EFFECT OF BRINE PRE-TREATMENT ON LIPID STABILITY OF
FROZEN HORSE MACKEREL (Trachurus trachurus)
Santiago P. Aubourg and Maurizio Ugliano
Instituto de Investigaciones Marinas (CSIC)
c/ Eduardo Cabello, 6
36208-Vigo (Spain)
Fax: +34 986 292762
e-mail: saubourg@iim.csic.es
ABSTRACT
The rancidity development during the frozen storage (-20ºC) of an underutilised
medium-fat fish species (horse mackerel; Trachurus trachurus) was investigated. A
special attention was given to a pre-freezing treatment consisting of an immersion in
NaCl solution (5%, 10% and 20%) and its effect on lipid damage during the fish frozen
storage. For it, lipid hydrolysis (free fatty acid content) and oxidation (conjugated
dienes formation; peroxide value, PV; thiobarbituric acid index, TBA-i; fluorescence
formation, FR) were studied up to 270 days of frozen storage. Oxidative rancidity
measured by the PV, TBA-i and FR showed to increase with the frozen storage time and
also as a result of an increasing salt content in fish muscle. A high peroxides formation
was observed at day 210 of frozen storage, specially in the case of 20% NaCl treated
samples. Lipid hydrolysis also increased with the frozen storage time; at the end of the
experiment (270 days), a decreasing effect of muscle salt content on lipid hydrolysis
was observed. Employment of appropriate antioxidant additions is recommended if
salting pre-treatment is to be needed to avoid a large lipid oxidation development and
ensure a longer shelf life time.
Running Title: Brine pre-treatment and lipid stability of frozen fish
Key Words: Underutilised fish, salt pre-treatment, frozen storage, lipid oxidation and
hydrolysis, shelf life time
2
INTRODUCTION
Most fish and other marine species give rise to products of great economic
importance in many countries. One of such products is frozen fish, which has been
largely employed to retain fish sensory and nutritional properties before it is consumed
or employed in other technological process (1,2). Before the freezing step is
accomplished, adequate storage techniques that efficiently cool the fish material should
be employed to reduce post-capture losses. In order to find the best quality fresh fish,
several on board handling systems have been encountered such as icing (3), refrigerated
see water (4) or chemical addition (5,6).
One of these previous handlings has been salt treatment, which can consist on a
NaCl direct addition to the ice used to cool fish (7), immersion of fish material in a
brine solution (8) or combination with other technological process such as freezing (9),
drying (10) and smoking (11). The preservative effect of salt has been recognised
according to a decrease in water activity, less availability to microbial attack and
enhancement of functional properties, leading to an increase of the shelf life time (12).
Although salt allows a prolonged storage, its contact with fish has been reported
to enhance lipid oxidation (13,14) of the highly unsaturated lipids (15) directly related
to the production of off flavours and odours (16,17), protein denaturation and texture
changes (18,19).
The fish industry is actually suffering from dwindling stocks of traditional
species as a result of drastic changes in their availability. Thus, fish technologists and
fish trade have turned their attention to some unconventional sources of raw material
(20,21). One of such species is horse mackerel (Trachurus trachurus), a medium-fat
content fish abundant in the Northeast Atlantic (22,23).
3
The present work concerns the horse mackerel and its commercialisation as a
frozen product. The study is aimed to investigate the effect of a previous brine treatment
on the stability of horse mackerel lipids during the frozen storage. For it, different
salting degrees are checked and different lipid damage indices are employed to assess
the rancidity development during the frozen storage.
