Lipid and Mineral Distribution in Different Zones of Farmed and Wild Blackspot Seabream

Lipid and mineral distribution in different zones of farmed and wild blackspot seabream (Pagellus bogaraveo)

Victoria Álvareza, Isabel Medinaa, Ricardo Pregob, and Santiago P. Aubourga,*

a Department of Food Technology; Instituto de Investigaciones Marinas (CSIC), C/ Eduardo Cabello 6, 36208-Vigo (Galicia, Spain)

b Department of Oceanography; Instituto de Investigaciones Marinas (CSIC), C/ Eduardo Cabello 6, 36208-Vigo (Galicia, Spain)

* Correspondent: Fax: +34 986 292762; e-mail:

SUMMARY

Lipid composition was studied in different white muscle zones (ventral, dorsal and tail) of wild and farmed blackspot seabream (Pagellus bogaraveo). The study was complemented by the moisture, trimethylamine oxide (TMAO) and trace mineral determinations. Farmed fish muscle showed higher lipid and triglyceride contents, but lower values for moisture, TMAO and α-tocopherol than its counterpart wild fish; no differences could be observed between both kinds of fish for the phospholipid, sterol and free fatty acid content. When compared to wild fish, a higher saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acid content was obtained in farmed fish, while lower values could be observed for the ω3/ ω6 and 22:6 ω3/ 20:5 ω3 fatty acid ratios. Most minerals analysed (Cu, Fe, Mn, Se and Zn) showed higher mean values in farmed fish muscle, except for Ca and Mg that provided higher mean contents in wild fish. Concerning muscle sites comparison, greater SFA, MUFA and PUFA contents could be detected in the dorsal zone than in the two other locations both for farmed and wild fish, according to a higher mean lipid content found in this site. Finally, the tail zone showed higher TMAO values than the two other locations.

Running Title: Lipid and mineral distribution in blackspot seabream

Keywords: Blackspot seabream, muscle zones, farmed, wild, lipid classes, fatty acids, trimethylamine oxide, minerals

1. INTRODUCTION

Marine species include relevant contents of different constituents such as nutritional and digestible proteins, lipid-soluble vitamins, microelements, non-protein nitrogen compounds and highly unsaturated fatty acids [1-3]. Most of these constituents have shown to support an important role both in human diet and in quality changes developed during processing and storage of the corresponding seafood product. Among such constituents, the lipid fraction is now the subject of a great deal of attention due to its high content on ω3 polyunsaturated fatty acids (PUFA), which has shown potential benefits to human health [4, 5] and negative effects on quality changes during the technological treatment [6].

Fish constituents have often been shown to be inhomogeneously distributed along the body of a fish, probably affected by physiological and anatomical factors [7]. In this sense, the lipid matter has been recognised as the most highly affected, showing wide differences in its content distribution in fatty [8, 9] and lean [10, 11] fish, depending on the tissue considered. Further, fatty fish studies have reported notorious differences concerning the lipid damages of different zones during processing [9, 12-13].

In recent years, the fishing sector has suffered from dwindling stocks of traditional species as a result of marked changes in their availability. This has prompted fish technologists and the fish trade to pay more attention to aquaculture techniques as a source of fish and other aquatic food products. Fish farming offers the possibility to control the quality of the entire productions processes, being one of the most important objectives to obtain a final product with sensory and nutritional quality attributes as close as possible to those of wild fish [14-16].

One of such fish species is blackspot seabream (Pagellus bogaraveo). This high value commercial species has long attracted a great interest because of its firm and flavourful flesh, so that remarkable efforts have recently been focused on its commercialisation as a farmed product [17]. Thus, most research concerning its aquaculture production has been carried out on farming conditions [18] and genetic differences [19]. However, previous information concerning its composition [20] and chemical changes during processing [21] can be considered scarce.

