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Determination of proximate composition, fatty acid profile and total amino acid contents in samples of the European eel (Anguilla anguilla) of different weights L. Gómez-Limia, N. Cobas, S. Martínez
PII: S1878-450X(21)00063-9
DOI: https://doi.org/10.1016/j.ijgfs.2021.100364 Reference: IJGFS 100364
To appear in: International Journal of Gastronomy and Food Science Received Date: 12 August 2020
Revised Date: 29 March 2021 Accepted Date: 10 May 2021
Please cite this article as: Gómez-Limia, L, Cobas, N, Martínez, S, Determination of proximate composition, fatty acid profile and total amino acid contents in samples of the European eel (Anguilla anguilla) of different weights, International Journal of Gastronomy and Food Science, https://
doi.org/10.1016/j.ijgfs.2021.100364.
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© 2021 Elsevier B.V. All rights reserved.
Author Statement
S Martínez and I. Franco Conceptualization, Methodology, Supervision, Validation, Writing- Reviewing and Editing; S Martínez and L. Gómez-Limia: Data curation, Writing- Original draft preparation; L. Gómez-Limia, N. Cobas,: Visualization, Investigation
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1
Determination of proximate composition, fatty acid profile
1
and total amino acid contents in samples of the European eel
2
(Anguilla anguilla) of different weights
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4
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Food Technology, Faculty of Science, University Campus As Lagoas s/n, 32004 6
Ourense, University of Vigo, Spain.
7 8 9 10
*Corresponding author:
11
Tel.: + 34-988- 12
Fax: + 34-988-387001 13
E-mail: [email protected] 14
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2 Abstract
20 21
European eel of different sizes were caught in the River Ulla (Galicia, NW Spain) 22
during winter. The proximate composition differed significantly between fish of 23
different sizes. Discriminant canonical analysis was used to identify the components 24
that enable specimens of different weights to be distinguished. Fat content was the most 25
variable parameter and was directly related to the weight of the fish. The fatty acids that 26
greater increased their content during the growth of the eel were linolenic acid, 27
arachidonic acid, eicosatrienoic acid, and tricosanoic acid. The lipid profiles and quality 28
indices of the eels indicated their high nutritional quality. The threonine, lysine, serine, 29
arginine, alanine and proline contents were highest in the smaller eels. The European eel 30
is a source of high-quality protein with a well-balanced amino acid profile. Moreover, 31
the ratio of essential to non-essential amino acids was highest in the larger eels.
32
33 34 35
Keywords: European eel; body weight; amino acids, fatty acids; proximate composition 36
37 38
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3 1. Introduction
39
The European eel (Anguilla anguilla) belongs to the order Anguilliformes and family 40
Anguillidae. The species is facultatively catadromous. Thus, the fish spawn in marine 41
habitats, where the eggs hatch, but migrate as pre-juveniles (elvers) to freshwater areas 42
(Tesch, 2003). The European eel was one of the most important and abundant species in 43
all rivers in the Iberian Peninsula in the early 20th century. However, at present, the 44
species is absent from more than 80 % of rivers in the region. The species has been 45
included in the International Union for the Conservation of Nature (IUCN)’s Red List as 46
critically endangered. (Pike et al., 2020).
47
The European eel is a commercially valuable species in Europe (mainly Spain, Portugal, 48
Italy and Netherlands) and Asia (mainly Japan, China, Korea and Taiwan). The eels can 49
live for up to 85 years, with females reaching a maximum length of 137 cm and weight 50
of 9 kg, and males reaching a maximum length of 51 cm and weight of 2.8 kg (FAO, 51
2020). However, the commercial weight is usually between 150 and 250 g, depending 52
on the country of sale. Small or medium sized specimens are in highest demand (FAO, 53
2018).
54
Although the European eel is a commercially important species, information on its 55
composition is scarce (Abrami et al. 1992; Degani et al. 1986; García-Gallego and 56
Akharbach, 1998; Heinsbroek et al. 2013). Most of the information available is related 57
to metabolism, growth, sexual maturation, biochemical factors associated with 58
migration, development of hatchery technologies for this species and the need for 59
adequate broodstock feed (Zied et al., 2013; Butts et al., 2015; Heinsbroek et al 2007;
60
Mazzeo et al., 2016; van Ginneken et al 2018; Kottmann et al., 2020). The nutrient 61
composition of the European eel can provide important insights into the physiological 62
and energetic status of the fish, which can help in predicting the conditions for recovery 63
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4 of the species. On the other hand, European eel is a potentially good candidate for 64
aquaculture. The nutritional properties are important in aquaculture in relation to 65
determining the quality and quantity of nutrients in fish diets. Commercial production of 66
the species in aquaculture depends on the capture of wild elvers (glass eels) (Støttrup et 67
al., 2016). The use of larger eels (less valued as a fresh product) could help to increase 68
the reproductive success of the species as well as enabling production of greater 69
numbers of elvers (glass eels) for aquaculture purposes. In addition, data on muscle 70
composition would help to clarify the changes that take place in body nutritional 71
composition during growth, which could help in the development of feed for eels at 72
different stages of aquaculture.
73
The composition of fish can provide information about its physiological condition, 74
energetic adaptation, habits, nutrititional value and commercial applications. It is 75
important to generate nutritional data in order to develop a suitable processing method 76
that enables eels to be consumed throughout the year, while respecting state-imposed 77
limitations aimed at protecting the species.
78
Therefore, the aim of this study was to evaluate how the proximate composition and the 79
fatty acid and amino acid profiles of muscle tissue from wild European eel are affected 80
by fish size (weight), in order to determine the nutritional value of the fish. In addition, 81
health-related lipid indices, including the atherogenic index (AI), the thrombogenic 82
index (TI) and the ratio of hypocholesterolemic to hypercholesterolemic fatty acids 83
(h/H), were determined. These indexes take into account the different effects that the 84
fatty acids can have on human health and in particular on the probability of increasing 85
the incidence of some coronary diseases (Turan et al., 2007). The ratio between 86
essential and non-essential amino acid (E/NE), which defines the quality of the protein, 87
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5 was also calculated. This information may be useful in relation to industrial processes, 88
culinary treatments or development of aquaculture.