MATERIALS AND METHODS
Raw fish, sampling, processing and chemicals
Fresh horse mackerel (Trachurus trachurus) were obtained in August 2000. The
length of the horse mackerel included was in the range 18-24 cm; the weight was in the
range 250-280 g. Whole horse mackerel fishes were divided into four groups;
individuals of the first group were left unsalted and immediately frozen at –80ºC. The
three remaining groups were immersed in 5%, 10% and 20% NaCl aq. solutions,
respectively, in an isothermal room at 15ºC. The fish were removed after two hours and
frozen at –80ºC. After 24 hours at –80ºC, all fish samples (salt treated and untreated)
were placed at –20ºC. Analyses were carried out on the white muscle of raw material
and of fish that had been kept frozen at –20ºC during 75, 150, 210 and 270 days. For
each kind of treatment, three different fish batches were considered and studied
separately to achieve the statistical study.
Chemicals employed along the present work (solvents, reagents) were reagent
grade (E. Merck; Darmstadt, Germany); NaCl employed included a maximum content
on iron and copper of 0.0001 % and 0.0002 %, respectively.
4
Composition analyses
Water content was determined by weight difference between the homogenised
fish muscle (1-2 g) and after 24 hr at 105 C. Results are expressed as g water/100 g
muscle.
Lipids were extracted by the Bligh and Dyer (24) method. Quantification results
are expressed as g lipids/100 g muscle.
NaCl content in white fish muscle was calculated from the amount of chlorine
by boiling in HNO3 with excess of AgNO3, followed by titration with NH4SCN (25).
Results are expressed as g NaCl / 100 g muscle.
Lipid damage measurements
Free fatty acids (FFA) content was determined on the Bligh and Dyer (24)
extract by the Lowry and Tinsley (26) method based on complex formation with cupric
acetate-pyridine. Results are expressed as g FFA/100 g lipids.
Conjugated dienes (CD) formation was measured at 233 nm (27) on the Bligh
and Dyer (24) extract. The results are expressed according to the formula: CD = B x V /
w, where B is the absorbance reading at 233 nm, V denotes the volume (ml) and w is
the mass (mg) of the lipid extract measured.
Peroxide value (PV) expressed as meq oxygen/kg lipid was determined by the
ferric thiocyanate method (28) on the Bligh and Dyer (24) extract.
The thiobarbituric acid index (TBA-i) was determined according to Vyncke (29)
on a 5% trichloracetic acid extract of the fish muscle. Results are expressed as mg
malondialdehyde/kg fish sample.
5
Interaction compounds formation
Fluorescence formation (Perkin-Elmer LS 3B) at 327/415 nm and 393/463 nm
was studied as described earlier (30,31). The relative fluorescence (RF) was calculated
as follows: RF = F/Fst, where F is the fluorescence measured at each excitation/emission
pair, and Fst is the fluorescence intensity of a quinine sulphate solution (1 g/ml in 0.05
M H2SO4) at the corresponding wavelength. The fluorescence ratio (FR) was obtained
from the lipid extract (24) analysis, according to the following calculation: FR =
RF393/463nm / RF327/415nm.
Statistical analyses
Data from the different lipid damage measurements were subjected to the
ANOVA one-way method (p<0.05) and correlation analysis (32); comparison of means
was performed using a least-squares difference (LSD) method.
RESULTS AND DISCUSSION
Composition analyses
Water contents ranged between 75% and 79% in all samples (Table 1); no
differences were obtained as a result of salt treatment or frozen storage time.
Comparison with previous research showed a higher water content than fattier species
(30) and a lower content than lean fish species (31,33), according to an inverse ratio
between water and lipid matter (34).
6
Lipid contents ranged between 1.5% and 3.0% on wet basis (Table 1);
differences obtained could be explained as a result of lipid content variations among
individual fishes and not as a result of salt treatment or frozen storage time.
An increasing salt content was found in the horse mackerel white muscle,
according to an increase of the brine solution concentration employed in the pretreatment (7,8). Salt contents obtained in the fish muscle were as follows: 0.10%–0.15%
(untreated samples), 0.37%–0.45% (5% NaCl treated samples), 0.78%–0.87% (10%
NaCl treated samples) and 1.02%–1.08% (20% NaCl treated samples) (Table 1). For
fish samples corresponding to the same salt treatment, no differences were obtained for
the muscle salt content as a result of the frozen storage time.
