Accepted Manuscript
Effects of temperature during frozen storage on lipid deterioration of saithe (Pollachius virens) and hoki (Macruronus novaezelandiae) muscles Magnea G. Karlsdottir, Kolbrun Sveinsdottir, Hordur G. Kristinsson, Dominique Villot, Brian D. Craft, Sigurjon Arason
PII: S0308-8146(14)00145-9
DOI: http://dx.doi.org/10.1016/j.foodchem.2014.01.113
Reference: FOCH 15338
To appear in: Food Chemistry Received Date: 1 November 2013 Revised Date: 7 January 2014 Accepted Date: 28 January 2014
Please cite this article as: Karlsdottir, M.G., Sveinsdottir, K., Kristinsson, H.G., Villot, D., Craft, B.D., Arason, S., Effects of temperature during frozen storage on lipid deterioration of saithe (Pollachius virens) and hoki (Macruronus novaezelandiae) muscles, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.
2014.01.113
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1 Effects of temperature during frozen storage on lipid deterioration of saithe (Pollachius 1
virens) and hoki (Macruronus novaezelandiae) muscles 2
3
(Abbreviated running title: Lipid deterioration of lean fish species during frozen storage) 4
5
Authors: Magnea G. Karlsdottira,b*, Kolbrun Sveinsdottira, Hordur G. Kristinssona,c, 6
Dominique Villotd, Brian D. Crafte, and Sigurjon Arasona,b 7
8
aMatis ohf. Icelandic Food and Biotech R&D, Biotechnology and Biomolecules, Vinlandsleid 9
12, IS-113 Reykjavík, Iceland.
10
bUniversity of Iceland, Department of Food Science, Vinlandsleid 12, IS-113 Reykjavík, 11
Iceland.
12
cDepartment of Food Science and Human Nutrition, University of Florida, 359 FSHN 13
Building, Newell Drive, Gainesville, FL 32611, USA.
14
dNestlé R&D, 5750 Harper Road, 44139, Solon, Ohio, USA.
15
eNestlé Research Center, Route du Jorat 57, 1000 Lausanne 26, Switzerland.
16 17
Corresponding author: Magnea G. Karlsdottir, Matis ohf., Icelandic Food and Biotech R&D, 18
Biotechnology and Biomolecules, Vinlandsleid 12, IS-113 Reykjavík, Iceland. Phone: +354 19
422 5088, e-mail: [email protected] 20
21 22 23 24 25
2 ABSTRACT
26
Lipid deterioration of two lean fish species, saithe (Pollachius virens) and hoki (Macruronus 27
novaezelandiae), during frozen storage at -20 and -30°C (up to 18 months) was studied. Lipid 28
composition, lipid oxidation and hydrolysis, and sensory attributes were evaluated on both 29
light and dark muscles of the fish species. Results showed significant lipid deterioration with 30
extended storage time, but lower storage temperature showed significantly more preservative 31
effects. A marked difference was observed between the composition of dark muscle of hoki 32
and saithe. Polyunsaturated fatty acids were the predominant lipids in dark muscle of saithe, 33
while monounsaturated fatty acids were predominant in dark muscle of hoki. Further, the 34
hydrolytic activity differed greatly between dark muscle of hoki and saithe, with significantly 35
lower activity observed in hoki. Present results indicate that both tertiary lipid oxidation and 36
hydrolysis products are appropriate for assessing lipid deterioration of saithe and hoki light 37
muscle during frozen storage.
38 39
Keywords: Frozen lean fish, lipid oxidation, lipid hydrolysis, sensory 40
41
3 42
1. Introduction 43
Although freezing is an effective method of preserving foods, some deterioration of food 44
quality occurs during frozen storage. The quality of frozen fish products during storage can be 45
influenced by several factors such as fish species, the biological status of fish at catch, 46
handling on-board, temperature and storage time before freezing, freezing rate, frozen storage 47
temperature, temperature fluctuations, thawing procedure and protection from light and 48
oxygen. (Pigott & Tucker 1987; Sörensen, Brataas, Nyvold & Lauritzsen 1995; Erikson, 49
Sigholt, Rustad, Einarsdottir & Jørgensen 1999). Marine lipids are natural and good sources 50
of polyunsaturated omega-3 fatty acids (PUFA), such as eicosapentaenoic acid (EPA, C20:5n- 51
3) and docosahexaenoic acid (DHA, C22:6n-3), which have been reported to have beneficial 52
health effects to consumers. However, due to the high amount of PUFA (Shewfelt 1981), 53
along with highly active pro-oxidants contained (Hultin 1994), marine lipids are highly 54
susceptible to oxidation. Lipid oxidation is therefore one of the primary causes of 55
deterioration of fish muscle during storage and can negatively affect colour (Wasasundara &
56
Shahidi 1994), odour and flavour (Bateman, Hughs & Morris 1953), protein functionality and 57
conformation (Gutteridge 1988) and overall nutritional content of fish muscle (Pearson, Gray, 58
Wolzak & Horenstein 1983).
59
Lipid oxidation is generally not regarded as a large problem in lean fish species due to 60
low lipid contents in the muscle and much less dark muscle than fatty fish species. Therefore, 61
past investigations on quality changes of lean fish species during frozen storage has mainly 62
focused on changing the physical properties of the muscles and of sensory acceptance.