89 90
2. Material and methods 91
92
2.1. Samples 93
94
European eel specimens were caught by professional eel fishermen operating in the 95
River Ulla (Galicia, NW Spain), during the authorized fishing season (winter). The eels 96
were caught in “nasa butrón” (funnel traps). The fish were transported to tanks 97
connected to freshwater recirculation modules, where they were held until slaughter, by 98
ice water immersion. They were then purchased, eviscerated and transferred to the 99
laboratory.
100
Each fish was weighed and the length was measured. The eels were classified into four 101
different groups (at least 15 fish per group) on the basis of their weight: (A) 10 - 100 g;
102
(B) 100 - 200 g; (C) 200 - 400 g; and (D) more than 400 g. The eels were washed, 103
packed in vacuum bags and stored at -20 °C until analysis. Before analysis, frozen eels 104
were defrosted at 4 ± 2 °C in a refrigerator. The head, skin and bone were removed for 105
analysis. The portion of tissue analysed was the muscle between the head and the anus.
106
The muscle sample was removed and homogenized in a grinder.
107 108
2.2. Proximate composition analysis 109
Fifteen eels from each size class were used in the different determinations.
110
Moisture, ash and protein content were determined as described in AOAC methods 111
(AOAC, 2006). The nitrogen (N) content of fish muscle samples was determined by the 112
Kjeldahl method, and the value was multiplied by 6.25 as an estimate of the crude 113
protein content. Fat was extracted according to Bligh and Dyer (1959) using a mixture 114
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6 of chloroform-methanol (1:2, v/v). The fat extract was weighed and the total lipid 115
content was expressed as percentage of the wet muscle weight.
116
The energy value, expressed as kcal/100 g edible part, was estimated using the factors 117
proposed by Merrill and Watt (1973): 9.02 for fat and 4.27 for protein (kcal/g).
118 119
2.3. Fatty acid profile analysis 120
Fatty acid methyl esters (FAMEs) were determined as described by Domínguez et al.
121
(2015). The fatty acids were identified and quantified by Gas Chromatography (GC) 122
using a Thermo Finnigan Trace (Thermo Finnigan, Austin, TX, USA) chromatograph, 123
equipped with a Split/Splitless AI 3000 Auto-injector and a flame ionization detector 124
(FID). Details of the chromatographic separation conditions and quantitative analysis 125
have been described by Domínguez et al. (2015).
126
All samples and patterns were injected into the carrier gas at least in duplicate.
127
Repeatability tests were performed by processing a pattern and a sample consecutively 128
six times each day. Reproducibility tests were also carried out by processing the pattern 129
and the sample twice a day for 3 days under the same experimental conditions described 130
by Domínguez et al. (2015).
131
There were no significant differences between the results obtained in either of the tests.
132
All fatty acids were expressed as a percentage of the total fatty acids. The content of 133
fatty acid was also expressed in mg/100 g muscle tissue using a fatty acid conversion 134
factor. For conversion of the percentile values to units of weight, the formula 135
recommended by Weihrauch et al. (1977) and FAO (2016) for fish is as follows:
136
Conversion factor = 0.933 - (0.143/total fat).
137
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7 Lipid quality indices were estimated according to the method used by Ulbricht and 138
Southgate (1991), based on fatty acid composition. Atherogenicity (AI) and 139
thrombogenicity (TI) indices were calculated using the following formulae:
140
AI = [(12:0) + (4 × 14:0) + (16:0)] × [PUFA (n-6 and n-3) + MUFA)]-1 141
TI = [(14:0) + (16:0) + (18:0)] × [(0.5 × MUFA) + (0.5 × n-6) + (3 × n-3) + (n-3 × n-6- 142
1)]-1. 143
The hypocholesterolemic and hypercholesterolemic index (h/H) was calculated 144
according to the equation described by Santos-Silva et al. (2002):
145
h/H = [(n-3 + n-6 + C18:1) × (C14:0 + C16:0)-1] 146
147
2.4.Quantification of total amino acids 148
Protein hydrolysis was carried out as described by Martínez et al. (2011). Amino acids 149
were identified and quantified by HPLC, under the conditions described by Martínez et 150
al. (2011). The liquid chromatography equipment consisted of a Thermo Finnigan 151
chromatograph with a UV/VISIBLE detector and photodiode matrix (Spectrasystem 152
UV6000LP). The column was a reverse phase C18 Ultrasphere 5–ODS (diameter of 4.6 153
mm and 25 cm of length) (Beckman, Fullerton, USA). The column temperature was 154
maintained at 50 ± 1 °C with a column heater (Spectrasystem 3000). The wavelength of 155
the detector was 254 nm. Standards of the different amino acids were supplied by Sigma 156
Chemical Co. (St Louis, MO). All samples and standards were injected into the column 157
at least in duplicate. Repeatability and reproducibility tests were performed as described 158
for fatty acid analysis. The results of the tests were not significantly different (P < 0.05).
159
The E/NE ratio (ratio between essential and non-essential amino acid) was also 160
calculated for evaluation of the quality of the fish protein.
161
162
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8 2.5. Statistical analysis
163
All analyses were carried out at least in triplicate. The data were examined by analysis 164
of variance (ANOVA), and the least squares test (LSD) was used (P 0.05) to compare 165
the mean values. The analysis were carried out by a forward stepwise method of 166
Statistica software version 7.1 (Statsoft © Inc., Tulsa, OK, USA). Correlations between 167
the different parameters analyzed were determined by multiple regressions with 168
confidence intervals of 95 % (P < 0.05), 99 % (P < 0.01) and 99.9 % (P < 0.001).
169
Canonical discriminant analysis (CDA) was used to classify eels into the different 170
groups according to weight. The CDA variables were selected by principal components 171
extraction and linear discriminant analysis, in order to identify the relations among these 172
data, according to the variability between weights. The variance was explained by each 173
canonical probability and by the analysis of the standardized scoring coefficients. The 174
variables with the highest discriminating capacity were selected to establish those 175
components capable of distinguishing and classifying eels according to their weight.
176
Results were analysed in terms of the absolute assignment of each eel to the pre- 177
assigned group.
178
179
3. Results and Discussion 180
181
3.1. Sample characteristics and proximate composition 182
The length (cm), and the proportion of skin and head (relative to the total gutted 183
weight), of fish in each group are shown in the Table 1. The length and weight of the 184
eels were closely correlated (r = 0.95). The proportions of head and skin did not differ 185
between eels of different weight.
