JOURNAL OF FOOD SCIENCE
CHEMISTRY/BIOCHEMISTRY
Lipolysis and Lipid Oxidation in Frozen
Minced Mackerel as Related to Tg9,
Molecular Diffusion, and Presence of Gelatin
N. C. Brake and O. R. Fennema
ABSTRACT
We determined the sub-zero temperature dependencies (TD)
of lipid hydrolysis (LH), lipid oxidation (peroxide value [PV]),
and diffusion of 14C-fructose in minced mackerel (MM) and
how these rates are influenced by gelatin (G) and MM Tg .
2 8
When 1% G was added, the rate of LH at 10 C decreased as
compared to that in controls, whereas the rate of PV and
2 2 2 8
TBARS increased ( 5, 15 and 20 C). The TD of 14C-fruc-
tose diffusion was similar to that of TBARS, but not to those
9
for LH or PV. Just below Tg , the rate decrease for TBARS
was abrupt, whereas rate decreases for LH and PV were
moderate to small, respectively.
Key Words: frozen mackerel, oxidation, lipid hydrolysis,
diffusion, gelatin
INTRODUCTION
ALTHOUGH FROZEN FOODS ARE MICROBIOLOGICALLY STABLE, THEY
are prone to chemical and physical (e.g., recrystallization, moisture
migration) deterioration during storage. Procedures for accurately pre-
dicting the stability of frozen foods do not exist. Water activity, an
effective measure for predicting the stability of unfrozen foods, is not
effective for frozen foods (Fennema, 1981). Subfreezing temperature
is also of little value for predicting stability because different foods
may deteriorate at different rates at a given temperature. It has been
suggested that the glass transition temperature (Tg) may be a good
indicator of stability, i.e., foods should exhibit good stability below
that temperature (Levine and Slade, 1988; Slade and Levine, 1991).
More specifically, Tg9, the glass transition achieved under conditions
of maximal ice formation, has been suggested as the temperature of
critical importance. An important restriction to this approach, howev-
er, is that it applies only to rates of events (physical and chemical) that
are diffusion-limited, i.e., only such events would be reduced to a rate
of practical insignificance at Tg9 or below. Physical events, being
uniformly diffusion-dependent, almost always approach practical ces-
sation at T,Tg9. Chemical reaction rates are not nearly so consistent;
some virtually stop near Tg9 whereas others do not (Lim and Reid,
1991; Simatos and Blond, 1991; Karel et al.,1993; Roos and Him-
berg, 1994). Such inconsistent behavior is probably related to wheth-
er or not the reaction rate is diffusion-limited. Some simple reactions
can be accurately predicted to be diffusion-limited, namely, those that
are “fast reactions” and/or those that have low activation energies (8-
25 kJ/mol; Fennema, 1996), but for many reactions, especially com-
plex ones, predictions are unreliable. Accurate assessment of the Tg9
hypothesis is further complicated by the fact that some low-molecular
weight molecules, most importantly water, retain all types of motion
below Tg9 if the glass matrix is composed of large polymers (Slade
Author Fennema is with the Dept. of Food Science, Univ. of Wisconsin-Madison,
1605 Linden Drive, Madison, WI 53706. Author Brake’s present address: Dreyer’s
Grand Ice Cream, Inc., 1250 Whipple Road, Union City, CA 94587. Address
inquiries to Dr. Owen R. Fennema.
© 1999 Institute of Food Technologists
and Levine, 1994).
Chemical reactions are a major cause of frozen food deterioration
and the Tg9 approach is the only potentially reliable measure for pre-
dicting chemical stability of foods at subfreezing temperatures. Thus,
9 technologists need to know which of the stability-governing chemical
reactions in frozen foods are diffusion-limited at Tg9. Lipid instability
is a major cause of frozen food instability, and fish are especially
susceptible to this mode of deterioration (Fennema, 1993). It should,
therefore, be informative to study oxidation and hydrolysis of lipids in
fish, giving special attention to changes in the rates of these reactions
at temperatures bracketing Tg9. It is currently not known whether
either of these reactions would be diffusion limited.
Mackerel was selected for study because: (1) oxidation and hy-
drolysis of lipids occur in mackerel at commercially important rates
during typical conditions of frozen storage (Hardy and Smith, 1976;
Ke and Woyewoda, 1978), and (2) results obtained in a real tissue
system would likely have greater relevance to frozen foods, in gener-
al, than would study of a simple model. Mackerel can be minced to
greatly improve inter-sample uniformity, and to enable additives to be
incorporated uniformly.
It would also be informative to conduct a parallel study on changes
in the diffusion rate of a small molecule at temperatures bracketing
Tg9. Knowledge about rates of molecular diffusion at subfreezing
temperatures is needed because of its fundamental relationship with
sample Tg9. Few data pertaining to diffusion in frozen samples are
available (Charoenrein and Reid, 1991; Nelson, 1993; Roos, 1995).
Discussions of techniques for measuring rates of molecular diffusion
have been reported but mention of frozen samples is usually brief or
nonexistent (Roos, 1995). Rapid, precise methods for measuring dif-
fusion in frozen systems do not exist. After considering the available
choices, a radiolytic method involving 14C-fructose was developed
for our use. Selection of water as a diffusant was not considered a
good choice because its small size would enable it to diffuse through
a hydrocolloid glassy matrix at T,Tg9 (Slade and Levine, 1994).
Hydrocolloids are known to raise Tg9 of samples containing small
molecules (Slade and Levine, 1991). This is advantageous because it
then becomes more likely that a food so altered will, at a given sub-
freezing temperature, be below its Tg9, and therefore more stable with
respect to diffusion-limited reactions. Some evidence in support of
this has been reported (Levine and Slade, 1986; Lim and Reid, 1991;
Kerr et al., 1993), but research in this area is not extensive. Hydrocol-
loid additives also have beneficial effects when added to minced fish
and many other foods at low concentrations (Da Ponte et al., 1985;
Glicksman, 1983, 1986; Pomeranz, 1985), but the mechanisms by
which they exert such effects are not well understood. For these rea-
sons we considered it important to determine the effects of an added
hydrocolloid on the temperature-dependence of reaction rates. After
evaluating many commercially available hydrocolloids, gelatin at 1%
(w/w) was selected for study.
The specific objectives of this study were to: (1) determine the
temperature-dependence of the rates of oxidation and hydrolysis of
lipids in frozen, minced mackerel, with and without the addition of 1%
w/w gelatin, (2) to determine the temperature-dependence of the rate
of diffusion of 14C-fructose in frozen, minced mackerel, (3) and to
ascertain whether the practical cessation temperature of the two lipid
Volume 64, No. 1, 1999 —JOURNAL OF FOOD SCIENCE 25
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Reaction Rates in Frozen Mackerel . . .