63
However, lipid oxidation and hydrolysis has been shown to occur and become an important 64
factor of lean fish approval in the recent past (Dulavik, Sörensen, Barstad, Horvli & Olsen 65
1998; Aubourg & Medina 1999; Roldán, Roura, Montecchia, Borla & Crupkin 2005;
66
4 Aubourg, Lago, Sayar & González 2007). Several methods have been developed to measure 67
different compounds as they form or degrade during lipid oxidation such as peroxides and 68
carbonyl compounds. Many of these compounds are unstable and are often affected by the 69
presence of pro-oxidants, antioxidants and the availability of oxygen among other factors.
70
Lipid hydroperoxides and their decomposition products such as aldehydes (e.g.
71
malondialdehyde) can interact further with proteins, phospholipids and nucleic acids resulting 72
in formation of fluorescent compounds, or so-called ‘tertiary oxidation products.’ These 73
compounds can be followed by fluorescence spectroscopy and such methods have been 74
demonstrated to effectively monitor lipid oxidation in biological materials such as fish (Lubis 75
& Buckle 1990; Aubourg, et al. 2007).
76
In the present study, lipid deterioration during frozen storage of two lean fish species 77
(saithe and hoki) was studied. The effects of storage time and temperature on sensory 78
properties, lipid oxidation and hydrolysis were analysed. A comparison between light and 79
dark muscle of the two species was performed. One of the main objectives of the study was to 80
investigate how two lean fish species, belonging to different families (Merlucciidae and 81
Gadidae), with similar type of commercial utilisation, differ in oxidative stability during 82
prolonged frozen storage. Furthermore, the various lipid quality indices measured in the study 83
were evaluated with specific emphasis on their capability to monitor fish decomposition 84
across the two species.
85 86
2. Materials and methods 87
2.1 Chemicals 88
All chemicals used in this study were of analytical grade and were purchased from Sigma- 89
Aldrich (St. Louis, MO, USA), Fluka (Buchs, Switzerland) or Sigma-Aldrich (Steinheim, 90
Germany).
91
5 92
2.2 Raw material, processing and sampling 93
Commercially available frozen blocks of saithe (Pollachius virens) and hoki (Macruronus 94
novaezelandiae) used for this study were provided by Nestec. The raw material of the saithe 95
and hoki used herein was caught in the Northeast Atlantic Ocean (FAO n°27) and Southwest 96
Pacific Ocean (FAO n°81), respectively, in March 2010. Upon arrival to the laboratory, the 97
frozen fish blocks were divided into four equal sized pieces, packed into waxed carton boxes 98
and distributed into two different storage containers set at -20 °C and -30 °C, and stored there 99
for up to 18 months. Experimental analysis was performed after 0, 3, 6, 12 and 18 months of 100
frozen storage. Prior to analysis, samples were thawed at refrigerated temperatures (4 ±1 °C) 101
for 24 hours. All analyses were performed separately on the light and the dark muscle of both 102
the hoki and the saithe. Six replicates (n=6) per sample were used for all chemical analyses 103
due to the potentiality for sample variation in the fish blocks. Any deviation from this 104
protocol is included below in the methods description.
105 106
2.3 Water and lipid content 107
Water content was determined by difference in weight of the homogenised muscle samples 108
before and after drying for 4 h at 102 to 104 °C (ISO 1993a). Results were calculated as g 109
water/100 g muscle. Total lipids (TL) were extracted from 25 g samples (80±1% water) with 110
methanol/chloroform/0.88 % KCl(aq) (at 1/1/0.5, v/v/v) according to the Bligh & Dyer (1959) 111
method. The lipid content was determined gravimetrically and the results were expressed as 112
grams lipid/100 g wet muscle.
113 114
2.4 Phospholipid content 115
6 Phospholipid content (PL) of the fish muscle was determined on the TL extracts by using a 116
colourimetric method (Stewart 1980) based on complex formation of phospholipids and 117
ammonium ferrothiocynate, followed by reading the absorbance of resultant solution at 488 118
nm (UV-1800 spectrophotometer, Shimadzu, Kyoto, Japan). A standard curve was prepared 119
with phosphatidylcholine in chloroform (5-50 µ g/ml) and results were expressed as a 120
percentage of the total lipid content.
121 122
2.5 Fatty acid profile 123
The fatty acid composition of the TL extracts was determined by gas chromatography of fatty 124
acid methyl esters (FAMEs). The methylation of fatty acids was carried out according to 125
AOCS (1998). The TL extract was vaporised at 55 °C under nitrogen to a constant weight. 70 126
mg of extracted lipids was dissolved in 1.5 ml 0.5N NaOH in methanol, and incubated for 7 127
min at 100 °C. After cooling to room temperature, 2 ml of BCl3/MeOH (12% boron 128
trichloride) were added and the samples incubated again for 30 min at 100 °C. After 129
subsequent cooling, 1 ml of internal standard (1 mg/ml of 23:0 methyl ester in isooctane) and 130
5 ml of concentrated NaCl solution were added. After phase separation, the isooctane layer 131
was transferred into a glass tube containing 1 mm bed of anhydrous Na2SO4. This was 132
repeated again with 1 ml of clean isooctane. The combined isooctane layers, containing the 133
FAMEs, were then transferred to GC vials. The FAMEs were separated on a Varian 3900 GC 134
equipped with a fused silica capillary column (HP-88, 100 m x 0.25 mm x 0.20 µm film, 135
Agilent Technologies), split injector and flame ionisation detector fitted with Galaxie 136
Chromatography Data System (Version 1.9.3.2 software). The oven ramp was programmed as 137
follows: 100 °C for 4 min, then increased to 240 °C at 3 °C/min and held at this temperature 138
for 15 min. The injector and detector temperature were 225 °C and 285 °C, respectively.