186
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9 Table 2 shows the proximate composition of muscle from European eel of different 187
weights.
188
The nutrient composition and nutritional value are associated with age and size of fish 189
(Heinsbroek et al., 2007). In this study, the moisture content ranged from 73.58 to 68.53 190
%, and it was higher in the smaller eels (groups A and B) than in the larger eels (groups 191
C and D). The protein content ranged from 19.54 to 19.20 %. The differences in protein 192
contents between large and small eels were not significant. The protein content 193
generally amounts to 15-20% of wet weight in the muscle (Erkan and Özden, 2007).
194
Some research has indicated that the protein content of some fish species increases 195
slightly or remains relatively stable as the body weight increases (Ramseyer, 2002).
196
However, Naeem and Salam (2010) reported that the protein content of Aristichthys 197
nobilis increases with body weight.
198
The lipid content ranged from 4.95 to 10.22 % and was higher in the larger eels. This is 199
consitent with observations made by other authors (Lupatsch et al., 2003;
200
Schreckenbach et al., 2001).Heinsbroek et al. (2007) reported that dry matter, lipid and 201
energy were closely related and increased with body weight in European eel weighing 202
between 10 and 130 g. These authors also pointed that there is a maximum body lipid 203
content, which again increases with body weight. Naeem and Salam (2010) observed 204
that the lipid content increased with increasing body weight in the Beluga sturgeon. The 205
lipid content was inversely correlated with moisture content (r = -0.90, P < 0.05), as is 206
commonly observed in fish (Gómez-Limia et al., 2020; Zhang et al., 2014). According 207
to Özogul et al. (2007), fatty fish usually contain at least 5- 8 % fat edible tissue.
208
Ackman (1990) classified fish on the basis of their fat content, as lean (lipid content < 2 209
%), low fat (2−4 %), medium fat (4-8 %) and high fat (> 8 %). In accordance with this 210
classification, the smallest eels (groups A and B) were classified as medium fat fish, and 211
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10 the larger eels (groups C and D) as fatty fish. Some authors have pointed out that lipids 212
tend to accumulate in eels prior to spawning migration and the lipid contents tend to 213
increase as the fish grow (Degani et al. 1986; Saito et al., 2015; van Ginneken et al., 214
2018). The fat content of the muscle tissue of the European eel increases from 8 to 28 % 215
between the yellow and silver stages (Larsson et al. 1990).
216
In the present study, the lipid content was negatively correlated with protein content (r = 217
- 0.37, P < 0.05).
218
The ash contents were higher in the smaller eels than in the larger eels. The values 219
obtained are similar to those observed by Wijayanti and Susilo (2018) in wild and 220
cultured eel (Anguilla bicolor). The ash content was significantly correlated with 221
protein content (r = 0.35; P = 0.05) but negatively correlated with lipid content (r = - 222
0.43; P = 0.05). Alaş et al. (2014) reported that the differences in the concentrations of 223
minerals among fish parts can be due to fish diet and environmental conditions. They 224
also pointed out that the mineral content may condition the suitability of the different 225
species to specific processing and storage conditions.
226
As expected, the energy values were lower in the smaller eels (128.05-146.87 kcal/g) 227
than in the larger eels (182.24 – 171.97 kcal/g).
228
In general, the proximate composition of European eel obtained in this study is similar 229
to values reported in sardines (Fernandes et al. 2014). However, Ersoy (2011) reported 230
lower moisture and protein contents and higher lipid and ash content for European eel 231
of average weight 270 - 300 g.
232
The different results may be attributed to available food resources in different regions or 233
to aquatic environmental factors, such as temperature and pH, in addition to species, 234
fishing season, size, reproduction status, age, diet, etc. Özogul et al. (2005) reported 235
lower protein, moisture and ash contents (17.50, 60.12 and 1.05 %, respectively) and 236
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11 higher lipid content (20.86 %) for European eel of average weight 228.5 g. Ozogul et al.
237
(2014) observed lower protein levels (15.79 %) and moisture (62.74 %), higher lipid 238
content (19.95 %), and similar ash content (1.41 %) for eels of average weight 464.59 g.
239
Oku et al. (2009) reported moisture, protein and lipid contents in muscle of wild 240
Japanese eels (average weight 195 g) of between 67.3 and 71.1 %, 18.3 and 19.4 %, and 241
8.1-13.5 %, respectively.
242
The body composition is affected by the composition of the fish diet. Suárez et al.
243
(1995) pointed out that the metabolism of the European eel can adapt to changes in 244
dietary composition. García-Gallego et al. (1995) indicated that the European eel is less 245
capable than rainbow trout of utilizing diets for growth.
246 247
3.2. Fatty acid composition 248
The fatty acid contents expressed as a percentage of total fatty acids and as g/100 g of 249
fish sample are shown in Table 3. Thirty-one fatty acids, ranging from myristic acid 250
(C14:0) to docosahexaenoic (C22:6n3, DHA) were identified and compared in 251
European eel of different weights. The fatty acids reported herein comprised over 99.57 252
% of the total fatty acids.
253
Fatty acid contents increased in muscle with fish size as its fat content increased. The 254
fatty acids that experienced the highest increases were linolenic acid (C18:3n6), 255
arachidonic acid (C20:4n6), eicosatrienoic acid (C20:3n3), and tricosanoic acid (C23:0).
256
The saturated fatty acid (SFA) content varied from 1500.85 (eels between 10 and 100 g) 257
to 3079.00-3179.24 mg/100 g (eels larger than 200 g). Palmitic acid (C16:0) was the 258
predominant SFA (between 1031.19 and 2043.21 mg/100 g). This fatty acid increased 259
in muscle with the weight of the fish but decreased its percentage of the total fatty acids 260
(22.95 %, A and 21.83 %, D), although the differences were not significant.
261
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12 Stearic acid (C18:0) (between 199.36 and 412.09 mg/100 g) and myristic acid (C14:0) 262
(between 182.74 and 438.20 mg/100 g) were other predominant SFA. The high content 263
of palmitic acid and stearic acid is attributed to their use as a major source of energy for 264
metabolism and growth (Sargent et al., 2002).