The present work was focused on the lipid composition of blackspot seabream. Its objective was to identify elements of differentiation that characterise wild and farmed fish. Such differentiation is considered important if cultured blackspot seabream is to be used as a replacement for the wild one in the seafood European marketing system. In addition, this study was addressed to different edible sites of the fish body and was complemented by the moisture, trimethylamine oxide and trace mineral assessments.

2. MATERIALS AND METHODS

2.1. Fish material and sampling

Farmed blackspot seabream specimens (12 individuals; 0.65-0.85 kg weight; 32-35 cm length) were obtained from a local aquaculture facility (La Coruña, Spain). Fish were cultivated in a single tank by employing a sand-bed-filtered seawater (salinity range 3.2-3.4 g/ 100 g). Feeding was carried out to satiety by employing the following commercial diet: moisture (9.5%), protein (44.5%), fat (13.5%), carbohydrate (23.5%) and ash (9.0%). Fish specimens were sacrificed in a water-ice mixture in the aquaculture facility and then kept in ice for 10 h until they arrived at the laboratory. The fish were distributed into six groups (two individuals per group). Each group was studied independently in order to carry out the statistical analysis (n = 6).

Wild blackspot seabream (12 individuals; 0.65-0.80 kg weight; 34-37 cm length) were caught near the Galician Atlantic coast and obtained in a local market 10 h after being caught. From catching till arrival to laboratory the fish were kept in ice. The fish were distributed into six groups (two individuals per group). Each group was studied independently in order to carry out the statistical analysis (n = 6).

In all cases, individual gonads were at the 5th/6th stage of Maier’s scale of gonad maturity. Both for farmed and wild fish groups, the white muscle of three different zones (ventral, dorsal and tail; Figure 1) was considered, carefully separated and studied independently in each fish group. The absence of bones, blood and dark muscle was verified. Chemical analyses were carried out separately on each of the selected zones.

All solvents and chemical reagents used in the experiments were reagent grade (Merck, Darmstadt, Germany).

2.2. Chemical analyses

Moisture content was determined by weight difference of the homogenised muscle (1-2 g) before and after 4 h at 105 ºC; the results were calculated as g water/ 100 g muscle.

The lipid fraction was extracted by the Bligh and Dyer [22] method. Quantification results were calculated as g lipid/ 100 g muscle.

Trimethylamine oxide (TMAO) content was determined by previous reduction of a trichloracetic acid extract of the muscle with titanium (III) chloride [23] and further assessment of trimethylamine (TMA) content by the spectrophotometric method described by Tozawa et al. [24]. Results are expressed as g TMAO-N/ 100 g muscle.

Total phospholipids (PL) were quantified by measuring the organic phosphorus on total lipid extracts according to the Raheja et al. [25] method based on a complex formation with ammonium molybdate. Results are expressed as g PL/ kg muscle.

Total sterols (ST) were determined on total lipid extracts by the method of Huang et al. [26] based on the Liebermann-Buchardt reaction. Results are expressed as g ST/ kg muscle.

Free fatty acid (FFA) content was determined following the Lowry and Tinsley [27] method, which is based on a complex formation with cupric acetate-pyridine. In this study, benzene was replaced by toluene. Results are expressed as g FFA/ 10 kg muscle.

To measure the triacylglycerol (TG) content, the lipid extract was first purified by 20 x 20 cm thin-layer chromatography plates coated with a 0.5 mm-layer of silica gel G from Merck (Darmstadt, Germany) using a mixture of hexane/ethyl ether/acetic acid (90:10:1, vol/vol/vol; two developments) as eluent [9]. Once the TG fraction was purified, the method of Vioque and Holman [28] was used for measuring the ester linkage content, according to their conversion into hydroxamic acids and further complexion with Fe (III). Results are expressed as g TG/ kg muscle.

Tocopherol isomers were analysed according to the Cabrini et al. [29] method. For this, lipophilic antioxidants were extracted from the muscle with hexane, dried under nitrogen flux, dissolved in isopropanol and injected into the HPLC system (ODS column, 15 cm x 0.46 cm i.d.); detection was achieved at 280 nm. The presence of the different tocopherol isomers was checked. Only the α-tocopherol isomer was detected, and its content was expressed as mg α-tocopherol/ kg muscle.