139
Helium was used as the carrier gas and the column flow rate was 0.8 ml/min; with a split ratio 140
7 of 200:1. This programme was based on the AOAC 996.06 (2001). Results were expressed as 141
percentage of total lipids. This analysis was performed on four replicates (n=4) per sample.
142 143
2.6 Free fatty acids evaluation 144
Free fatty acid (FFA) content was determined on the TL extracts according to Lowry &
145
Tinsley (1976), with modifications from Bernardez, Pastoriza, Sampedro, Herrera & Cabo 146
(2005). The FFA concentration was calculated as µM quantities of oleic acid based on a 147
standard curve spanning a 2-22 µ mol range. Results were expressed as grams FFA / 100 g of 148
total lipids.
149 150
2.7 Lipid oxidation measurements 151
A modified method of Lemon (1975) was used for measuring thiobarbituric acid reactive 152
substance (TBARS). A fish muscle sample (5.0 g) was homogenised with 10.0 ml of 153
trichloroacetic acid (TCA) extraction solution (7.5% TCA, 0.1% propyl gallate and 0.1%
154
EDTA mixture prepared in ultra-pure water) using a homogeniser at maximum speed for 10 155
seconds (Ultra-Turrax T-25 basic, IKA, Germany). The homogenised samples were then 156
centrifuged at 5100 rpm for 20 min (TJ-25 Centrifuge, Beckmann Coulter, USA). Supernatant 157
(0.5 ml) was collected and mixed with an equal volume of thiobarbituric acid (0.02 M) and 158
heated in a water bath at 95 °C for 40 min. The samples were cooled down on ice and 159
immediately loaded into a 96-well microplate reader (NUNC A/S Thermo Fisher Scientific, 160
Roskilde, Denmark) and the absorbance measured at 530 nm (Tecan Sunrise, Austria). A 161
standard curve was prepared using tetraethoxypropane. The results were expressed as µmol of 162
malonaldehyde diethylacetal / kg of wet muscle.
163
Lipid hydroperoxides (PV) were determined with a modified version of the ferric 164
thiocyanate method (Santha & Decker Eric 1994). Total lipids were extracted from 5.0 g of 165
8 samples with 10 ml ice-cold chloroform:methanol (1:1) solution, containing 500 ppm BHT to 166
prevent further peroxidation during the extraction process. Sodium chloride (0.5 M) was 167
added (5.0 ml) in to the mixture and homogenised for 30 sec before centrifugation at 5100 168
rpm for 5 min (TJ-25 Centrifuge, Beckmann Coulter, USA). The chloroform layer was 169
collected (500 µL) and matched with 500 µL chloroform:methanol solution. A total amount 170
of 5 µL of ammonium thiocyanate (4 M) and ferrous chloride (8 mM) mixture (1:1) was 171
finally added. The samples were then brought to room temperature for 10 min and read at 500 172
nm on a spectrometer (Tecan Sunrise, Austria). A standard curve was prepared using cumene 173
hydroperoxides. The results were expressed as mmol lipid hydroperoxides / kg of wet muscle.
174
Formation of fluorescent compounds (OFR) was determined with a Perkin Elmer LS 175
50B fluorescent spectrometer by absorbance measurements at 393/463 and 327/415 nm 176
excitation/emission maxima according to other researchers (Aubourg & Medina 1999). The 177
relative fluorescence (RF) was calculated as RF=F/Fst, where F is the sample fluorescence 178
intensity at each excitation/emission maximum and Fst is the fluorescence intensity of a 179
quinine sulphate solution (1 µg/ml in 0.05M H2SO4) at the corresponding wavelength. The 180
fluorescence shift (OFR) was calculated as the ratio between the two RF values, i.e. OFR 181
=RF393/463nm / RF327/415nm, and was analysed on the organic phase resulting from the lipid 182
extraction previously described (Bligh & Dyer 1959).
183 184
2.8 Sensory evaluation 185
Generic descriptive analysis (GDA) (Stone & Sidel 2004) was used to assess cooked samples 186
of the two lean fish species. Ten panellists, all trained according to international standards 187
(ISO 1993b); including detection and recognition of tastes and odours, trained in the use of 188
scales and in the development and use of descriptors, participated in the sensory evaluation.
189
The panel was trained in recognition of sensory characteristics of the samples and describing 190
9 the intensity of each attribute for a given sample using an unstructured scale (from 0 to 191
100%). Thirty sensory attributes for appearance (3), odour (10), flavour (9) and texture (8) 192
were evaluated using an unstructured scale (0–100%) based on Sveinsdottir, et al. (2009).
193
Most of the attributes were analysed for the light and the dark muscles together, but some 194
were evaluated separately for the dark and light muscles. These attributes were: colour (dark 195
and light muscle); frozen storage flavour (light muscle) and rancid flavour (dark muscle).
196
The blocks were cut into equal sample portions (~40 g pieces) and cooked for 5 197
minutes in a pre-warmed oven (Convotherm Elektrogeräte GmbH, Eglfing, Germany) at 95- 198
100 °C with air circulation and steam, and then served to the panel (the serving temperature 199
were 65-75 °C). Each panellist evaluated duplicates of each sample in a random order per 200
session (4 samples for each session). A computerised system (FIZZ, Version 2.0, 1994-2000, 201
Biosystèmes) was used for data recording. GDA data was corrected for level effects (effects 202
caused by level differences between assessors and replicates) by the method of Thybo &
203
Martens (2000).