265
The percentage of myristic acid (C14:0) was lower in the smallest eels. C14:0 has been 266
implicated in hypercholesterolemia in humans, although low amounts of this amino acid 267
have also been reported to be beneficial to human health (Fernandes et al. 2014).
268
The content of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids 269
(PUFAs) increased with the weight, although their total percentage in the fat did not 270
differ between eels of different weights. MUFAs ranged between 2264.79 and 4809.42 271
mg/100 g, whereas PUFAs between 740.74 and 1522.63 mg/100 g.
272
Oleic acid (C18:1n9) was the predominant fatty acid (between 1546.59 and 3451.49 273
mg/100 g). There was no variation in the percentage of oleic acid between eels of 274
different weights (34.42-35.98 %). Oleic acid is of external origin and its content is 275
related to dietary fatty acid content and depends on the metabolism of each fish species 276
(Ackman, 1990). Oleic acid is the major MUFA in the lipids of many species of 277
freshwater and marine fish species (Özogul et al. 2007).
278
The proportion of n-3 PUFAs was high in the muscle tissue of the eels.
279
Eicosapentaenoic (C20:5n3, EPA), docosahexaenoic (C22:6n3, DHA) and 280
docosapentaenoic (C22:5n3) acids were dominant in the PUFAs. The content of n-3 was 281
higher in the larger eels (724.26 mg/100 g -C-; 717.53 mg/100 g -D-) than the smaller 282
eels (320.53 mg/100 g-C-; 454.28 mg/100 g –D-). The levels of n-6 also were higher in 283
the larger eels (C and D) than in the smallest eels (A and B).
284
Of the n-3 with nutritional implications, EPA and DHA are present at higher levels in 285
larger fish. DHA and EPA acids are nutritionally important because of their value in 286
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13 preventing several diseases, mainly those affecting the cardiovascular system 287
(Simopoulos, 2008). However, these fatty acids are highly susceptible to autoxidation 288
because of the high degree of unsaturation.
289
The linoleic acid (C18:2n6) ranged from 43.75 to 112.14 mg/100 g and its percentage of 290
the total fatty acids ranged from 0.94 to 1.18 %. The content of linoleic acid in fish is 291
influenced by diet, with higher concentrations in wild fish that usually feed on 292
zooplankton, small fish or/and insects (Salimon and Rahman, 2008). Arachidonic acid 293
is also influenced by variations in the fatty acid composition of the diet. This fatty acid 294
is important in fish metabolism as it is known to be the main precursor fatty acid of 295
eicosanoids in fish (Passi et al., 2002). Levels of arachidonic acid (20:4 n-6) were 296
higher in the larger eels (127.60 mg/100 g in group C and 162.21 mg/100 g in group D) 297
than in the smaller eels (44.17 mg/100 g in group A and 66.97 mg/100 g in group B).
298
The ratio of polyunsaturated to saturated fatty acids (PUFA/SFA) is used to assess the 299
nutritional quality of fish, and the value should be higher than 0.45 (de Melo et al.
300
2013). The PUFA/SFA ratio of the eel muscle tissue ranged from 0.48 to 0.52, and did 301
not differ significantly in relation to weight of the fish. These values indicated a higher 302
beneficial effect of consumption of larger eels than smaller eels.
303
The n-3/n-6 ratio is used to identify the high n-3 PUFA foodstuffs. In the present study, 304
the average n-3/n-6 ratio in eels ranged between 2.07 (A) and 1.80 (D). The comparison 305
of eels by weight revealed that the n-3/n-6 ratios were similar in all eels. Valfré et al.
306
(2003) reported that the n-3/n-6 ratio ranged from 1 to 4 in freshwater fish species, and 307
from 5 to more than 10 in marine fish. Other studies have shown that PUFA contents 308
are lower in freshwater fish than in marine fish, because freshwater fish mainly feed on 309
vegetation and plant materials, whereas marine fish generally feed on zooplankton, 310
which are rich in PUFAs (Vlieg and Body, 1988). In the case of the eels, differences in 311
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14 freshwater and marine environments also affect the fatty acid composition. In our study, 312
all of the eels were caught in freshwater.
313
The differences in fatty acid content during growth found in the present study might 314
correspond to the diet. In the ontogeny, a major change in diet occurred. The diet 315
change play an important role in determining the migration patterns. Butts et al. (2015) 316
reported that diet had a significant effect on most fatty acids in the neutral lipid and 317
phospholipid fractions of muscle of male European eel. Heinsbroek et al. (2013) and 318
Støttrup et al. (2016) reported changes in fatty acids in female eels of different sizes.
319
The nutritional quality of the eels of different weights is also shown in Table 3.
320
The atherogenicity index (AI) and thrombogenicity index (TI) can provide information 321
about the potential capacity of foodstuffs to promote atherosclerosis and platelet 322
aggregation (Turan et al. 2007). Low values of the AI and TI (< 1) are assumed to be 323
beneficial to human health. The AI and TI values in the eels varied between 0.64 and 324
0.66 and between 0.58 and 0.64, respectively. In this study, AI and TI values were less 325
than 1, which indicates that the consumption of eels may reduce the risk of coronary 326
heart disease.
327
The hypocholesterolemic/hypercholesterolemic ratio (h/H) is associated with 328
cholesterol metabolism, and higher values of this index are considered beneficial to 329
human health (Chen and Liu, 2020). The h/H values obtained in this study ranged from 330
1.80 to 2.05. These values indicate that regular intake of European eel may have a 331
hypocholesterolemic effect. Similar results have been reported for other fish (Fernandes 332
et al. 2014).
333 334
3.3. Amino acid composition 335
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15 Fish protein is an essential source of nutrients and is rich in essential amino acids such 336
as lysine, methionine, cystine, threonine and tryptophan (Mohanty et al., 2014). Amino 337
acids are important for fish metabolism. They also play an important role as taste and 338
flavour components.
339
The method used in this study allowed the analysis of 17 amino acids. The amino acid 340
compositions of the eel samples are shown in Table 4. The values are expressed as 341
g/100 g of fish sample. Glutamic acid was the most abundant amino acid (1.81-2.00 342
g/100 g), followed by leucine (1.38-1.55 g/100 g), relative to other amino acids. Both of 343
these amino acids have important roles. Glutamic acid plays an important role in amino 344
acid metabolism, and leucine can stimulate muscle protein synthesis and has a 345
therapeutic role in stress conditions such as trauma, burn and sepsis (De Bandt and 346
Cynober, 2006). Our data were similar to those reported by Mohanty et al. (2014), for 347
different marine species, in which glutamic acid and leucine were also the most 348
abundant amino acids.