Total lipid extracts were converted into fatty acid methyl esters (FAME) by employing acetylchloride and then analysed by gas-liquid chromatography (Perkin-Elmer 8700 chromatograph), according to previous procedure [13]. For it, a fused silica capillary column SP-2330 (0.25 mm i.d. x 30 m, Supelco, Inc., Bellefonte, Pa, USA) was employed. Peak areas were automatically integrated, 19:0 fatty acid being used as internal standard for quantitative analysis. Content of each fatty acid is expressed as g/ kg muscle.

Seven essential minerals (Ca, Cu, Fe, Mg, Mn, Se and Zn) were chosen for the present study as being included among the most abundant oligoelements [1] and according to previous farmed/ wild fish comparative studies [15, 30].

Edible flesh samples were dried in a stove at 50º C until constant weight and later ground in a mortar. Then, a fraction of ca. 500 mg of each sample was weighed in a Teflon vessel of 40 ml and a mixture of 4 ml of 65 % HNO3 and 1 ml of H2O2 was added to carry out the microwave digestion. To be digested, the different muscle mixtures were introduced in a Milestone 1200 Mega microwave groupedin series of seven samples plus one blank and one certified material reference (DORM-2, National Research Council Canada)to verify the correct sample solution. Concentration values (mg/ kg muscle) for the different minerals in the reference material (found and certified, respectively) were as follows: Ca (1.08±0.07 and not certified), Cu (2.03±0.57 and 2.34±0.16), Fe (127±20 and 142±10), Mg (1.02±0.09 and 1.01±0.04), Mn (3.00±0.23 and 3.66±0.34), Se (1.49±0.05 and 1.40±0.09) and Zn (23.3±0.9 and 25.6±2.3).

Mineral contents of the digested sampleswere determined by atomic absorption spectrometry (AAS). Ca, Fe, Mg and Zn were analysed by means of flame atomic absorption spectrometry (FAAS) using a Varian 220 FS apparatus. Cu, Mn and Se were analysed by means of electrothermal atomic absorption spectrometry (ETAAS) using a Varian 220 apparatus equipped with Zeeman background correction. Quantification results are expressed as mg/ kg muscle, except for Ca and Mg (g/ kg muscle).

2.3. Statistical analysis

Data (n = 6) obtained from the different chemical analyses were subjected to the ANOVA method (p<0.05) to explore differences by two different ways: fish origin (farmed/ wild comparison) and body zone (ventral/ dorsal/ tail comparison) (Statsoft Inc., Statistica, version 6.0, 2001). Comparison of means was performed using a least-squares difference (LSD) method. For parameters where a non-homogeneous variation was detected, the non-parametric Kruskal-Wallis test was employed.

3. Results and Discussion

3.1. General composition parameters

In all muscle zones studied, moisture content (Table 1) was found higher for wild fish than for its counterpart obtained from farming conditions. However, no significant differences could be concluded among muscle sites for both kinds of fish material.

A higher lipid content (Table 1) was obtained in farmed fish than in wild one, this according to the known inverse ratio between water and lipid constituent values [1]. This higher lipid content can be explained as a result of the different diet and live conditions corresponding to both kinds of fish and agrees to previous comparative research on other fish species such as turbot [14, 30], gilthead seabream [16] and seabass [31]. As for moisture content, no differences could be observed among muscle sites for both wild and farmed fish.

Water and lipid contents obtained for the white muscle of wild blackspot seabream agree to those reported for other lean fish species [1, 11]. Contrary, contents on such constituents for the farmed fish can be considered as belonging to a fattier-type fish pattern than the wild one [21]. However, lipid content values for farmed fish muscle can be considered low when compared to previous studies on related farmed fish species such as seabass [15, 31] and sea bream [16, 31], where fattier diets were employed. Such differences can be explained on the basis of the strong and direct influence of lipid content in diet on lipid content of the farmed product [14, 32].