204 205
2.9 Data analysis and correlations 206
Statistical analyses were performed in Microsoft Office Excel 2010 (Microsoft Inc, Redmond, 207
Wash., U.S.A.) and SigmaStat 3.5 (Dundas Software Ltd., GmbH, Germany). One way 208
ANOVA, Duncan´s test and Pearson´s correlation were performed on the means of the values 209
obtained. The significance level cut-off was set at 95% (p < 0.05).
210 211
3. Results and discussions 212
3.1 Chemical composition 213
The water content of saithe dark and light muscle was similar, ranging from 79.7-81.0%. The 214
dark muscle of hoki had significantly lower (74.2-75.3%) water content than its light 215
10 counterpart (79.8-81.6%). No significant changes in water content were observed during the 216
frozen storage for both species. In average, total lipids (TL) in the light and dark muscle of 217
saithe were determined to be 0.6±0.1% and 1.1±0.1%, respectively. Corresponding values for 218
hoki light and dark muscles were 0.6±0.1% and 7.6±2.3%, respectively. The total lipid 219
contents of the light muscle of both hoki and saithe is in agreement with previously reported 220
values for white fish species (Huss 1995). For the dark muscle, however, the present results 221
were considerably lower compared with previously reported values, which showed saithe 222
lipids at 5.5% (Dulavik, et al. 1998) and hoki at 20-30% (MacDonald, Hall & Vlieg 2002), 223
respectively. It is important to note, however, that the study of MacDonald, et al. (2002) 224
showed very wide seasonal fluctuation and extremely high variation between individual fish 225
with the lipid content of dark muscle varying from 1.9% to 53.7%. This may explain the 226
discrepancy observed in total lipids contents of dark muscle shown above.
227
In the initial raw material, phospholipids were the major component of the total lipids 228
in both light and dark muscle of saithe (60.2±4.0%, 40.1±6.0% respectively) and in light 229
muscle of hoki (50.1±6.1%). The ratio of phospholipids was much lower in the dark muscle 230
of hoki or 5.0±1.2% of the total lipids. This low ratio of phospholipids may indicate that the 231
majority of the lipids in the dark muscle of hoki are bound to triacylglycerols. During frozen 232
storage, a significant loss of phospholipid content occurred in all samples (data is not shown).
233
Phospholipid content was affected by storage duration and temperature. After 18 months of 234
frozen storage at -20 °C, light muscle from hoki and saithe showed 89.1% and 88.2% loss in 235
phospholipid content, respectively, compared with initial values. Corresponding losses for 236
samples stored at -30 °C were 63.3% and 39.5%, respectively. Similar behaviour was found 237
for dark muscle where higher loss of phospholipids was observed for samples stored at -20 °C 238
compared with -30 °C. The decrease in phospholipid content correlated significantly with 239
storage time for both saithe and hoki light muscles (r= -0.86 and r = -0.86, respectively). The 240
11 present results indicate that phospholipids are highly affected by storage time and 241
temperature.
242 243
3.2. Fatty acid profile 244
The fatty acid composition of saithe and hoki were rather similar in MUFA/PUFA/SFA 245
levels, with the exception of the hoki dark muscle (Table 1). The main difference between the 246
dark and light muscle of hoki was the higher amount of monounsaturated fatty acids (MUFA) 247
(40.1>20.2%) and lower amount of polyunsaturated fatty acids (PUFA) (30.1<51.5%) in the 248
dark and light muscles, respectively. In saithe and hoki, both dark and light muscles, 249
unsaturated fatty acids (MUFA and PUFA) were more abundant than saturated fatty acids 250
(SFA). Among SFAs, palmitic acid (C16:0) was largely predominant in both species and 251
muscles, followed by stearic acid (C18:0). The amount of palmitic acid was significantly 252
higher in the light muscle compared to the dark muscle for both species tested. No significant 253
change in palmitic acid content was detected during the storage time for all samples. Stearic 254
acid contents were significantly higher in the light muscle of hoki compared with the dark 255
muscle. After 18 months of frozen storage the amount of stearic acid increased slightly, but 256
significantly, in hoki and saithe light muscle and a higher increase was observed at -20 °C 257
compared to -30 °C.
258
Among the MUFAs, oleic acid (C18:1n9) was the most predominant fatty acid, 259
followed by eicosanoic acid (C20:1n9) for both hoki and saithe. The MUFAs were at 260
significantly higher level in the dark muscles compared to the light muscle for both saithe 261
(20.7% and 15.1%, respectively) and hoki (40.1% and 20.2%, respectively). Hoki dark muscle 262
had significantly higher amounts of MUFAs compared with the other sample groupings. A 263
slight increase in oleic acid was observed in hoki dark muscle after 18 months storage at -20 264
12
°C, but no significant changes were detected among the MUFAs content during the storage 265
period for the sample groupings.
266
The proportion of PUFAs was higher in saithe lipids compared to hoki regardless of 267
muscle type. Further, the proportion of PUFAs was significantly higher in light muscle when 268
compared to the dark muscle (p<0.05) across the two fish species. Hoki dark muscle was 269
especially low in PUFAs compared to all the other sample groupings. In terms of total PUFA 270
contents, the following order was found: saithe light muscle (59.1%) > saithe dark muscle 271
(55.0%) > hoki light muscle (51.5%) >> hoki dark muscle (30.1%). The highest quantity of 272
PUFA in both species was associated with n-3 compounds, with docosahexaenoic acid (DHA, 273
C22:6n-3) being the most abundant followed by eicosapentaenoic acid (EPA, C20:5n-3).