reactions occurs at Tg9, and whether the temperature-dependence of
these reactions parallels that of 14C-fructose diffusion.
MATERIALS & METHODS
Fish
Atlantic mackerel (Scomber scombrus; ca 0.7-1 kg) was shipped
whole on ice from Boston, MA, to Madison, WI (total shipping time
3–4 days; postmortem age not known). After gutting, filleting, debon-
ing, and removing heads, tails and skin, the fish were minced and
processed for storage studies.
Processing and storage
For coarse mincing, fillets at 08C were passed at high speed through
a die with 5 mm-diam. holes (Kitchen Aid, Model D45, Hobart Mfg.
Co., Troy, OH). All equipment used was either stainless steel or plastic.
Finely minced fish was obtained by chopping the fillets into 2.5 3
2.5 3 0.5 cm pieces and freezing them in liquid nitrogen. The pieces
were then ground for 1 min at low speed in a Waring Blendor (CB-6,
Model 91–215, Dynamics Corporation of America, New Hartford,
CT). The resulting fish powder was thawed overnight in a cold room
at 68C before further use.
Gelatin was added to some samples. A 6% (w/w) aqueous gelatin
solution was prepared by dissolving gelatin (type A, 300 bloom, Gray-
slake Gelatin Company, Grayslake, IL), in 30% of the distilled water
(90°C), mixing for 3 min, and then adding the rest of the water (cold).
The solution was warmed to 408C, added to minced fish at 68C to
provide a gelatin concentration of 1% (w/w), and thoroughly mixed.
Distilled water, equivalent in amount to that added to gelatin-contain-
ing samples, was added to the control samples.
All samples were packaged, stored at 68C overnight to allow the
gelatin to gelatinize, frozen on solidified carbon dioxide (278.58C)
and placed in a 270°C air freezer (618C) before being stored in
circulating water/ethylene glycol baths (2095 series (6 0.38C) or 2325
series (60.18C); Forma Scientific Inc., Marietta, OH) at the various
subfreezing temperatures selected (25, 28, 212.5, 215, 220 or
2708C).
The experimental plan for the primary study of chemical changes
in minced mackerel at various subfreezing temperatures is shown in
Fig. 1. A large batch (13 kg) of minced Atlantic mackerel (caught in
March, 1995) was equally divided, and 1% w/w gelatin was added to
one portion (Fig. 1). Each batch was then further divided into 10-g
and 50-g packages. The 10-g packages were intended for measure-
ment of TBARS (thiobarbituric acid reactive substances) and the
50-g packages for measurement of peroxide value and fatty acids.
Following freezing of all packages on solid carbon dioxide, they were
placed at the desired storage temperatures, and then withdrawn at
times dependent on the temperature (see Fig. 2-5).
temperature (25, 28, 210, 212.5, 215 or 2208C). At predeter-
mined times, chosen based on the storage temperature (See Fig. 2-5),
samples were analyzed for lipid oxidation and hydrolysis. Time zero
was established as 6h following jar submersion based on the time
required for the geometric center of a 50-g sample of fish mince at
2408C to reach 258C. This was determined by means of a thermo-
couple and potentiometer.
Upon removal from storage at 215 or 2208C, samples were held
at 2158C for 3-6h and then extracted without prior thawing. Upon
removal from storage at 25 or 2108C, samples were placed in a
2408C freezer overnight (to inhibit chemical reactions), stored at
2158C (9h), then extracted.
The method of Bligh and Dyer (1959), as modified by Woyewoda
et al. (1986), was used to extract lipids. Both the PV and TBARS tests
were used to monitor lipid oxidation. For PV, the ferric thiocyanate
method of Schmedes and Holmer (1989) was used. For TBARS, the
method of Lemon (1975) was used.
For determination of FFA, an appropriate amount of lipid extract
(depending on the degree of hydrolysis) was pipetted into a 125 mL
Erlenmeyer flask which contained 30 mL propanol and 15 mL meth-
anol. Chloroform was added to bring the total volume of solvent to 75
mL. Metacresol purple (3-5 drops) was added and the yellow solution
was titrated with 0.05 N NaOH to a violet end point. FFA content was
calculated and expressed as oleic acid equivalents.
All chemicals were ACS reagent grade, except ethanol (95%, lab-
oratory grade) and isopropyl alcohol (laboratory grade). Analytical
tests on a single sample were performed in duplicate, and the mean of
these duplicate values was used when computing means and variances
for sample replicates (Fig. 1).
Diffusion study
Muscle from fish caught in April of 1995 was coarsely minced,