Lipid hydrolysis
FFA content of the raw material (1.15 %) showed rather similar values to fatty
fish species (tuna, sardine) (30,35) and lower than lean fish (blue whiting, haddock,
cod) (31,33).
A gradual increase in free fatty acid formation was obtained for all kinds of
samples as a result of the frozen storage time (Table 2) according to previous research
(31,33). Up to 210 days, no significant differences could be assessed as a result of the
previous salt treatment. However, at 270 days a higher hydrolysis development could be
observed in untreated samples and those that had been immersed in 5% NaCl solution
than in the two other treatments. Accordingly, a decreasing effect of muscle salt content
could be inferred on free fatty acid formation at the end of the experiment.
7
Lipid oxidation
Different and complementary lipid oxidation indices were carried out to assess
the rancidity development in the different stages of the present experiment.
The effect of the frozen storage time on primary oxidation measured by the
conjugated diene formation showed (Table 2) a general increase from raw material (CD
= 0.78) till day 150, followed by a slight decrease at day 210 and no changes till the end
of the experiment. CD formation has already been reported to increase during the initial
steps of oxidation (36) and then CD levels decreased as a result of lipid hydroperoxides
breakdown (37,38). No significant differences could be assessed as a result of the salt
pre-treatment, so that no conclusions could be done about the effect of the muscle salt
content on this kind of primary oxidation products.
Peroxides formation (Table 2) showed a general increase from raw material (PV
= 1.51) till day 75, followed by little changes at day 150. Then, a sharp increase was
observed (day 210), that led in all cases to peroxide values above 15. At the end of the
experiment, a general decrease was observed, that could be explained as a result of
peroxides decomposition during this latest step (37,38). Comparison of the different
kinds of samples showed in most cases higher values for fish samples that had been
immersed in 20% NaCl solutions than in the three other conditions; a very high value
was obtained at day 210 for 20% NaCl treated samples.
Secondary lipid oxidation measured by the TBA-i provided a general and
gradual increase with the storage time for all fish samples (Table 2), according to
literature concerning fatty and lean fish species (30,31,39). A strong effect of the salt
presence could be concluded since 20% NaCl treated samples showed in most cases
higher TBA-i values than the other kinds of fish material. At the end of the experiment a
8
very high TBA-i value was obtained for samples that had been treated with 20% NaCl;
this was in accordance with the low PV in this sample after 270 days (Table 2).
Interaction compounds formation
Fluorescence formation (Table 2) expressed as FR showed an increase in all salt
treated samples at day 75 compared to raw material (FR = 0.71). Then, a general
increase could be assessed at day 210, followed by no changes at the end of the
experiment. At high salt concentrations an increasing effect of salt presence in muscle
on fluorescent compounds formation can be inferred since fish samples pre-treated with
20% NaCl solution showed in most cases higher FR values than the untreated samples.
Formation of fluorescent products as a result of interaction between lipid
oxidation compounds and protein like molecules has been reported to depend on lipid
oxidation products formation (peroxides and carbonyls, namely) (38,40,41). In the
present experiment, fluorescence formation developed stronger in the latest stages (210
and 270 days) than in previous steps, according to values obtained for peroxides and
thiobarbituric acid reactive substances (Table 2).
Correlation analyses
The different lipid damage indices were tested for linear correlation with the salt
content of the white muscle at each frozen storage time (Table 3).
In accordance with previous results (Table 2), the FFA and CD formation did
not led to good correlations with the salt content of the muscle along the whole
experiment. The CD value provided some satisfactory results at the beginning (75
days), when lipid damage was still relatively low (36). In the same way, FR only
9
provided accurate results at the beginning (75 days) and at the end of the experiment
(270 days), according to data on Figure 5.