349
The major essential amino acids in the tissues were leucine (1.38-1.55 g/100 g), lysine 350
(0.81-0.94 g/100 g), threonine (0.69-0.82 g/100 g), valine (0.77-0.80 g/100 g) isoleucine 351
(0.74-0.77 g/100 g) and phenylalanine (0.68-0.72 g/100 g). In addition to glutamic acid, 352
the major non-essential amino acids were arginine (0.95-1.31 g/100 g), aspartic acid 353
(1.10-1.19 g/100 g), alanine (0.98-1.15 g/100 g) and glycine (0.78-1.02 g/100 g).
354
Arginine plays an important role in cell division, wound healing, hormone release, 355
neurotransmission and maintance of blood pressure, etc. (Mohanty et al., 2014). The 356
hydroxyproline content (0.12-0.16 g/100 g) was low. This amino acid is present almost 357
exclusively in collagen and in low amounts in other proteins. Osibona (2011) found that 358
hydroxyproline was absent in marine species but present in freshwater species.
359
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16 Significant (P < 0.05) differences in the quantities of some amino acids were found in 360
relation to fish weight. Relative to the large eels, the small eels contain higher levels of 361
histidine, threonine, lysine, hydroxyproline, serine, glycine, arginine, alanine and 362
proline. The larger eels had lower levels of total essential and non-essential amino acids 363
than the smaller eels. The total essential amino acids ranged from 6.21-6.23 g/100 g in 364
the larger eels to 6.60-6.68 g/100 g in the smaller eels, while the total non-essential 365
amino acid content ranged from 7.23-7.53 g/100 g in the larger eels to 7.99-8.40 g/100 g 366
in the smaller eels. These different patterns suggest different amino acid requirements in 367
the different sizes of eels. Therefore, the feed for farmed European eel should be 368
formulated according to the specific amino acid considered.
369
Some authors have observed differences in amino acids profiles between juvenile and 370
adult fish. Sankar et al. (2013) observed that protein content was high in medium sized 371
anchovies, and that the essential amino acid content was higher in small and medium 372
size anchovies than in the larger fish.
373
In the European eel, the total amino acid content of the muscle contains between 45 and 374
46 % of essential amino acids. The ratio between essential and non-essential amino 375
acids (E/NE) is used as an indicator of protein quality. The E/NE ratio was between 376
0.81 and 0.86. The amounts of essential and non-essential amino acids were higher in 377
the larger eels. The findings show that European eel protein is of high quality and has a 378
well-balanced amino acid profile.
379
The differences between small and large eels may be attributed to ontogenetic changes 380
in the amino acid requirements of different growth stages. Amino acids can be used for 381
energy production, transformed into other amino acids and also used in gluconeogenesis 382
and lipogenesis, as well as in the synthesis of other nitrogen compounds (Hochachka 383
and Mommsen, 1995).
384
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17 385
3.4 Canonical discriminant analysis 386
Multivariate statistical techniques were used with the aim of discriminating between 387
eels of different weights. All variables studied were used in the analysis and factorial 388
analysis to obtain the variables that contributed most to the classification. Canonical 389
discriminant analysis was applied to these variables, and the coefficients (correlation 390
discriminant functions) are shown in Table 5. Canonical discriminant analysis was 391
applied to selected variables, and the classification percentage was 100 % accurate for 392
all fish (Fig. 1). The eels were classified by groups according to their weight.
393
The F1 factors were the most significant (P < 0.05). In agreement with the values of 394
Pearson's correlation between the original variables and the linear combinations inside 395
of each data group, the fatty acids with the highest discriminating power (Table 5) were 396
C14:0, C16:0, C18:3n6 and C22:6n3. The aspartic acid and threonine contents also had 397
a significant discriminating power in this root. According to F2, the amino acids with 398
the highest discriminating power were aspartic acid, threonine and ratio of essential and 399
non-essential amino acid (E/NE). These results indicate that some amino acids and fatty 400
acids changes significantly during growth.
401
Discriminant analysis was important in identifying those components that distinguish 402
between fish of different weights. However, fatty acid and amino acid profiles are 403
expected to differ depending on the fish weight. This suggests a relationship between 404
growth, and fatty acid and amino acid requirements, which may also be driven by other 405
growth factors not evaluated here.
406
The changes reflect the different requirements of the fish throughout its growth and the 407
changes that take place in its muscle tissue.Proteins, lipids, amino acids and fatty acids 408
are important chemical compounds involved in the energy metabolism of fish, and they 409
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18 are related to feeding, migratory movement and sexual changes associated with 410
spawning. Therefore, these changes could be a general pattern in eels, although it would 411
be necessary to study a greater number of factors.
412 413
4. Conclusion 414
The findings of our research showed that the European eel is a source of high quality 415
protein and lipids, with a well balanced composition of essential fatty acids and amino 416
acids. In summary, in the European eel the body weight influences proximate body 417
composition as well as the fatty acid and amino acid contents. The total protein and ash 418
contents decreased as the body weight increased, but the moisture and fat contents 419
increased. The results of present study provide important nutritional information about 420
the European eel, which could be useful in relation to industrial processes or culinary 421
treatments. The chemical composition of fish indicates its quality and influences its 422
technological and culinary proprieties. The composition can determine the suitability of 423
specific cooking and processing techniques and determine processing and storage 424
conditions. The nutritional value of the larger eels, often undervalued relative to small 425
eels, can also highlighted. In addition, the eels are show a high potential for aquaculture.
426
Information about the muscle composition of fish and the influence of body weight is 427
also important to determine the quality and quantity of different components required in 428
feeds.
429
Moreover, several combinations of fatty acids and amino acids could be used to 430
discriminate between eels of different weights.
431 432
Acknowledgements 433
Thanks to the Xunta de Galicia for financial support under the Consolidation and 434
restructuring program of competitive research units: Strategic Research Partnerships 435
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19 (2009/060). The first author acknowledges financial support from the University of 436
Vigo through a pre-doctoral fellowship.