Lipid oxidation and hydrolysis studies

For determination of peroxide value (PV) and free fatty acids
(FFA), 50-g samples were prepared. A stainless steel tube (height 5.1
cm, inner diam. 3.8 cm) was filled with minced fish, the sample was
then gently pushed out, double-wrapped in Saran and frozen to
278.58C on solidified carbon dioxide. Samples were then placed in
zipper-type freezer bags and stored in air at 2708C (618C) until
adjusted to the desired study temperature.
For determination of thiobarbituric acid-reactive substances
(TBARS), 10-g samples were prepared. Plastic cups (height 1.2 cm,
inner diam 4.0 cm, Decagon Devices, Inc., Pullman, WA) were filled
with fish mince and then sealed with a plastic cap and frozen to 78.58C
on solidified carbon dioxide. All samples were then further packaged
and stored as described.
For studies at the various subfreezing temperatures, samples were
removed from 2708C storage and tempered for 1 day at 2408C. They
were then placed in glass jars (2.4L, W.A. Hammond Drierite Compa-
Fig. 1—Experimental plan for chemical evaluation of minced mack-
ny, Xenia, OH) and the jars were submerged in a temperature-con- erel. PV is peroxide value; TBARS is thiobarbituric acid reactive sub-
trolled, circulating ethylene glycol bath (60.38C) set at the desired stances; FFA is free fatty acids.