TMAO molecule has been recognised as an ubiquitous constituent of marine species, supporting an important role in the osmoregulation during the live animal period [2]. After the fish death, TMAO can develop different kinds of breakdown damages during the processing/ storage process that lead to different spoilage molecule formation such as TMA, dimethylamine and formaldehyde, this implying an important effect on quality loss [33, 34].

In the present study, TMAO-N content (Table 1) for wild fish was found largely higher than for its counterpart obtained from farming conditions. This result agrees to previous comparative research on cod [35] and olive flounder [36] and may be related to the different live conditions. Accordingly, a higher formation of the above mentioned spoilage molecules is to be expected in wild fish muscle than in its counterpart from farming condition when technological treatments are applied. When the comparative zone study is considered, both kinds of fish showed higher mean values in the tail site than in the two others, being this difference significant in the case of farmed fish. To our knowledge, no previous research accounts for the distribution of TMAO in different muscle zones of the fish body. The higher value obtained in the tail zone in the present study may be explained as a result of a greater interchange surface with the live medium in this zone; thus, in agreement to the osmoregulation role accorded to TMAO, a higher TMAO content would be necessary in this zone to prevent dehydration.

Previous research has shown that the highest TMAO-N contents are described in elasmobranches, squids and gadoid fish (750-2500 mg TMAO-N/ kg muscle) [2, 33, 34]. Present results prove that TMAO-N content in farmed blackspot seabream is relatively low [21], being similar to that reported for flat and pelagic fish species [33, 34].

3.2. Lipid composition

Table 2 shows the lipid group contents obtained in the three muscle locations of wild and farmed blackspot seabream.

When PL, ST and FFA are considered, no differences between farmed and wild fish are observed. Such lipid groups are considered as functional ones, so that their content and presence in the muscle should not be specially influenced by external factors such as water temperature, feeding availability and its composition [7]. Additionally, differences among muscle zones were not obtained in both kinds of fish in the present work. Again, this result agrees to the fact that such kinds of lipid groups develop a structural role in living bodies, so that its presence in the muscle is hardly affected by internal factors such as anatomical and physiological aspects [7, 14]. This difference lack among muscle sites agrees to previous research on PL [37] and cholesterol [31] contents related to other fish species.

Free fatty acid content has been recognised as a good indicator of lipid hydrolysis during processing and storage of fish species [38]. Value ranges found in the present study correspond to a very low lipid hydrolysis development [11, 21], according to the fact that fish was analysed immediately after catching/ slaughtering. Thus, values obtained in the present research should correspond to the in vivo metabolic action of lipases and phospholipases on high-molecular-weight lipids such as TG and PL, respectively. According to data shown in Table 2, a different hydrolytic enzyme activity in the different muscle zones was not observed for both kinds of fish.

Results are different when a depot lipid class like TG is concerned. In this case, a higher content was obtained for farmed fish than for its counterpart from wild condition. This higher level can be explained as a result of the diet and the live conditions, and agrees to the higher lipid content mentioned in Table 1 for farmed fish. As for the other lipid groups, no differences among zones could be detected for both wild and farmed fish.

Alpha-tocopherol contents obtained in fish muscle are also expressed in Table 2. Values were included in the range reported for most fish species [1, 39]. However, higher values were obtained for wild fish than for its counterpart from farming condition. This difference could be explained as a result of a different diet intake between both kinds of blackspot seabream, according to previous comparative research [40, 41]. Indeed, if the α-tocopherol/ total lipid ratio is considered, an even lower value is observed in each zone in the farmed fish when compared with its corresponding site in the wild one (30-43 and 120-180 mg/ 100 g lipids for farmed and wild fish, respectively). Concerning the muscle zone comparison, lower mean values could be detected for the tail site than in the two others; such differences were significant in the case of wild fish between ventral and tail zones. Some different distribution of α-tocopherol in muscle was also observed in higher-size farmed fish species such as turbot (Scophthalmus maximus) and Atlantic halibut (Hippoglossus hippoglossus) [42].