437 438
Conflicts of Interest 439
The authors declare no conflict of interest 440
441
5. References 442
443
Abrami, G., Natiello, F., Bronzi, P., McKenzie, D., Bolis, L., Agradi, E., 1992. A 444
Comparison of highly unsaturated fatty acid levels in wild and farmed eels (Anguilla 445
anguilla). Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 101, 79-81.
446
https://doi.org/10.1016/0305-0491(92)90161-J 447
Ackman, R.G., 1990. Seafood lipids and fatty acids. Food Rev. Int. 6, 617-646.
448
https://doi.org/10.1080/87559129009540896 449
Alaş, A., Özcan, M.M., Harmankaya, M., 2014. Mineral contents of the head, caudal, 450
central fleshy part, and spinal columns of some fishes. Environ. Monit. Assess. 186, 451
889–894. https://doi.org/10.1007/s10661-013-3429-3 452
AOAC International (2006). Official Methods of Analysis of AOAC International. Md.
453
AOAC International, Gaithersburg.
454
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification.
455
Can. J. Biochem. Physiol. 37, 911-917.
456
Butts, I.A.E., Baeza, R., Stottrup, J.G., Kruger-Johnsen, M., Jacobsen, C., Pérez, L., 457
Asturiano, J.F., Tomkiewicz, J., 2015. Impact of dietary fatty acids on muscle 458
composition, liver lipids, milt composition and sperm performance in European eel.
459
CBP - Part A: Molecular and Integrative Physiology. 183, 87-96.
460
doi:10.1016/j.cbpa.2015.01.015 461
Journal Pre-proof
20 Chen, J., Liu, H., 2020. Nutritional indices for assessing fatty acids: A mini-review. Int.
462
J. Mol. Sci., 21, 5695. https://doi.org/10.3390/ijms21165695 463
De Bandt, J., Cynober, L., 2006. Therapeutic use of branched-chain amino acids in 464
burn, trauma, and sepsis. J. Nutr. 136, 308S-313S.
465
https://doi.org/10.1093/jn/136.1.308S 466
de Melo, A., de Oliveira, H.H., dos Santos, R.C., 2013. Omega-6/omega-3 and 467
PUFA/SFA in Colossoma macropomum grown in Roraima, Brazil. Orbital: Electron.
468
J. Chem., 5, 30-35.
469
Degani, G., Hahamu, H., Levanon, D., 1986. The relationship of eel Anguilla anguilla 470
(L.) body size, lipid, protein, glucose, ash, moisture composition and enzyme activity 471
(aldolase). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 84, 739-745.
472
https://doi.org/10.1016/0300-9629(86)90398-1 473
Domínguez, R., Martínez, S., Gómez, M., Carballo, J., Franco, I., 2015. Fatty acids, 474
retinol and cholesterol composition in various fatty tissues of Celta pig breed: Effect 475
of the use of chestnuts in the finishing diet. J. Food Compos. Anal. 37, 104-111.
476
https://doi.org/10.1016/j.jfca.2014.08.003 477
Erkan, N., Özden, Ö., 2007. Proximate composition and mineral contents in aqua 478
cultured sea bass (Dicentrarchus labrax), sea bream (Sparus aurata) analyzed by 479
ICP-MS. Food Chem. 102, 721-725. https://doi.org/10.1016/j.foodchem.2006.06.004 480
Ersoy, B., 2011. Effects of cooking methods on the proximate, mineral and fatty acid 481
composition of European eel (Anguilla anguilla). Int. J. Food Sci. Tech. 46, 522-527.
482
https://doi.org/10.1111/j.1365-2621.2010.02546.x 483
FAO (Food and Agriculture Organization of the United Nations), 2018. FishFinder.
484
Species Fact Sheets Anguilla anguilla.
485
http://http://www.fao.org/fishery/species/2203/en (accessed 26 November 2020).
486
Journal Pre-proof
21 FAO (Food and Agriculture Organization of the United Nations), 2020. FishFinder.
487
Species Fact Sheets Anguilla anguilla. http://www.fao.org/fishery/species/2203/en 488
(accessed 11 August, 2020).
489
FAO (Food and Agriculture Organization of the United Nations), 2016.
490
FAO/INFOODS Databases. FAO/INFOODS global food composition database for 491
fish and shellfish, version 1.0 - uFiSh1.0. http://www.fao.org/3/i6655e/i6655e.pdf 492
(accessed 11 March 2021).
493
Fernandes, C.E., da Silva Vasconcelos, M.A., de Almeida Ribeiro, M., Sarubbo, L.A., 494
Andrade, S.A.C., de Melo Filho, A.B., 2014. Nutritional and lipid profiles in marine 495
fish species from Brazil. Food Chem. 160, 67-71
496
https://doi.org/10.1016/j.foodchem.2014.03.055 497
García-Gallego, M., Akharbach, H., 1998. Evolution of body composition of European 498
eels during their growth phase in a fish farm, with special emphasis on the lipid 499
component. Aquacult. Int. 6, 345-356.
500
García-Gallego, M., Bazoco, J., Suárez, M.D., Sanz, A., 1995. Utilization of dietary 501
carbohydrates byfish: a comparative study in eel and trout. Anim. Sci. 61, 427–436.
502
Gómez-Limia, L., Cobas, N., Franco, I., Martínez, S., 2020. Fatty acid profiles and lipid 503
quality indices in canned European eels: Effects of processing steps, filling medium 504
and storage. Food Res. Int. 136, 109601. 10.1016/j.foodres.2020.109601.
505
Heinsbroek, L.T.N., Van Hooff, P.L., Swinkels, W., Tanck, M.W.T., Schrama, J.W., 506
Verreth, J.A.J., 2007. Effects of feed composition on life history developments in 507
feed intake, metabolism, growth and body composition of European eel, Anguilla 508
anguilla. Aquac. 267, 175–187.
509
Heinsbroek, L.T.N., Støttrup, J.G., Jacobsen, C., Corraze, G., Kraiem, M.M., Holst, 510
L.K., Tomkiewicz, J., Kaushik, S.J., 2013. A review on broodstock nutrition of 511
Journal Pre-proof
22 marine pelagic spawners: the curious case of the freshwater eels (Anguilla spp.).
512
Aquac. Nutr. 19, 1-24.
513
Hochachka, P.W., Mommsen, T.P., 1995. Metabolic Biochemistry, Volume 4 1st 514
edition. Elsevier, Amsterdam.