26 JOURNAL OF FOOD SCIENCE—Volume 64, No. 1, 1999


mixed with fructose (0.18% w/w), and gelatin (1% w/w) was thor-
oughly mixed into half of the samples. Distilled water equivalent in
amount to that added to the gelatin-containing samples was added to
the controls. All samples were frozen into cylinders (2.5 cm long, 3.5
cm diam) with a 1-cm-diam hollow core. Freezing was accomplished
on solidified carbon dioxide (2h). Finely minced fish (unfrozen) con-
taining 14C-fructose (1.92 mCi/g of fish) and added water (5 parts fish
to 1 part water; to compensate for the aqueous solution of gelatin
added to some of the samples outside the core region) was used to fill
the hollow cores of the frozen samples. The samples were packaged,
and then allowed to equilibrate at 2708C. Samples intended for study
at 258C were tempered at 2158C for 24h, then transferred to 258C.
Samples intended for study at 2158C were placed directly at that
temperature. Time zero was regarded as the time at which samples
attained 215°C.
At selected time intervals, samples were removed from their re-
spective baths and stored in the 2158C freezer for about 4.5h. The fish
sample was pushed slightly from the metal cylinder allowing a thin
disc (about 0.5 cm thick) to be manually sliced from the protruding
end, using the end of the cylinder as a guide. The first slice, consisting
of the rough outer surface was discarded. Two disks were taken from
each cylinder, the smoothest surface of each disk was flattened face
down, the samples were placed in freezer bags, then stored on solidi-
fied carbon dioxide until they were exposed to X-ray film. Fish disks,
wrapped in plastic (to avoid film contamination) were placed on top of
a sheet of X-ray film (Fuji Medical X-Ray Film, RX, 13 3 18 cm; Fuji
Photo Film Co., Ltd., Japan) with the smooth side of the disks facing
the film. The samples and X-ray film were enclosed in a vinyl expo-
sure cassette (Sigma Chemical Co., St. Louis, MO) and then clamped
between two prechilled glass plates (20 cm 3 20 cm 3 0.3 cm) using
four 2" office binder clips. After wrapping with aluminum foil the
samples were exposed for 60 days at 2708C. This unusually low
exposure temperature was used to minimize fructose diffusion during
exposure. A time period of 60 days was needed to obtain a suitable
image. Following exposure, samples were transported on solidified
carbon dioxide to a dark room and the film was developed.
Diffusion of 14C-fructose was evident as a semi-dark region ex-
tending outward from the intensely black original core. The outer
perimeters of these regions were measured using CollageTM (Photo-
dyne, Hartland, WI) to quantify the extent of migration. Samples were
analyzed in triplicate or quadruplicate.
Statistical analysis
Using the analytical mean of duplicates from each sample at a
Fig. 2—Formation of FFA in finely and coarsely minced mackerel at
-10°C, with and without 1% gelatin. Error bars are standard devia-
tions for 3 to 4 replicates.
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given time and temperature, the mean of replicate samples and the
corresponding standard deviation were calculated (Fig. 1). One-way
Anova was used to determine significance of differences between
treated and untreated samples at a given time and temperature. Signif-
icant differences were defined as p#0.05.
RESULTS & DISCUSSION
Selection of a hydrocolloid
Several hydrocolloids (alginates, gelatin [100 and 300 bloom, gelled
and ungelled], xanthan, carrageenan, chitosan, and methyl cellulose)
were selected for initial study based on their solubility in water, ability to
impart viscosity at low concentrations, Tg9 (high desirable), and previ-
ous indications of effectiveness in frozen systems. The efficacy of these
hydrocolloids, all tested at 1% w/w, was judged based on their ability to
inhibit formation of fatty acids in minced mackerel at 2108C. Gelatin
caused the greatest inhibition among the hydrocolloids tested, with alg-
inates ranking second (data not shown). Gelatin (300 bloom, gelled)
was selected for further testing. The effect of 1% w/w gelatin on Tg9 of
minced mackerel (213.38C; Brake and Fennema, 1998) was deter-
mined by DSC and a change could not be detected.
Rate of fatty acid formation in minced, frozen mackerel,
with and without gelatin
FFA formation in minced mackerel at 2108C was dependent on
time, degree of mincing and presence of gelatin (Fig. 2). FFAs in-
creased steadily over the 2-mo period, and the degree of lipolysis was
more rapid in finely minced mackerel than in coarsely minced. The
likely explanation is that finely minced samples incurred a greater
degree of decompartmentalization, and had larger interfacial area per
unit mass, than coarsely minced samples, therefore providing greater
exposure of enzyme to substrate.
Gelatin (1% w/w) exerted an inhibitory effect (p,0.05 or p,
0.01) on lipolysis in both finely minced and coarsely minced mackerel
at all storage periods beyond 0-time (Table 1). The inhibitory effect of
gelatin tended to increase with storage time in finely minced samples
and this effect was more pronounced in finely minced fish. Fine minc-
ing facilitated uniform distribution of gelatin probably enhancing its
inhibitory effect. This would enable it to associate more intimately
with sites of lipolysis and to better establish diffusion-retarding matri-
ces. The degree of inhibition increased with time which may indicate
that reactants, presumably closely positioned initially, had to diffuse
increasingly greater distances to reaction sites, and reaction products
encountered increasing difficulty diffusing away from reaction sites.
Fig. 3—Temperature dependence of FFA formation in coarsely minced
mackerel with and without 1% gelatin. Error bars are standard devia-
tions for 3 to 4 replicates. Time zero values for all temperatures
were averaged.
Volume 64, No. 1, 1999—JOURNAL OF FOOD SCIENCE 27