274
DHA was similar in all the sample groupings, except the hoki dark muscle, in which it was 275
markedly lower than the other samples. EPA contents were much higher in saithe compared to 276
hoki regardless of muscle type. Also, the amount of EPA was significantly higher in saithe 277
light muscle compared with dark muscle (p<0.05).
278
A definite loss of long-chain n-3 PUFA was observed in both saithe and hoki, light 279
and dark muscle after 18 months of frozen storage, with greater losses observed in the 280
samples stored at -20 °C. The amount of DHA and EPA in both muscle types decreased with 281
longer storage time (data not shown). DHA was especially unstable in all the samples tested 282
and higher losses were observed for the higher storage temperature (-20 °C). Both the saithe 283
light and dark muscle showed significantly higher losses (p<0.05) of DHA compared to the 284
samples stored at -20 °C. The study of Dulavik et al. (1998) showed a rather small decrease of 285
DHA in saithe light muscle, while DHA in the dark muscle was more easily destroyed.
286
Similarly, Xing, Yoo, Kelleher, Nawar & Hultin (1993) did not observe any changes in the 287
fatty acid composition of cod muscle after storage at -20 °C for 32 weeks. Since the material 288
used in the present study was frozen as a block, the loss of the n-3 PUFA observed in both the 289
13 light and dark muscle could be attributed to the orientation of the fillets within the frozen fish 290
blocks. In general, the frozen fish blocks tested herein came with the fillets oriented with the 291
dark muscle inward and the light muscle outward. Further, it is worth mentioning that the 292
content of total fatty acids was not significantly affected during the storage period.
293 294
3.3. Lipid deterioration 295
Lipid hydroperoxides data for all the sample groupings was summarised and appears in 296
Figure 1. The formation of peroxides in the fish fillets tested appears to be strongly species 297
dependant. Saithe (Figure 1A) was very stable during the first 12 months of storage 298
regardless to storage temperature (p>0.05). After 18 months of frozen storage, however, a 299
slight, yet significant, increase of peroxide was observed in both muscle types. Hydroperoxide 300
formation was slightly more pronounced in the samples stored at -20 °C compared to -30 °C 301
(p<0.05). In comparison to saithe, the hoki muscles (Figure 1B) showed a much more 302
pronounced and more progressive peroxide formation over time at both storage temperatures.
303
After 18 months of storage, a sharp increase of peroxides was observed with considerably 304
higher levels attained in the samples stored at -20 °C compared with their counterparts stored 305
at -30 °C (p<0.05). Hoki dark muscle stored at -20 °C showed a near linear increase of 306
peroxides over the storage period (r = 0.87). The dark muscle of hoki proved to be much more 307
prone to lipid oxidation when compared with its light muscle at both storage temperatures.
308
This behaviour in frozen storage is similar to that obtained from a fatty fish species 309
(Undeland, Stading & Lingnert 1998).
310
The marked difference in peroxide formation between hoki light and dark muscle is 311
likely due to the considerably higher lipid contents in the dark muscle (recall from above 312
7.6±2.3% vs. 0.6±0.1% in the light muscle). This may also be why the saithe appears to be 313
more durable over frozen storage when compared to the hoki; its total lipids for light and dark 314
14 muscle were 0.6±0.1% and 1.1±0.1, respectively. The presence of active pro-oxidants such as 315
hemoglobin (Richards & Hultin 2002) could also explain the high peroxide value in the dark 316
muscle of hoki, since bleeding on-board is not common practice for this fish species. The 317
amount of haem iron content of the initial material was evaluated in the framework of a 318
parallel study and was six-fold higher in the hoki dark muscle compared to the light muscle.
319
Haem iron content in the saithe light and dark muscle was similar to the levels contained in 320
the hoki light muscle (data not shown).
321
The results for TBARS analysis, a marker for the decomposition of secondary lipid 322
oxidation products, appear also in Figure 1. TBARS results for both the saithe dark and light 323
muscle (Figure 1C) showed low/no formation of secondary oxidation products up to month 6, 324
followed by a sharp increase up to month 12, after which only the dark muscles values 325
continued to increase. A significantly higher (p<0.05) increase of TBARS formation appeared 326
in the samples stored at -20 °C, for both muscle types, indicating a protective effect for the 327
samples stored at -30 °C. The study by Dulavik, et al. (1998) on saithe dark and light muscle 328
showed similar results where more aggressive oxidation formation was obtained at a higher 329
frozen storage temperature. Results for the hoki light muscle samples stored at -20 and -30 °C 330
(Figure 1D) showed little formation of TBARS over the storage term. The hoki dark muscle 331
samples, however, showed a noticeable bloom of TBARS at 3 and 6 months, for the samples 332
stored at -20 and -30 °C, respectively. These results are in-line with the peroxide results for 333
the hoki dark muscle samples (Figure 1B).