515
Kottmann, J.S., Jørgensen, M., Bertolini, F., Loh, A., Tomkiewicz, J., 2020. Differential 516
impacts of carp and salmon pituitary extracts on induced oogenesis, egg quality, 517
molecular ontogeny and embryonic developmental competence in European eel.
518
PloS one, 15(7), e0235617. https://doi.org/10.1371/journal.pone.0235617 519
Larsson, P., Hamrin, S., Okla, L., 1990. Fat content as a factor inducing migratory 520
behavior in the eel (Anguilla anguilla L.) to the Sargasso Sea. Sci. Nat.-Heidelberg.
521
77, 488-490. https://doi.org/10.1007/BF01135929 522
Lupatsch, I., Kissil, G. Wm., Sklan, D., 2003. Comparison of energy and protein 523
efficiency among three fish species: gilthead seabream (Sparus aurata), European 524
seabass (Dicentrarchus labrax) and white grouper (Epinephelus aeneus): energy 525
expenditure for protein and lipid deposition. Aquac. 225, 175-189.
526
Martínez, S., Losada, P., Franco, I., Carballo, J., 2011. Protein, amino acid, ash and 527
mineral contents in Brassica spp. grown in Northwest Spain. Int. J. Food Sci. Tech.
528
46, 146-153. https://doi.org/10.1111/j.1365-2621.2010.02463.x 529
Mazzeo., I., Giorgini, E., Gioacchini, G., Maradonna, F., Vilchez Olivencia, M.C., 530
Baloche, S., Dufour, S., 2016. A comparison of techniques for studying oogenesis in 531
the European eel Anguilla anguilla. J. Fish Biol. 89, 2055-2069.
532
doi:10.1111/jfb.13103 533
Merrill, A., Watt, B., 1973. Energy value of foods: basis and derivation. Agriculture 534
Handbook, 74. United States Department of Agriculture, Washington.
535
Journal Pre-proof
23 Mohanty, B., Mahanty, A., Ganguly, S., Sankar, T.V., Chakraborty, K., Rangasamy, A., 536
Paul, B., Sarma, D., Mathew, S., Asha, K.K., Behera, B., Aftabuddin, M., Debnath, 537
D., Vijayagopal, P., Sridhar, N., Akhtar, M.S., Sahi, N., Mitra, T., Banerjee, S., 538
Paria, P., Das, D., Das, P., Vijayan, K.K., Laxmanan, P.T., Sharma, A.P., 2014.
539
Amino acid compositions of 27 food fishes and their importance in clinical nutrition.
540
J. Amino Acids, 2014, 1-7. http://dx.doi.org/10.1155/2014/269797 541
Naeem, M., Salam, A., 2010. Proximate composition of fresh water bighead carp, 542
Aristichthys nobilis, in relation to body size and condition factor from Islamabad, 543
Pakistan. Afr. J. Biotechnol. 9, 8687-8692. https://doi.org/10.5897/AJB10.888 544
Oku, T., Sugawara, A., Choudhury, M., Komatsu, M., Yamada, S., Ando, S., 2009.
545
Lipid and fatty acid compositions differentiate between wild and cultured Japanese 546
eel (Anguilla japonica). Food Chem. 115, 436-440.
547
https://doi.org/10.1016/j.foodchem.2008.12.032 548
Osibona, A.O., 2011. Comparative study of proximate composition, amino and fatty 549
acids of some economically important fish species in Lagos, Nigeria. Afr. J. Food 550
Sci. 5, 581-588.
551
Ozogul, I., Polat, A., Özogul, Y., Boga, E.K., Ozogul, F., Ayas, D., 2014. Effects of 552
laurel and myrtle extracts on the sensory, chemical and microbiological properties of 553
vacuum‐packed and refrigerated European eel (Anguilla anguilla) fillets. Int. J. Food 554
Sci. Tech. 49, 847-853. https://doi.org/10.1111/ijfs.12374 555
Özogul, Y., Özogul, F., Alagoz, S., 2007. Fatty acid profiles and fat contents of 556
commercially important seawater and freshwater fish species of Turkey: A 557
comparative study. Food Chem. 103, 217-223.
558
https://doi.org/10.1016/j.foodchem.2006.08.009 559
Journal Pre-proof
24 Özogul, Y., Özyurt, G., Özogul, F., Kuley, E., Polat, A., 2005. Freshness assessment of 560
European eel (Anguilla anguilla) by sensory, chemical and microbiological methods.
561
Food Chem. 92, 745-751. https://doi.org/10.1016/j.foodchem.2004.08.035 562
Passi, S., Cataudella, S.F., Di Marco, P., De Simone, F., Rastrelli, L., 2002. Fatty acid 563
composition and antioxidant levels in muscletissue of different Mediterranean marine 564
species of fish and shellfish. J. Agric. Food Chem. 50, 7314−7322.
565
Pike, C., Crook, V., Gollock, M., 2020. Anguilla anguilla. The IUCN Red List of 566
Threatened Species 2020: e.T60344A152845178.
567
https://dx.doi.org/10.2305/IUCN.UK.2020-2.RLTS.T60344A152845178 (accessed 568
22 January 2021).
569
Ramseyer, L.J., 2002. Predicting whole-fish nitrogen content from fish wetweight using 570
regression analysis. N. Am. J. Aquac. 64, 195–204.
571
Saito, H., Kurogi, H., Chow, S., Mochioka, N., 2015. Variation of lipids and fatty acids 572
of the Japanese freshwater eel, Anguilla japonica, during spawning migration. J.
573
Oleo Sci. 64, 603-616. 10.5650/jos.ess14293.
574
Salimon, J., Rahman, N., 2008. Fatty acids composition of selected farmed and wild 575
freshwater fishes. Sains Malaysiana. 37, 149-153.
576
Sargent, J.R., Tocher, D.R., Bell, J.G., 2002. The lipids, in: Halver J.E., Hardy R.W.
577
(Eds.), Fish Nutrition. Academic, San Diego, pp. 181–257.
578
Sankar, T.V., Anandan, R., Mathew, S., Asha, K.K., Lakshmanan, P.T., Varkey, J., 579
Aneesh, P.A., Mohanty, B.P., 2013. Chemical composition and nutritional value of 580
anchovy (Stolephorus commersonii) caught from Kerala Coast, India. Eur. J. Exp.