Reaction Rates in Frozen Mackerel . . .
Table 1—Statistical significance of gelatin on accumulation of fatty
acids in finely and coarsely minced mackerela
Sample Time(days)
0 27 45 63
Fine mince NS ** ** b 215
Coarse mince NS ** b
* 220
aNA=not significant; *p < 0.05; **p < 0.01. Data from Fig. 2.
bNo data.
This inhibitory mechanism, would result in samples with higher vis-
cosity (those containing gelatin), as observed, exhibiting greater inhi-
bition with time than those without.
The possibility also must be considered that gelatin could bind
fatty acids so that the titration procedure did not accurately reflect the
true extent of hydrolysis. To test this possibility, the fatty acid contents
of coarsely minced mackerel, with and without gelatin, were measured
immediately after preparation. Differences between the samples were
not significant (p,0.05)(data not shown), thus precluding gelatin/
fatty acid binding as an influence.
If a gelatin-induced increase in viscosity contributed to lipolysis
inhibition, then a decrease in temperature should greatly enhance this
effect. This possibility was tested using coarsely minced fish stored at
25, 215 and 2208C (Fig. 3). Coarsely minced fish, rather than finely
minced, was used since the former more closely represented some
products of commerce. Although the inhibitory effect of gelatin was
highly significant (p,0.01; Table 2) after 30.5 days at 258C and after
213 days at 2208C, the degree of inhibition did not appear to increase
with decreasing temperature. Consequently, the action of gelatin to
reduce the rate of lipolysis in mackerel may not be caused by its ability
to increase viscosity.
Rate of lipid oxidation in minced, frozen mackerel, with
and without gelatin
Lipid oxidation in minced mackerel, with and without gelatin, was
monitored by measuring peroxide value (PV; Fig. 4) and formation of
thiobarbituric acid reactive-substances (TBARS; Fig. 5). Increases in
these values were found with increasing storage times at 25, 215 and
2208C, and rates of both reactions exhibited a pronounced tempera-
ture dependence.
With a few exceptions (mostly at 258C), the presence of gelatin
increased (p,0.05; Tables 3 and 4) lipid oxidation as compared to that
in corresponding control samples (Fig. 4 and 5). This immediately
raised the possibility that the gelatin used was contaminated with
minerals (Cu and Fe) that catalyze oxidation. The mineral content of
Fig. 4—Temperature dependence of PV formation in coarsely minced
mackerel with and without 1% gelatin. Error bars are standard devia-
tions for 3 to 4 replicates.
28 JOURNAL OF FOOD SCIENCE—Volume 64, No. 1, 1999
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Table 2—Statistical significance of gelatin on accumulation of fatty
acids in coarsely minced mackerela
Temp (°C) Significance at various times (dayssignificance)b
25 0ns
5.5ns 11.5ns 16.5* 20.5ns 30.5**
0ns 25ns 48ns 74** 99* 206ns
0ns 23.5ns 62ns 90ns 137ns 213*
aData from Fig. 3.
b NS=not significant; *p < 0.05; **p < 0.01.
the gelatin was analyzed by an independent laboratory and found to
contain 2.5 ppm Fe and ,2.5 ppm Cu (limit of detection method), as
well as other minerals. Since an Fe concentration in this range is
known to catalyze lipid oxidation in muscle (Ke and Ackman, 1976),
it seemed likely that metals in the gelatin were responsible for the
enhanced lipid oxidation.
Lipolysis can sometimes affect the rate of lipid oxidation (Castell
et al., 1966; Shewfelt, 1981). In our study, rates of lipid oxidation in
the absence and presence of gelatin remained virtually constant over
the study time (Figs. 4 and 5). This suggested that the rate of oxidation
was not influenced to a detectable degree by concurrent lipolysis.
Temperature dependence of the diffusion rate of
14
C-fructose, with and without gelatin
Determination of molecular diffusion was accomplished using
autoradiography of 14C-fructose. This method was selected for use
because minute quantities of a radiolabeled material could be detected,
and results of good precision are attainable. The disadvantage of this
method is that film exposure at subfreezing temperatures requires
many weeks.
14C-Fructose was considered to be representative of relatively
small, water-soluble molecules that are not expected to interact appre-
ciably with other constituents under our conditions. A lipid was con-
sidered unsuitable as a test diffusant because it would not be expected
to diffuse significantly under our conditions. Water was also regarded
as unsuitable because it would partition between the ice and liquid
phase and its diffusibility was not characteristic of other small mole-
cules of interest, e.g., it can continue to diffuse slowly at T,Tg9
(Slade and Levine, 1994). To decrease any tendency of labeled fruc-
tose to interact with other organic molecules during testing, a small
amount of unlabelled fructose (0.18 % w/w) was added to the unla-
belled portion of the minced fish. Carbon-14 was chosen as the label
because it has a long half-life and could be easily monitored.
The diffusion patterns of 14C-fructose in minced mackerel at 25
and 2158C (no gelatin) were monitored photographically (Fig. 6).
Fig. 5—Temperature dependence of TBARS formation in coarsely
minced mackerel with and without 1% gelatin. Error bars are stan-
dard deviations for 3 to 4 replicates.
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Table 3—Statistical significance of gelatin on accumulation of perox- Table 4—Statistical significance of gelatin on accumulation of
ides in coarsely minced mackerela malonaldehyde in coarsely minced mackerela
Temp (°C) Significance at various times (dayssignificance)b Temp (°C) Significance at various times (dayssignificance)b
25 0ns
5.5 ns
11.5 ns
16.5** 20.5* 30.5** 25 0ns
5.5ns 11.5ns 20.5ns 30.5ns —
215 0** 25* 48** 74ns 99ns 206** 215 0** 25* 48* 74ns 99** 206**
220 0* 23.5** 62** 90* 137** 213** 220 0ns 23.5ns 90** 95** 139* 213**
aData from Fig. 4. aData from Fig. 5.
bNS=not significant; *p<0.05; **p<0.01. bNS=not significant; *p < 0.05; **p < 0.01.
cNo data.

