Oxidation kinetics of linoleic acid in the presence of saturated acyl L-ascorbate

Article abstract of DOI:10.1007/s11746-005-1082-5

The oxidation processes of linoleic acid (LA) in the presence of L-ascorbic acid or saturated acyl L-ascorbate additives were measured at various temperatures and molar ratios of the additive to LA. Higher oxidative stability of LA was observed at higher additive levels for all additives. The addition of the ascorbates lengthened the induction period for the oxidation of LA. An autocatalytic kinetic rate equation was used to model the oxidation processes of LA mixed with the ascorbates, and the dependence of the rate constant, k, on acyl-chain carbon number was determined. At any temperature, the use of ascorbate additives decreased the K value for LA, and there was a slight tendency for K values to decrease with increasing acyl-chain length. The apparent activation energy, E_a, and the frequency factor, k ₀, for the rate constant were determined from Arrhenius plots. The calculated E_a and k₀ values also decreased with increasing ascorbate acyl-chain length. Copyright

Full text of DOI:10.1007/s11746-005-1082-5

Oxidation Kinetics of Linoleic Acid in the Presence
of Saturated Acyl -Ascorbate
L
Yoshiyuki Watanabe, Eiichiro Ishido, Xu Fang, Shuji Adachi*, and Ryuichi Matsuno
Division of Food Science and Biotechnology, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan
ABSTRACT: The oxidation processes of linoleic acid (LA) in the abilities (9–12). Although there have been some studies of the
presence of L-ascorbic acid or saturated acyl L-ascorbate addi- antioxidative ability of palmitoyl ascorbate for the oxidation of
tives were measured at various temperatures and molar ratios of
the additive to LA. Higher oxidative stability of LA was observed
at higher additive levels for all additives. The addition of the
ascorbates lengthened the induction period for the oxidation of
LA. An autocatalytic kinetic rate equation was used to model the
oxidation processes of LA mixed with the ascorbates, and the de-
pendence of the rate constant, k, on acyl-chain carbon number
was determined. At any temperature, the use of ascorbate addi-
tives decreased the k value for LA, and there was a slight tendency
oils (13–17), the application of the acyl ascorbates with
medium chain lengths has not been reported.
In this study, we analyzed the oxidation processes of linoleic
acid (LA) in the presence of
L-ascorbic acid or saturated acyl
L-ascorbate by applying a kinetic equation of the autocatalytic
type to the processes. The dependence of the rate constant on
the acyl-chain carbon number of the ascorbates at various tem-
peratures was investigated. Furthermore, the apparent activa-
for k values to decrease with increasing acyl-chain length. The tion energy, E , and the frequency factor, k , for the rate con-
a
0
apparent activation energy, E , and the frequency factor, k , for stant were determined, and the effects of the carbon number on
a
0
the rate constant were determined from Arrhenius plots. The cal- them were demonstrated.
culated E and k values also decreased with increasing ascor-
a
0
bate acyl-chain length.
EXPERIMENTAL PROCEDURES
Materials. (+)-Ascorbic acid and palmitoyl L-ascorbate were
Paper no. J10440 in JAOCS 82, 389–392 (May 2005).
L
KEY WORDS: Apparent activation energy, autocatalytic-type
kinetics, frequency factor, rate constant, saturated acyl
bate.
obtained from Nacalai Tesque (Kyoto, Japan). Acetone, caprylic
acid, and methyl myristate were purchased from Wako Pure
Chemical Industries (Osaka, Japan). Lauric acid and trimethyl-
silyl diazomethane solution were purchased from Sigma Chemi-
L-ascor-
®
cal (St. Louis, MO). Immobilized lipase, Chirazyme L-2 C2
Lipid oxidation in foods causes rancidity and nutritional deteri-
oration. Oxidation is a complicated process including the steps
of initiation, propagation, and termination. The kinetics of the
oxidation in a bulk lipid have been extensively studied (1–3),
and the entire lipid oxidation process has been described by a
kinetic expression of the autocatalytic type in terms of the frac-
tion of unoxidized substrate (2). In previous studies, we also
applied the expression to the entire oxidation process of n-6
FFA, their esters, and acylglycerols, and we evaluated their ki-
netic parameters (4–6).
from Candida antarctica, was purchased from Roche Molecular
Biochemicals (Mannheim, Germany). Linoleic acid (purity
>90%) was purchased from Tokyo Kasei Kogyo (Tokyo, Japan).
All the other chemicals of analytical grade were obtained from
either Wako Pure Chemical Industries or Nacalai Tesque.
Enzymatic synthesis and purification of saturated acyl L-
ascorbate.
L-Ascorbic acid (0.04 mol) and caprylic or lauric acid
(
2
0.2 mol) were added to a screw-capped amber glass bottle, and
0 g of Chirazyme L-2 C2 and 400 mL of acetone were further
®
added to the bottle. The headspace of the bottle was filled with
L-Ascorbic acid is a hydrophilic vitamin with high reducing
N , and the bottle was tightly sealed. The bottle was then im-
activity due to its enediol-lactone resonant structure; it is
widely used as an additive in foods and cosmetics. Its lipophilic
derivatives with long acyl chains such as 6-O-palmitoyl and 6-
2
mersed in a water bath at 55°C with vigorous shaking. After ca.
24 h, each 6-O-acyl L-ascorbate was isolated from the reaction
mixture according to the reported procedures (8), with a slight
modification. The immobilized lipase was removed by filtration
from the reaction mixture, and the solvent was removed by ro-
tary evaporation. The concentrate was washed three times with
O-stearoyl L-ascorbates are also used as an additive in foods
rich in lipids. Recently, some studies have appeared on the li-
pase-catalyzed synthesis of acyl ascorbates in an organic sol-
vent (7,8). We also synthesized various 6-O-acyl ascorbates by
using an immobilized lipase and reported their antioxidative
50 mL of n-hexane to remove the unreacted FA. The mixture was
dissolved in 150 mL of ethylmethylketone, and water (3 × 50
mL) was added to remove the unreacted ascorbic acid. The or-
ganic phase was separated, and excess solvent was removed by
rotary evaporation. The purified acyl ascorbate was dried in a
desiccator with a petri dish containing phosphorus pentoxide.
*
To whom correspondence should be addressed at Division of Food Science
and Biotechnology, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan.
E-mail: adachi@kais.kyoto-u.ac.jp
Copyright © 2005 by AOCS Press
389
JAOCS, Vol. 82, no. 5 (2005)

