reactions which
would each require the requisitely substituted amino ketone.
The pattern of methylated pyrazines depends upon the partic-
ular amino acids present although the sugar also has a bearing
3
(2H)-Furanone. This product, formed both at pH 7 and at
pH 10 again builds up to a maximum after 60 min and then
decays away, the maximum yield being strongly reduced by
pressure. 3(2H)-Furanone is formed in the retro-aldol frag-
mentation of xylose which loses a molecule of formaldehyde on
treatment with base, Scheme 7. The reaction appears to be
18
19
on this. Ammonia has been suggested as being involved and
it has been reported that only the nitrogens originate in the
amino acids, the remainder of the ring being built up from
20
sugar material. Conceivably the pyrazine which is also formed
is then methylated by a formaldehyde equivalent since fre-
quently numerous methylated pyrazines are formed in similar
reactions. Aminoketones can arise from an amino acid such as
lysine by transamination to a 1,2-dione which in turn arises by
retro-aldol cleavage of 1-deoxyosone and 2-methylpyrazine is a
major product from heating glycerol with ammonia. The ratio
of 2-methyl- to 2,5-dimethylpyrazines appears to be very sen-
sitive to conditions.
It is not clear in this immensely complex system which of or
even whether these reactive intermediates are available as such
but a ring-forming reaction of this type would be expected to
have a large negative volume of activation (e.g. Ϫ18 cm mol
for imine formation) so no such reaction appears to be rate-
controlling.
Scheme 7 Formation of 3(2H)-furanone.
3
Ϫ1
unrelated to the ARP so it may be coincidence that the reaction
profile is similar to those of ARP-derived products.
3
-Methylcyclopentane-1,2-dione. Building up to a maximum
concentration also after 60 min, the maximum yield of this
compound is also suppressed by pressure; at 1 kbar only about
1
0% of that formed at atmospheric pressure is observed. This
product is observed in reactions between glucose and amino-
16
acids but in the present case xylose would need to acquire a
further carbon atom so little can be said about the mechanism
with any certainty.
Scheme 8 Formation of methylpyrazine.
In conclusion it appears that the volatile products of the
Maillard reaction are generally suppressed by the application
of high pressure and that many of them appear to be controlled
by a common precursor such as the ARP whose decomposition
has a positive volume of activation. If food processing is
carried out at high pressure and at temperatures high enough
Products formed at pH 10
At higher pH, products separated and identified are shown in
Fig. 2 and Table 2. There are significant differences between
these and the neutral products since at pH 10 nitrogen hetero-
J. Chem. Soc., Perkin Trans. 2, 1999, 2213–2218
2217

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for Maillard chemistry to occur, then cooked flavours are likely
to be diminished.
7 N. S. Isaacs and M. Coulson, J. Phys. Org. Chem., 1996, 9, 639.
8
9
A. Apriyantono and J. M. Ames, J. Sci. Food Agric., 1993, 61, 477.
R. Hayashi, Agric. Biol. Chem., 1991, 55, 2071.
1
0 N. S. Isaacs, High pressure techniques in chemistry and physics,
W. Holzapfel and N. S. Isaacs (eds.), Oxford University Press, 1997,
p. 324.
Acknowledgements
We are grateful for funding of this work to M. A. F. F. and an
industrial consortium through a LINK studentship to M. J. B.
11 From a data base made available by courtesy of D. S. Mottram
whose help in identification we acknowledge with gratitude.
2 I. Blank and L. B. Fay, J. Agric. Food Chem., 1996, 44, 531.
3 V. Hill, PhD Thesis, Reading University, 1998.
1
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