These patterns, and those of minced mackerel containing gelatin, were
quantified using an image analyzer (Fig. 7). Clearly evident are the
good precision of the data, the pronounced temperature-dependence
of diffusion rate, and an inability of 1% gelatin to influence (p,0.05)
rates of diffusion. The fact that 1% gelatin did not have a significant
effect on the diffusion rate of fructose was in accord with the observa-
tion that this concentration of gelatin did not significantly alter the Tg9
value of mackerel. Also, it confirmed that the inhibitory effect of
gelatin on rates of lipolysis did not increase with decreases in sub-
freezing temperature, and that small molecules could diffuse through a
glassy hydrocolloid matrix (Slade and Levine, 1994).
No study of diffusion similar to that conducted here has been
reported. The temperature dependence of fructose diffusion rate ap-
pears to be considerably greater than that of Zn diffusion in frozen
NaCl/starch gel, as reported by Charoenrein and Reid (1991). How-
ever, comparison of those results with ours may not be reliable be-
cause of the many experimental differences, and the absence of infor-
mation about Tg9 of their sample.
Note that ethanol (1.7% w/w) was inadvertently added (present in
radiolabelled fructose as received) when the radiolabelled fructose
solution was combined with fish mince used for the center core. If
ethanol enhanced fructose diffusion, then the effect would be more
pronounced in the early stages and diminish with time as concentra-
tion decreased due to diffusion of ethanol in a radial manner. Such
behavior was not obvious (Fig. 7).
Relationship of rates of chemical reactions to Tg9
Since the reported Tg9 for mackerel (ca. 213.38C; Brake and Fen-
nema, 1998) was within the temperature range studied (25 to 2208C),
it was of interest to determine how rates of lipolysis, lipid oxidation,
and diffusion of 14C-fructose changed near mackerel Tg9. Additional
reaction rates at 28 and 212.58C were measured to better bracket Tg9
and better improve our understanding of the results. For these two
temperatures, only 3–4 time points were used to determine rates.
Several equations are available for plotting rate versus temperature
data, and the equations of Arrhenius and Williams-Landel-Ferry (WLF)
are most common. In the zone beginning with the onset of freezing
and concluding with glass formation, neither equation would be ex-
pected to yield plots with good linearity because changes of state
violate assumptions underlying both (Fennema, 1975; Le Blanc et al.,
1988; Levine and Slade, 1989). Furthermore, proper employment of
the WLF equation requires that values for the two constants be deter-
mined for the specific product and conditions being studied. Suitable
constants were not available for our products and these could not be
readily determined.
The Arrhenius approach, although not expected to yield linear

B
14
Fig. 6—Diffusion pattern of C-fructose in coarsely-minced, frozen mackerel (no gelatin): (A) at -5°C, (B) at -15°C. All images are proportion-
ately scaled (the original diameter at time 0 was 1 cm).