390
Y. WATANABE ET AL.
Oxidation of linoleic acid. The autoxidation processes of
LA mixed with -ascorbic acid or saturated acyl -ascorbate at
various molar ratios were measured as follows: First, LA and
-ascorbic acid or capryloyl, lauroyl, or palmitoyl -ascorbate
were dissolved in methanol at concentrations of 0.120 and
.012 mol/L, respectively. The LA solution (200 µL each,
which corresponded to 0.024 mmol of LA) was placed in
amber glass vials. Then, 50, 100, 150, 200, or 400 µL of
L
L
L
L
0
L
-
ascorbic acid or acyl ascorbate solution was added to the vials
to give the molar ratio of 0.025, 0.05, 0.075, 0.1, or 0.2 (ascor-
bate/LA), respectively. Then 150, 100, and 50 µL of methanol
was added to the samples in the molar ratios of 0.025, 0.05, and
0
.075, respectively. For the oxidation process of LA without
any additive, 200 µL of methanol only was added to the LA so-
lution. The mixture (25 µL each) was placed in flat-bottomed
glass cups (1.5 cm i.d. and 3.0 cm height), and the methanol
was then evaporated under reduced pressure. The cups were
placed in a plastic container in which a petri dish filled with a
saturated lithium chloride solution was placed to maintain the
relative humidity (RH) at 12%. The container was stored in the
dark at a given temperature (37, 50, 65, or 80°C). Samples were
periodically taken, and 300 µL of methyl myristate solution
FIG. 1. Oxidation processes of linoleic acid mixed with (a)
L-ascorbic
acid or (b) capryloyl -ascorbate at various molar ratios and at 37°C and
L
[
methanol/benzene/methyl myristate = 20:80:0.05 (by vol)] 12% RH. The ratios of the additives to linoleic acid were (G) 0, (N)
was added. Methyl myristate was used as the internal standard 0.025, (I) 0.05, (L) 0.075, and (M) 0.1. The solid curves were obtained
from the estimated values of k and Y for the oxidation processes.
for GC analysis of unoxidized LA. Thirty microliters of a 2.0
mol/L trimethylsilyl diazomethane solution (hexane as a sol-
vent) was then poured into the cup to convert unoxidized LA
0
Applicability of the kinetic equation of the autocatalytic type
to methyl linoleate (18). The mixture was left for 30 min at to the oxidation process of linoleic acid mixed with a saturated
room temperature. After evaporation of methanol, benzene, acyl -ascorbate. Figure 2 shows the oxidation processes at
and hexane under reduced pressure, the remainder was then 80°C and 12% RH of LA with no additive and of LA mixed
-ascorbic acid or capryloyl, lauroyl, or palmitoyl ascor-
L
dissolved with 500 µL of hexane, which was used for the GC with
L
analysis with an FID (19).
bate at the molar ratio (additive/LA) of 0.05. LA with no addi-
tive was quickly oxidized. When ascorbic acid was added to
LA, the oxidative stability of LA was slightly improved.
Capryloyl, lauroyl, and palmitoyl ascorbates retarded the oxi-
RESULTS AND DISCUSSION
Oxidation process of linoleic acid mixed with
L-ascorbic acid or dation of LA more than ascorbic acid. There seemed to be little
capryloyl
7°C and 12% RH of LA mixed with (a)
capryloyl ascorbate at various molar ratios of the ascorbates to
L
-ascorbate. Figure 1 shows the oxidative stabilities at difference in the antioxidative ability among the three acyl
-ascorbic acid and (b) ascorbates.
We showed previously (4,6) that the entire oxidation process
3
L
LA. The higher the molar ratio was, the more strongly the oxida- of LA could be expressed by the following kinetic equation of
tion of LA was suppressed in both cases. The induction period the autocatalytic type:
for the oxidation of LA was lengthened by the addition of ascor-
bates. The oxidation processes of LA mixed with lauroyl and
palmitoyl ascorbates were similar (data not shown). Capryloyl
dY
= −kY(1−Y)
[1]
dt
ascorbate had a greater antioxidative ability than ascorbic acid. where Y is the fraction of the unoxidized substrate, t is the time,
The oxidation of LA when mixed with capryloyl ascorbate at the and k is the rate constant. Under the condition of Y = Y at t =
0
molar ratio of 0.1 was suppressed for at least 130 h. Frankel et 0, the integration of Equation 1 gives
al. (15) reported that hydrophilic ascorbic acid was more effi-
1
−Y
Y
1−Y0
Y0
cient as an antioxidant than lipophilic palmitoyl ascorbate for the
oxidation of oil in a bulk system. Our results are contradictory to
ln
= kt +ln
[2]
theirs. A possible reason for the inconsistency is a difference in where Y is the initial fraction of unoxidized substrate and de-
0
the concentration of the ascorbate. The concentrations of the termines the induction period due to the mathematical format
ascorbates in our experiments were higher by two to three orders of the equation. The applicability of Equation 2 to the oxida-
of magnitude than those in the experiments by Frankel et al. (15). tion processes of LA mixed with various ascorbates was exam-
Another possible reason is a difference in the substrate. We used ined. Linear plots of ln[(1 − Y)/Y] vs. t for the oxidation process
LA, whereas they used soybean oil.
are shown in Figure 3. Based on a linear regression analysis,
JAOCS, Vol. 82, no. 5 (2005)