334
Tertiary lipid oxidation products were investigated by means of fluorescent properties 335
in the organic phases (OFR) resulting from the Bligh and Dyer (1959) extraction (results are 336
summarised in Figure 2). Of particular notice in the OFR results is the behaviour of the hoki 337
dark muscle (Figure 2B), which is relatively flat over the entire storage period. This could 338
potentially be due to the aforementioned difference in chemical composition of the lipids in 339
15 hoki dark muscle (triglyceride-bound), compared to the three other sample types 340
(phospholipid-bound). According to other lipid researchers (Frankel 1998), lipid-soluble 341
fluorescence compounds are believed to be produced from oxidised phospholipids, or from 342
oxidised fatty acid esters in the presence of phospholipids. In contrast to the dark muscle, the 343
light muscle of hoki showed a markedly higher OFR as well as a higher increase in the sample 344
stored at the higher storage temperature -20 °C. Comparable OFR results were obtained for 345
the saithe light and dark muscle (Figure 2A) as for hoki light muscle. A similar finding on the 346
preservative effect of storage temperature during frozen storage have been reported in the 347
literature (Dulavik, et al. 1998; Aubourg, et al. 2007).
348
The evolution of free fatty acid (FFA) in the stored fish samples over the 18 months 349
period were summarised and appear also in Figure 2. As with the OFR data, the FFA data 350
followed a similar overall pattern between sample types with the hoki dark muscle (Figure 351
2D) standing apart. Unlike the hoki dark muscle, the hoki light muscle as well as the saithe 352
light and dark muscle (Figure 2C) showed a steady increase in FFA content over the entire 353
storage period. The observed difference between the hoki dark muscle and the other three 354
sample types could be explained by the different lipid composition contained. Recall only 355
around 5% of the total lipids of the hoki dark muscle were comprised of phospholipids, 356
whereas the hoki light muscle and the saithe light and dark muscle contain from 40-60%
357
phospholipids. It has been suggested in the literature (Hardy, McGill & Gunstone 1979) that a 358
decrease in phospholipid content during frozen storage of lean fish is the main factor driving 359
the accumulation of FFA. The present results indicate that the muscle type with the lowest 360
phospholipid ratio (hoki dark muscle) shows the lowest hydrolytic activity. Higher enzymatic 361
activity in the light muscle could also contribute to this difference since FFAs have been 362
reported to be widely produced during frozen storage as a result of enzyme catalyst (Shewfelt 363
1981; Aubourg & Medina 1999). In parallel with the OFR data, the FFA data exhibited a 364
16 partial protective affect with the samples stored at the lower storage temperature -30 °C, for 365
all the samples tested. According to other research carried out on frozen lean fish species 366
(Aubourg & Medina 1999; Aubourg, Rey-Mansilla & Sotelo 1999), formation of FFA has 367
been shown to be sensitive to both storage time and temperature. Further, FFA accumulation 368
has been correlated with lack of acceptability of frozen fish since FFA are known to cause 369
texture deterioration by interacting with proteins (Mackie 1993).
370 371
3.4. Sensory evaluation and correlation analyses 372
Generic descriptive analysis (GDA) results for the saithe and hoki, light and dark muscles, 373
over the storage period were summarised and appear in Figure 3. As the GDA attributes that 374
relate most to lipid degradation are ‘rancid’ flavour and order as well as ‘frozen storage’
375
flavour and odour, these were the only attributes presented. As aforementioned, the frozen 376
storage and rancid flavour were evaluated separately on the light and dark muscle, 377
respectively. An average GDA score above 20 on scale from 0 to 100, indicates that the 378
samples are becoming rancid (Sveinsdóttir, Martinsdottir, Hyldig, Jorgensen & Kristbergsson 379
2002). As seen in Figure 3, both the saithe (Figure 3B) and the hoki (Figure 3D) dark 380
muscle samples stored at -20 °C were significantly (p<0.05) more rancid in flavour and odour 381
after the 18 months period than their respective samples stored at -30 °C. Similar results were 382
obtained for the frozen storage flavour and odour attributes for the both saithe light (Figure 383
3A) and hoki light (Figure 3C). These results could to some extent be related to protein 384
denaturation and the formation of dimethylamine and formaldehyde in the light muscle during 385
frozen storage (Huss 1995). It is important to note that the hoki light and dark samples were 386
extremely rancid after 18 months of storage and their tasting was rejected by the sensory 387
panel (i.e. as seen in the lack of a bar for f-frozen and f-rancid at 18 months in Figure 3C and 388
17 3D, respectively). These results are in agreement with the chemical lipid oxidation data 389
summarised above.
390
Sensory descriptive analysis with a trained panel is often regarded as the most 391
powerful tool to monitor lipid oxidation (Broadbent & Pike 2003; Timm Heinrich, Xu, 392
Nielsen & Jacobsen 2003), but such analyses are often quite costly and time consuming. It is 393
therefore of great interest to discover chemical markers that correlate well with the results of 394
sensory evaluations. Therefore, in the present study, correlation analyses were performed 395
between the sensory data and the physicochemical data obtained in order to determine if such 396
markers exist for the fish muscles assayed (see Table 2 and Table 3). For the saithe light and 397
dark samples, strong significant (p<0.05) correlations with frozen storage odour were 398
obtained for storage time (r = 0.94 for both), phospholipid decomposition (r = -0.92 and - 399
0.77), FFA (r = 0.88 and 0.89), OFR (r = 0.68 and 0.78), and TBARS (r = 0.88 and 0.94).