Volume 64, No. 1, 1999—JOURNAL OF FOOD SCIENCE 29


Reaction Rates in Frozen Mackerel . . .
plots in the range from the initial melting point to Tg9, should do so
below Tg9, provided no further changes of state occur (Fennema,
1996). However, the lack of sub-Tg9 changes of state cannot be as-
sumed because other investigators have reported Tg values for fish in
the temperature range of 240 to 2778C (Nesvadba, 1993; Simatos
and Blond, 1993; Inoue and Ishikawa, 1997). Furthermore, the neces-
sary information to accurately determine order-of-reaction and reac-
tion rate constants was not available, so Arrhenius plots could not be
prepared.
Plots of the logarithm of reaction rate vs. temperature were pre-
pared (Fig. 8) and found to adequately distinguish differences in the
temperature-dependence of rates in the temperature zone of primary
interest, i.e., just below mackerel Tg9. Dashed lines are interpolations
over the broad range of temperature (2208C to 2708C) where data
were not collected, and where, consequently, the true shape of the
curve was not known.
In the absence of light, and with constant pressure and initial com-
position, four factors exert primary control over the rate of a reaction:
temperature, the diffusion factor, D, a frequency-of-collision factor,
A, and the chemical activation-energy factor, Ea For the rate of a
reaction to be diffusion-limited factors A and Ea must not be rate-
limiting, i.e., properly oriented reactants must collide with great fre-
quency and Ea must be sufficiently small that collisions have a high
probability of resulting in a reaction. Viscosity dependency, because it
is inversely related to translational molecular mobility, is a property of
crucial importance when considering rate-limiting mechanisms at sub-
freezing temperatures. Because A is not strongly viscosity dependent
it will be discussed no further (Bull, 1964).
Ea is lowered greatly if enzyme catalysts are involved (such reac-
tions are more likely to be diffusion-limited). Thus we needed to
determine which of these reactions were enzyme catalyzed. Enzymes
capable of catalyzing lipid oxidation (lipoxygenases) and lipid hydrol-
ysis (lipases and phospholipases) are present naturally in fish (Ger-
man and Kinsella, 1985; Haard et al., 1994). Their presence, however,
does not necessarily mean they would be active at temperatures near
mackerel Tg9 (213.38C). Lipases and phospholipases have been re-
ported to be capable of functioning in fish and other types of samples
at or below 2208C (Olley and Lovern, 1960; Lovern, 1962; Mullenax
and Lopez, 1975; Geromel and Montgomery, 1980). Furthermore, it
is well established that hydrolysis of lipids in fish will not occur at
significant rates in the absence of enzyme catalysis (Olley and Lovern,
1960; Lovern, 1962). It is reasonable, therefore, to assume that gener-
ation of fatty acids in our samples was enzyme catalyzed.
Fig. 7—Temperature dependence of 14C-fructose diffusion in coarsely-
minced mackerel with and without 1% gelatin.
30 JOURNAL OF FOOD SCIENCE—Volume 64, No. 1, 1999
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The effect of lipoxygenases on oxidation of fish lipids at subfreez-
ing temperatures is somewhat less clear. However, unlike lipases and
phospholipases, lipoxygenases are relatively unstable at 08C or be-
low. German et al. (1992) studied 12-lipoxygenase in carp and trout at
08C and found a half-life of ,3h; 15-lipoxygenase had a half-life of
about 10h in carp at 08C and exhibited very little activity in trout. It is
also well known that lipid oxidation can occur readily in the absence
of enzymes. Based on this information, it is reasonable to assume that
lipoxygenases did not have a major influence on the two kinds of
oxidation we monitored.
Assuming constant pressure, temperature, and sample composi-
tion, the diffusion factor, D, would be influenced primarily by the
hydrodynamic radii of the diffusing entities. As the steps in a reaction
are increased, it becomes more likely that a large reactant (or product)
will dominate D for the reaction sequence, causing D to be small
(slow diffusion).
A further point of consideration is that the reactions under study
are atypical of most reactions that cause deterioration of food since the
substrates (lipids) are not soluble in water. The reactions under study
therefore would occur at a lipid-water interface. This may be of impor-
tance when considering how a polymer-based glassy matrix would
influence the translational mobility of reactants and products to and
from the reaction site.
Based on these considerations, qualitative explanations for the
results (Fig. 8) can be suggested. With PV development, the change in
reaction rate near Tg9 was essentially negligible (Fig. 8B). The only
factor favoring a diffusion-limited rate for PV development is the low
activation energy that is attributable to metal (hematin) catalysis. This
mode of catalysis has an activation energy similar to that of lipoxyge-
nase catalysis (Tappel, 1962). Factors tending to cause the PV reac-
tion rate not to be diffusion-limited are: (1) this reaction is a measure
of early oxidation, involving only two steps (abstraction of hydrogen
and addition of oxygen), (2) the molecules are small and highly mo-
bile (large D), and (3) the reaction site (lipid-water interface) may
lessen the mobility-restricting effects of the polymeric glassy matrix,
provided hematin is present at the site in a catalytically-effective con-
centration. If these assumptions are valid, and the latter three factors
more than counterbalanced the low activation energy, then the rate of
the PV reaction would not be diffusion-limited.
Development of FFA exhibited a pattern very similar to that of PV
except for the rate acceleration in the zone of 25 to 288C (two left-
most data points, Fig. 8A). This was not surprising since freeze-
concentration of reactions often causes such an effect (Fennema, 1975).
Two factors favoring a diffusion-limited rate for lipid hydrolysis
are: 1) the low activation energy that is attributable to enzyme-cataly-
sis, and 2) the reactants (enzymes and lipids) are moderately large.
Factors tending to cause the rate of lipid hydrolysis not to be diffu-
sion-limited are: (1) the reaction sequence is short, and (2) the reaction
occurs at a water/lipid interface. The reaction site may be a factor of
key importance in understanding this reaction. We assumed that lipas-
es were originally present at concentrations sufficient to sustain the
reaction for a long period, and that migration of FA away from the
reaction site occurred at a rate sufficient to avoid inhibition of lipoly-
sis. Then, as discussed previously, the high viscosity conditions cre-
ated by the polymeric glassy matrix might not have a rate-controlling
effect on this reaction. Lipases are originally located in tissue or-
ganelles (e.g., lysosomes) and these would be disrupted during sam-
ple mincing and cooling. Because lipases function only at a water-
lipid interface, they would tend, during sample preparation, to migrate
to water-lipid interfaces, thereby allowing lipolysis to continue at
mackerel Tg9 without the need for significant diffusion of lipases.
Thus, one would not expect the rate of this reaction to be diffusion-
limited.
The results with TBARS (Fig. 8C) were quite different than those
for the other two reactions. Here, the reaction rate decreased abruptly
immediately below Tg9. At 2208C, the TBARS rate was much closer
to the near-cessation rate (2708C value) than were the rates of the
other two reactions (Fig. 8 A and 8B). Although the TBARS rate
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Table 5—Comparative rates of lipid oxidation, lipid hydrolysis and
diffusion of 14C-Fructose
Event Rate at: 2
[Rate 15°C/
25 C
°
215 C°
2
rate 5°C]
3 100
Fatty acid 0.05g oleic acid eq. 0.0116g oleic acid eq. 19%
developmenta (100g oil)21 (day)21 (100g oil)21 (day)21
Peroxide valueb 0.0195 meq peroxide 0.0028 meq peroxide 14%
(kg fish)21 (day)21 (kg fish)21 (day)21
TBARS
developmentc 0.00125 mol 0.00006 mol 4.8%
malonaldehyde malonaldehyde
(g fish)21 (day)21 (g fish)21 (day)21
Diffusion of 0.047 increase in 0.00175 increase in 3.7%
14
C-fructosed (circumference 4 (circumference 4
circumference @ zero circumference @ zero
time)(day)21 time)(day)21
aValues calculated from data in Fig. 3.
bValues calculated from data in Fig. 4.
cThiobarbituric acid reactive substances. Values calculated from data in Fig. 5.
d Values calculated from data in Fig. 7.