The PV showed satisfactory results along the whole experiment. However, linear
correlation values were better in the beginning of the experiment (75 days), when
peroxide decomposition was still low (Table 2).
The TBA-i provided the best linear correlation values along the whole
experiment. Indeed, as long as frozen storage time and lipid damage increased (days
210 and 270), linear correlation values were better according to data on Table 2.
Non linear fittings (exponential and logarithmic) were also studied. As a whole,
little differences were obtained in the case of FFA, CD, TBA-i and FR. In the case of
PV, better correlation values were obtained by considering an exponential fitting.
CONCLUSIONS
In accordance with previous research on lean (31,33,42) and fatty (30,43) fish
species, horse mackerel has shown an important lipid hydrolysis and oxidation
development during the frozen storage. This lipid damage caused by the storage
conditions has been satisfactorily assessed by traditional chemical indices such as FFA,
PV, TBA-i and FR.
Results have shown a strong effect of NaCl content of fish muscle on the
rancidity development, according to the primary (PV) and secondary (TBA-i) oxidation
compounds produced during the frozen storage (Table 2). These results agree with
previous research carried out on fattier species such as sardine (8,13) and mackerel (44).
Sodium chloride has been reported to act as prooxidant by enhancement of the
10
prooxidant effect of chelatable iron ions (45) widely present in fish muscle, specially in
the dark one (15,34).
Present results on lipid hydrolysis do not show a clear tendency of FFA
formation caused by the salt content of white muscle, since relatively poor correlation
values could be found at each frozen storage time (Table 3). At the end of the
experiment, untreated samples and those that were immersed in 5% NaCl solution
showed higher (p<0.05) values than the two other kinds of samples (Figure 1). This
inverse relationship between salt content and FFA formation has been already
mentioned for fatty species such as sardine (8,13), mackerel (6) and salmon (46), so that
NaCl presence led to an advantage from a sensory point of view.
For the present medium-fat content species, in cases where salting pre-treatment
would be needed for further frozen commercialisation, employment of appropriate
antioxidant additions is recommended (2,47) to avoid a large lipid oxidation
development and thus, guarantee a longer shelf life when consumed as frozen product.
In such studies, chemical lipid oxidation indices should be employed in addition to
sensory properties so that a more complete view of changes produced can be obtained.
ACKNOWLEDGEMENTS
The authors acknowledge Mr. Marcos Trigo and Mrs. Janet Ares for technical
assistance and the Comisión Interministerial de Ciencia y Tecnología (CICyT) for
financial support through the research project ALI 99-0869 (2000-2002).
11
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14
TABLE 1
Results obtained on water, lipid and NaCl contents in fish muscle of the different
samples1
Frozen Storage Brine solution
Time (days)
employed
Raw material
Water content
Lipid content
NaCl content
(%)
(%)
(%)
78.2 (0.91)
1.58 (0.400)
0.11 (0.033)
75
0%
76.0 (0.20)
2.16 (0.387)
0.10 (0.086)
75
5%
76.9 (0.97)
1.80 (0.414)
0.42 (0.111)
75
10 %
75.4 (1.96)
2.42 (0.902)
0.80 (0.231)
75
20 %
77.2 (0.20)
1.56 (0.389)
1.07 (0.361)
150
0%
77.0 (1.56)
1.65 (0.491)
0.14 (0.074)
150
5%
76.4 (1.34)
2.60 (1.338)
0.44 (0.096)
150
10 %
77.5 (0.26)
1.54 (0.229)
0.78 (0.211)
150
20 %
77.1 (0.48)
1.89 (0.152)
1.05 (0.433)
210
0%
76.1 (1.95)
2.46 (1.115)
0.12 (0.102)
210
5%
77.1 (0.70)
1.91 (0.757)
0.37 (0.124)
210
10 %
76.6 (1.64)
2.22 (0.814)
0.86 (0.186)
210
20 %
76.4 (0.63)
2.27 (1.439)
1.02 (0.424)
270
0%
78.6 (1.30)
1.52 (0.629)
0.12 (0.066)
270
5%
76.1 (2.48)
2.16 (1.520)
0.40 (0.079)
270
10 %
76.8 (1.51)
2.57 (1.560)
0.81 (0.264)
270
20 %
77.3 (1.20)
2.02 (1.078)
1.05 (0.261)
15
1
Values are means of three independent determinations. Standard deviations are
expressed in brackets.