400
Significant relationships were also found for the rancid odour attributes with saithe light and 401
dark muscle samples being correlated with storage time (r = 0.68 for both), FFA (r = 0.74 and 402
0.78), OFR (r = 0.69 and 0.78), and peroxides (r = 0.72 and 0.74). Rancid flavour (dark 403
muscle) and frozen storage flavour (light muscle) attributes shared significant relationships 404
across storage time (r = 0.77 and 0.79), phospholipid decomposition (r = -0.74 and -0.76), 405
FFA (r = 0.84 and 0.78) and OFR (r = 0.80 and 0.79).
406
The results of the correlations generated between chemical and sensory data for the 407
hoki samples tested appear in Table 3. In hoki light and dark samples, frozen storage odour 408
attributes were significantly (p<0.05) correlated with storage time (r = 0.71 for both), and 409
phospholipids decomposition (r = -0.76 and -0.69). Rancid odour attributes were significantly 410
(p<0.05) correlated with storage time (r = 0.85 for both), phospholipids content (r = -0.85 and 411
-0.86), OFR (r = 0.75 and 0.79), and PV (r = 0.69 and 0.70). Rancid flavour (dark muscle) and 412
frozen storage flavour (light muscle) attributes did not share correlations across any of the 413
18 parameters tested (i.e. opposite case with the saithe samples). This is logical as the light and 414
dark muscles of hoki were clearly made up of very different material as described above.
415
Rancid flavour (dark muscle) attributes of hoki were significantly (p<0.05) correlated with 416
storage time (r = 0.72) and phospholipids (r = -0.70), whereas frozen storage flavour (light 417
muscle) was correlated with FFA (r = 0.93) and OFR (r = 0.87).
418
Based on the present results, no clear chemical test is consistently correlated to both 419
frozen and rancid, flavour and odour attributes across the two species evaluated. It can be 420
noted, however, that use of FFA and OFR in combination does have the potential to predict 421
many of the selected sensory attributes of frozen saithe and hoki. FFA and OFR increase in 422
saithe samples showed a strong correlation with all the selected sensory attributes tested, 423
whereas they were only significant correlated with frozen flavour in the hoki samples. This 424
difference between hoki and saithe can be explained by the different nature of the hoki dark 425
muscle and the low formation of FFA and OFR therein over the storage period. Good 426
correlations have also been obtained in the literature between the sensory acceptance criteria 427
of frozen horse mackerel and FFA / OFR data (Aubourg, Pineiro & González 2004), giving 428
credence to such an approach.
429 430
4. Conclusions 431
The present study demonstrated a beneficial effect of long term frozen storage of fish fillets at 432
-30 °C vs. -20 °C based on the preservation of phospholipids as well as the reduced formation 433
of free fatty acids (FFA), hydroperoxides, fluorescent tertiary oxidation products (OFR) and 434
negative sensory rancidity attributes. Therefore, it can be recommended to store these fish 435
products at -30 °C rather than -20 °C in order to gain longer storage life. Further, although 436
saithe and hoki are both lean fish species from the order of gadiformes, their lipids 437
composition and stability were significantly different; especially between the dark muscles of 438
19 the two species. Polyunsaturated fatty acids (PUFA) were the predominant lipids in the dark 439
muscle of saithe, while monounsaturated fatty acids (MUFA) levels were significantly higher 440
than PUFA in the dark muscle of hoki. Further, hoki dark muscle contained six-fold higher 441
lipid contents and the majority of which were bound to triacylglycerols, as opposed to being 442
bound to phospholipids which were common to all the other muscle tissues tested. Correlation 443
analyses of the different parameters measured in present study indicated that FFA and OFR 444
could serve as chemical markers for negative sensory attributes such as frozen storage and 445
rancid flavour; this is especially the case for the light muscle tissues across species.
446 447
Acknowledgements 448
The authors would like to acknowledge Nestlé for their financial support and for providing 449
the raw fish material for this research study.
450 451
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22 Figure Captions
559 560
Figure 1. Lipid hydroperoxide formation (mmol/kg muscle) and thiobarbituric acid reactive 561
substances formation (TBARS; µmol MDA/kg samples) in light and dark muscle of saithe (A 562
and C) and hoki (B and D), stored at -30 °C and -20 °C for 18 months. Bars represent the 563
standard deviation for the samples tested (n=6).
564 565
Figure 2. Evolution of the fluorescent shift ratio (OFR) and free fatty acid (FFA) in light and 566
dark muscles of saithe (A and C) and hoki (B and D) during 18 months of frozen storage at - 567
30 °C and -20 °C. Bars represent the standard deviation for the samples tested (n=6).
568 569
Figure 3. Changes in the GDA scores for rancid and frozen storage flavour (f) and odour (o) 570
developed in saithe (A and B) and hoki (C and D) light and dark muscle, respectively, during 571
18 months of frozen storage at -20 °C and -30 °C. Parameter f-frozen was only evaluated on 572
the light muscle, whereas f-rancid was only evaluated on the dark muscle of both fish species.
573
a-b A different letter within sensory attributes indicates significant difference between the two 574
storage temperatures at (p<0.05).
575 576
Figure 1
Figure 2
Figure 3
2 Table 1. Fatty acid composition (g/100g of total lipids) of light and dark muscle of saithe and hoki stored at -20 °C and -30 °C for 18 months 2
(n=4; mean±stdv.).