reduction may not be sufficient to conclusively categorize this reaction

as diffusion-limited, it was clearly more diffusion-dependent than the
other two. All factors previously considered as affecting rate work in
favor of causing the rate of this reaction to be diffusion-limited (low
activation energy, moderate size of many reactants, relatively long
reaction sequence, and reaction site). The reaction would begin in a
manner identical to that of peroxide development. However, addition-
al steps would involve diffusion of moderately-sized reactants and
products to and from the original reaction site. Some of the reactions
would perhaps continue at significant distances from the original site.
Thus, the polymeric glassy matrix may interfere to an important de-
gree. Although unproven, this reasoning is in accord with the results.

Relationship of rates of chemical reactions to rate of

14
C-fructose diffusion
A comparison of the temperature-dependencies of rates of the
chemical reactions with that for diffusion of 14C-fructose can be made
only at 25 and 2158C since diffusion was studied only at these two
temperatures (Table 5). Data in the last column, rate at 2158C ex-
pressed as a percentage of the rate at 258C, are the values of most
interest. It is clear that the TBARS value corresponds closely to that of
the diffusion data, whereas values for PV and FFA do not. This
confirms the earlier conclusions that the TBARS reaction was at or
very near to being, rate-limited by diffusion, whereas the other two
reactions were not.

A useful perspective
Some chemical reactions are influenced to a greater degree than
others by increasing viscosity and decreasing translational mobility of
molecules. Categorization of various chemical reactions as being, or
not being, rate-limited by diffusion is complex. As a further complica-
tion, others have shown that the rate of a given reaction may be diffu-
sion-limited in one environment and not in another (Karel et al., 1993;
Roos and Himberg, 1994). It is important to know which reactions,
among those that are primary causes of deterioration in foods of com-
mercial importance, are rate-limited by diffusion. Although some of
Fig. 8—Plots of log of chemical changes versus temperature in the factors that influence this categorization are known (and others,
coarsely-minced, frozen mackerel: (A) fatty acid concentration (as
oleic); (B) peroxide value; (C) malonaldehyde concentration (a mea- such as the nature of the glassy matrix need to be explored), their
sure of thiobarbituric acid reactive substances). Dashed lines are application in a quantitative manner has not been achieved. Such an
major interpolations over zones where data were not collected. Each achievement would greatly improve our ability to accurately predict
point was obtained from the slope of a plot of chemical value vs time
at a specific temperature. Except for temperatures of -8 and -12.5°C (rather than measure) the diffusion-dependence of reaction rates. The
(data not shown) slope values are from data in Fig. 3 to 5. Tg9 approach to predicting the chemical stability of frozen foods sim-
Regression coefficients for slope values ply cannot be reliably applied until such information becomes avail-
Temp (°C) Plot A Plot B Plot C
2 5 0.92 0.98 0.97
able. Without the Tg9 approach, temperature is the only measure we
2 8 0.95 0.99 0.58 have for predicting the chemical stability of frozen foods, and that
2 12.5 0.98 0.99 0.96 measure alone is inadequate. For the well being of the frozen food
2 15 0.93 0.97 0.70
2 20 1.00 0.94 0.70
industry, it is imperative that the uncertain relationship between rates
2 70 0.66 0.34 0.96 of chemical reactions and Tg9 be clarified.

Volume 64, No. 1, 1999—JOURNAL OF FOOD SCIENCE 31


Reaction Rates in Frozen Mackerel . . .
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