OXIDATION KINETICS OF LINOLEIC ACID
391
FIG. 2. Oxidation processes of (G) linoleic acid with no additive and
that mixed with (N) -ascorbic acid, (I) capryloyl -ascorbate, (L) lau- the autocatalytic type at 80°C and with the molar ratio of 0.05. The sym-
royl -ascorbate, or (M) palmitoyl -ascorbate at the molar ratio of 0.05 bols are the same as those in Figure 2. Y denotes the fraction of unoxi-
FIG. 3. Determination of the rate constant, k, in the rate expression of
L
L
L
L
and at 80°C. The solid curves were drawn using the k and Y values es- dized linoleic acid. The solid curves were drawn based on Equation 2.
0
timated in Figure 3.
with no additive (closed symbols) was greater than that for LA
with ascorbic acid or ascorbate (open symbols). When capryl-
oyl, lauroyl, or palmitoyl ascorbate was added to LA, there was
a tendency for the k value in the presence of ascorbates with
larger acyl-chain carbon numbers to be slightly smaller. The k
value for LA mixed with ascorbic acid (acyl-chain carbon num-
ber equal to zero) seemed to be greater than that for LA mixed
with the acyl ascorbates at any temperature.
Figure 5 shows the dependence of the acyl-carbon number of
the ascorbates on the ln[(1 − Y )/Y ] value in the rate expression
0
0
of the autocatalytic type (Eq. 2). As mentioned above, the Y0
value reflects the initial state of the substrate, which affects the
induction period. The large Y value indicates the elongation of
0
the induction period. The Y values for the additives were greater
0
FIG. 4. Relationship between acyl-chain carbon number of ascorbates
and the rate constant k at (G) 37, (I) 50, (L) 65, and (M) 80°C. Open
symbols represent the rate constants (mean ± SE) for the oxidation of
linoleic acid mixed with various ascorbates, and the closed symbols
that for no additive.
than that for LA with no additive although there were some ex-
ceptions. This indicated that the addition of an additive delayed
the induction period in the oxidation process of LA.
The temperature dependence of the rate constant k was ana-
lyzed based on the Arrhenius equation:
the k and Y values were determined from the slope and the in-
k = k exp(−E /RT)
[3]
0
0
a
tercept, respectively (Table 1). The k and Y values for the oxi-
0
dation processes of LA at 37, 50, and 65°C were also estimated where k is the frequency factor, E is the apparent activation en-
0
a
in the same manner.
ergy, R is the gas constant, and T is the absolute temperature. The
†
Dependence of the rate constants on acyl-chain carbon Arrhenius plots using the results shown in Figure 4, in each case,
number of ascorbates. Figure 4 shows the relationship between produced a straight line for use in evaluating E and k from the
a
0
the acyl-chain carbon number of the ascorbates and the k value slope and the intercept of the line, respectively. Figure 6 shows
at various temperatures. At any temperature, the k value for LA the relationship between the acyl-chain carbon number and the
TABLE 1
a
The Rate Constant k and the Parameter Y for the Oxidation of Linoleic Acid at 80°C
0
Ascorbic
acid
Capryloyl
ascorbate
Lauroyl
ascorbate
Palmitoyl
ascorbate
Additive
None
k [h−1]
ln[(1 − Y )/Y ]
3.81 ± 0.49
−3.78 ± 0.62
0.978
1.44 ± 0.065
−3.83 ± 0.16
0.979
0.634 ± 0.043
−4.36 ± 0.31
0.987
0.571 ± 0.081
−4.36 ± 0.56
0.987
0.405 ± 0.071
−3.53 ± 0.43
0.972
0
0
Y (mean)
0
a
The values are mean ± SE expected for the Y value.
0
JAOCS, Vol. 82, no. 5 (2005)