16
TABLE 2
Measurement of the lipid damage1 during the frozen storage of horse mackerel that was
previously immersed in a NaCl solution2
Storage time
and NaCl
treatment
Raw material
Day 75
Untreated
5% NaCl
10% NaCl
20% NaCl
Day 150
Untreated
5% NaCl
10% NaCl
20% NaCl
Day 210
Untreated
5% NaCl
10% NaCl
20% NaCl
Day 270
Untreated
5% NaCl
10% NaCl
20% NaCl
FFA
CD
PV
TBA-i
FR
1.15
0.78
1.51
0.12
0.71
3.57
3.29
3.21
2.69
a
a
a
a
0.90
0.97
1.05
0.98
a
a
a
a
5.61 a
4.73 a
7.64 ab
10.45 b
0.26 a
0.35 ab
0.36 ab
0.42 b
0.72 a
0.98 ab
0.98 ab
1.14 b
4.24
4.63
4.84
4.38
a
a
a
a
1.47
1.38
1.45
1.32
a
a
a
a
8.03 a
5.27 ab
11.68 b
10.77 b
0.26 a
0.27 a
0.37 ab
0.54 b
0.87
0.83
0.86
1.06
4.60
5.32
4.96
5.06
a
a
a
a
1.21
1.31
1.18
1.06
a
a
a
a
18.91
20.66
21.23
28.72
a
a
a
b
0.42
0.47
0.70
0.77
a
a
b
b
1.09 a
1.22 ab
1.16 ab
1.36 b
7.80
7.75
6.27
6.73
b
b
a
a
1.31
1.20
1.17
1.23
a
a
a
a
10.47 ab
8.15 a
13.58 ab
17.29 b
0.68
0.75
0.87
1.39
a
a
a
b
1.10 a
1.16 a
1.21 ab
1.36 b
a
a
a
a
1
Abbreviations: FFA (free fatty acids), CD (conjugated dienes), PV (peroxide value),
TBA-i (thiobarbituric acid index), FR (fluorescence ratio).
2
Values are means of three independent determinations. Values in the same column and
for the same frozen time followed by different letters are significantly different
(p <0.05).
17
TABLE 3
Linear correlation values between the NaCl content of the white muscle and values
obtained for the different lipid damage indices1 at each frozen storage time2
Frozen Storage Time (days)
―——————————————————————————
Lipid damage
index
FFA
CD
PV
TBA-i
FR
75
150
─ 0.36
(─ 0.41; ─ 0.32)
0.60
(0.48; 0.67)
0.86
(0.90; 0.82)
0.68
(0.62; 0.73)
0.68
(0.57; 0.73)
0.23
(0.13; 0.28)
─ 0.20
(─ 0.16; 0.21)
0.51
(0.57; 0.47)
0.69
(0.72; 0.64)
0.34
(0.39; 0.30)
210
270
0.10
─ 0.44
(0.07; 0.13)
(─ 0.47; ─ 0.44)
─ 0.30
─ 0.57
(─ 0.34; ─ 0.25) (─ 0.49; ─ 0.62)
0.54
0.62
(0.60; 0.49)
(0.72; 0.55)
0.90
0.85
(0.88; 0.89)
(0.87; 0.82)
0.40
0.62
(0.48; 0.36)
(0.64; 0.60)
1
Abbreviations as specified in Table 2.
2
Results in parentheses correspond to non linear fittings (exponential and logarithmic,
respectively). Significant values (p<0.05) are in bold print.
18
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