581
Biol. 3, 85-89.
582
Santos-Silva, J., Bessa, R.J.B., Santos-Silva, F., 2002. Effect of genotype, feeding 583
system and slaughter weight on the quality of light lambs: II. Fatty acid composition 584
Journal Pre-proof
25 of meat. Livest. Prod. Sci. 77, 187-194. https://doi.org/10.1016/S0301- 585
6226(02)00059-3 586
Schreckenbach, K., Knosche, R., Ebert, K., 2001. Nutrient and energy content of 587
freshwater fishes. J. Appl. Ichthyol. 17, 142–144.
588
Simopoulos, A.P., 2008. The importance of the omega-6/omega-3 fatty acid ratio in 589
cardiovascular disease and other chronic diseases. Exp. Biol. Med. 233, 674-688.
590
https://doi.org/10.3181/0711-MR-311 591
Støttrup, J.G., Tomkiewicz, J., Jacobsen, C., Butts, I.A.E., Holst, L.K., Krüger-Johnsen, 592
M., Graver, C., Lauesen, P., Fontagné-Dicharry, S., Heinsbroek, L.T.N., Geneviève, 593
C., Kaushik, S., 2016. Development of a broodstock diet to improve developmental 594
competence of embryos in European eel, Anguilla anguilla. Aquacult. Nutr. 22, 725- 595
737.
596
Suárez, M.D., Hidalgo, M.C., García-Gallego, M., Sanz, A., De la Higuera, M., 1995.
597
Influence of therelative proportion of energy yielding nutrients on liver intermediary 598
metabolism of the European eel. Comp. Biochem. Physiol. 111A, 421–428.
599
Tesch, F.W., 2003. The Eel. Blackwell Science, Oxford.
600
http://dx.doi.org/10.1002/9780470995389 601
Turan, H., Sönmez, G., Kaya, Y., 2007. Fatty acid profile and proximate composition of 602
the thornback ray (Raja clavata, L. 1758) from the Sinop Coast in the Black Sea. J.
603
Fish. Sci. 1, 97-103. https://doi.org/10.3153/jfscom.2007012 604
Ulbricht, T., Southgate, D., 1991. Coronary heart disease: seven dietary factors. Lancet, 605
338, 985-992. https://doi.org/10.1016/0140-6736 (91)91846-M 606
Valfré, F., Caprino, F., Turchini, G.M., 2003. The health benefit of seafood. Vet. Res.
607
Commun. 27, 507. https://doi.org/10.1023/B:VERC.0000014208.47984.8c 608
Journal Pre-proof
26 van Ginneken, V.J.T., De Vries, E., Verheij, E., 2018. The lipid composition and 609
biochemistry of the migrating European eel (Anguilla anguilla L.): A LCMS-study 610
following a lipidomics based systems biology approach. Adv. Biochem. Biotehcnol.
611
(April), 162. https://doi.org/10.29011/2574-7258 612
Vlieg, P., Body, D.R., 1988. Lipid contents and fatty acid composition of some New 613
Zealand freshwater finfish and marine finfish, shellfish, and roes. N. Z. J. Mar.
614
Freshwat. Res. 22, 151-162.
615
Weihrauch, J.L., Posati, L. P., Anderson, B.A., Exler, J., 1977 Lipid conversion factors 616
for calculating fatty acid contents of foods. J. Am. Oil Chem. Soc. 54, 36–40.
617
Wijayanti, I., Susilo, E.S., 2018. Proximate content of wild and cultured eel (Anguilla 618
bicolor) in different part of body. IOP Conf. Ser.: Earth Environ. Sci. 116, 012091.
619
Zhang, Z., Liu, L., Xie, C., Li, D., Xu, J., Zhang, M., Zhang, M., 2014. Lipid contents, 620
fatty acid profiles and nutritional quality of nine wild caught freshwater fish species 621
of the Yangtze Basin, China. J. Food Nut. Res. 2, 388-394. 10.12691/jfnr-2-7-10.
622
Zied, R., Allam, S.M., Sadek, M.A., 2016. Effect of fish meal type and its percentage in 623
diet on growth performance, feed utilization and body chemical composition of 624
European eel (Anguilla anguilla).EJNF, 16, 181-187.
625 626 627
Journal Pre-proof
27 Figure captions
628
Fig. 1 629
Plot of the different eels depending on their weight on the axes representing the values 630
of the two discriminating functions. A: from 10 and 100 g; B: from 100 and 200 g; C:
631
from 200 and 400 g; D: more than 400 g 632
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28 Table 1.
633
Physical parameters of European eel 634
A B C D
Weight (g) 32.48 ± 13.01a 172.94 ± 22.76b 302.78 ± 55.25c 503.55 ± 118.22d Length (cm) 29.30 ± 3.57a 37.95 ± 2.47b 54.70 ± 1.84c 67.55 ± 0.78d Skin (%) 8.45 ± 2.36a 9.02 ± 1.35a 9.30 ± 1.58a 9.35 ± 0.86a Head (%) 13.05 ± 3.27a 12.16 ± 1.22a 12.62 ± 2.74a 10.48 ± 1.82a
a-d.- Mean values of at least three determinations ± standard deviation with different superscripts in the same column
635
were significantly different (P <0.05).
636 637 638
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29 Table 2.
639
Proximate composition of European eel 640
A B C D
Moisture (%) 73.58 ± 0.99 a 72.73 ± 4.07a 68.53 ± 3.96b 68.77 ± 4.67b Protein (%) 19.54 ± 0.52 19.20 ± 1.09 19.20 ± 1.05 19.62 ± 1.06 Fat (%) 4.95 ± 0.64a 6.99 ± 2.89a 10.22 ± 4.14b 9.97 ± 3.58b Ash (%) 1.50 ± 0.19a 1.37 ± 0.16a.b 1.26 ± 0.25b.c 1.23 ± 0.16c Energy values 128.05 ± 4.42a 146.87 ± 28.93a 182.24 ± 33.12b 171.97 ± 32.32b
A: from 10 and 100 g; B: from 100 and 200 g; C: from 200 and 400 g; D: more than 400 g
641
a-c.- Mean values of at least three determinations ± standard deviation with different superscripts in the same column
642
were significantly different (P <0.05).
643