392
Y. WATANABE ET AL.
FIG. 5. Dependence of the ln[(1 − Y )/Y ] value on acyl-chain carbon
0
0
number of ascorbates at various temperatures. The symbols are the
same as those in Figure 4. Open symbols represent the ln[(1 − Y )/Y ] parent activation energy, E , or (I) the frequency factor, k , for the rate
FIG. 6. Effect of acyl-chain carbon number of ascorbates on (G) the ap-
0
0
a
0
values (mean ± SE) in the autocatalytic-type rate expression for the oxi- constant (mean ± SE). Open symbols represent the oxidation processes
dation of linoleic acid mixed with various ascorbates, and the closed of linoleic acid mixed with various ascorbates, and the closed symbols
symbols that for no additive.
that for no additive.
E or k value for the rate constant. The E values for LA mixed
with every ascorbate were 50 to 70 kJ/mol, and there was a ten-
9. Watanabe, Y., S. Adachi, K. Nakanishi, and R. Matsuno, Conden-
a
0
a
sation of L-Ascorbic Acid and Medium-Chain Fatty Acids by Im-
mobilized Lipase in Acetonitrile with Low Water Content, Food
Sci. Technol. Res. 5:188–192 (1999).
dency for E value to decrease with increasing acyl-chain carbon
a
number. The k values for LA mixed with ascorbates were smaller
0
10. Watanabe, Y., Y. Minemoto, S. Adachi, K. Nakanishi, Y. Shi-
mada, and R. Matsuno, Lipase-Catalyzed Synthesis of 6-O-Eicosa-
than that for LA with no additive or mixed with ascorbic acid.
The k value also decreased with increasing acyl-chain length.
pentaenoyl L-Ascorbate in Acetone and Its Autoxidation, Biotech-
nol. Lett. 22:637–640 (2000).
1. Watanabe, Y., S. Adachi, K. Nakanishi, and R. Matsuno, Lipase-
0
This would indicate that the presence of acyl ascorbate lowers
both the height of the energy barrier for the oxidation of LA and
the probability of the reaction resulting in the oxidation of LA.
1
Catalyzed Synthesis of Unsaturated Acyl L-Ascorbates and Their
Ability to Suppress the Autoxidation of Polyunsaturated Fatty
Acids, J. Am. Oil Chem. Soc. 78:823–826 (2001).
1
2. Watanabe, Y., X. Fang, Y. Minemoto, S. Adachi, and R. Matsuno,
ACKNOWLEDGMENT
Suppressive Effect of Saturated Acyl L-Ascorbate on the Oxida-
tion of Linoleic Acid Encapsulated with Maltodextrin or Gum
Arabic by Spray-Drying, J. Agric. Food Chem. 50:3984–3987
This study was supported by the Program for the Promotion of Basic
Research Activities for Innovative Biosciences (PROBRAIN),
Japan.
(2002).
1
1
1
1
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Pierce, Effects of Tocopherols, Ascorbyl Palmitate, and Lecithin
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[Received September 6, 2002; accepted April 27, 2005]
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