3
Muscle type
Storage
cond. C16:0 C16:1n7 C18:0 C18:1n7 C18:1n9 C20:1n9 C20:4n3 C20:4n6 C20:5n3 C22:1n9 C22:5n3 C22:6n3 ∑MUFA ∑PUFA ∑SFA ∑n3 ∑n6
Saithe light muscle
Control 17.74 0.82 3.88 2.37 6.78 1.75 0.60 1.66 13.44 0.61 1.25 40.05 15.06 59.05 23.54 56.08 2.89
±0.32a ±0.14a ±0.13a ±0.12a ±0.26a ±0.12a ±0.03a ±0.15a ±0.53a ±0.04a ±0.05a ±2.01a ±0.21a ±0.34a ±0.24a ±0.25a ±0.21a
-30 °C 17.44 0.72 4.17 2.28 6.78 1.73 0.63 1.70 11.80 0.53 1.28 39.44 14.37 56.87 23.29 55.52 2.73
±0.79a ±0.12a ±0.01b ±0.29a ±0.54a ±0.41a ±0.14a ±0.02a ±0.71b ±0.05a ±0.25a ±2.44a ±1.66a ±1.01b ±0.72a ±1.27a ±0.00a
-20 °C 17.47 0.78 4.34 2.28 7.43 1.81 0.65 1.85 11.00 0.51 1.20 33.55 15.25 55.45 23.66 54.12 3.01
±0.58a ±0.20a ±0.04c ±0.31a ±0.70a ±0.01a ±0.01a ±0.20a ±0.33b ±0.05a ±0.10a ±4.56b ±1.19a ±1.79b ±0.73a ±1.99a ±0.35a
Saithe dark muscle
Control 14.60 0.87 4.41 3.53 9.25 2.75 0.68 1.50 9.33 1.06 1.56 38.81 20.68 55.01 20.48 52.95 3.14
±0.33a ±0.08a ±0.18a ±0.23a ±0.15a ±0.22a ±0.04a ±0.06a ±0.22a ±0.07a ±0.16a ±0.38a ±0.59a ±0.42a ±0.25a ±0.22a ±0.06a
-30 °C 15.15 0.78 4.41 3.18 8.49 2.50 0.70 1.58 9.28 0.92 1.51 38.14 18.81 54.62 20.99 52.85 3.07
±0.10b ±0.02a ±0.01a ±0.17a ±0.35b ±0.25a ±0.13a ±0.07a ±0.59a ±0.01b ±0.23a ±1.20a ±0.26b ±0.13a ±0.08a ±0.12a ±0.16a -20 °C 14.50a 0.86 4.42 3.44 9.76 2.72 0.70 1.64 7.90 1.03 1.44 36.41 20.84 53.04 20.40 51.28 3.21
±0.01 ±0.11a ±0.05a ±0.16a ±0.57a ±0.05a ±0.01a ±0.12a ±0.56b ±0.06a ±0.00a ±1.47b ±0.75a ±0.61b ±0.07a ±0.75b ±0.23a
Hoki light muscle
Control 20.17 1.68 4.06a 2.32 9.18 3.39 0.85 1.19 6.49 1.90 1.52 38.46 20.17 51.5 26.42 48.43 2.75
±0.35a ±0.37a ±0.16 ±0.13a ±1.21a ±0.83a ±0.12a ±0.08a ±0.20a ±0.64a ±0.07a ±0.87a ±3.29a ±3.38a ±0.09a ±0.33a ±0.10a
-30 °C 20.90 1.56 4.43 1.93 8.22 3.02 0.85 1.39 6.18 1.73 1.54 35.26 18.03 47.87 27.35 46.15 2.86
±0.25a ±0.25a ±0.25b ±0.25a ±0.25a ±0.25a ±0.25a ±0.25ab ±0.25a ±0.25a ±0.25a ±0.25b ±0.25a ±0.25b ±0.25b ±0.25b ±0.25a
-20 °C 20.07 1.86 4.64 1.97 9.74 3.18 0.85 1.73 5.91 1.75 1.53 31.00 20.23 46.22 26.89 44.42 2.80
±0.32a ±0.29a ±0.15b ±0.09a ±1.93a ±0.05a ±0.01a ±0.32b ±0.23b ±0.25a ±0.01a ±1.94c ±2.19a ±2.08b ±0.13b ±1.99b ±0.15a
Hoki dark muscle
Control 18.09 4.05 2.85 3.59 16.04 8.31 1.60 0.71 6.99 5.26 1.57 12.43 40.06 30.10 26.00 26.31 3.20
±0.40a ±0.14a ±0.09a ±0.08a ±0.47a ±0.44a ±0.11a ±0.05a ±0.39a ±0.68a ±0.05a ±0.73a ±1.31a ±1.05a ±0.37a ±0.45a ±0.09a
-30 °C 17.49 4.07 2.93 2.91 15.07 8.20 1.73 1.19 6.85 5.48 1.59 12.60 38.29 30.04 25.28 26.88 3.44
±0.12a ±0.05a ±0.03a ±0.08b ±0.27a ±0.21a ±0.02a ±0.02b ±0.14a ±0.53a ±0.05a ±0.07a ±0.36b ±0.07a ±0.13b ±0.16b ±0.05b -20 °C 17.36 4.52 2.76 3.00 17.48 8.19 1.68 1.30 6.05 4.93 1.52 9.73 40.8 27.86b 25.19b 24.67 3.40
±0.03a ±0.54a ±0.15a ±0.24b ±3.88a ±0.02a ±0.01a ±0.19b ±0.66b ±0.92a ±0.01a ±1.39b ±1.98a ±1.41 ±0.32 ±3.30a ±0.08b a-c Different letters within column of each muscle type indicate a significant difference at (p<0.05).
4
