**2. Materials and methods**

#### **2.1. Sample collection**

All mollusk samples were purchased from two mussel farms in spring (March 2015), summer (July 2015), and autumn (October 2015). The mussel farms are located in one of the most ecologically non-polluted areas along the Northern part of the Bulgarian Black Sea coast (Kavarna). The samples were immediately frozen at −20°C and stored in a fridge. The biometric characteristics as mean weight (g) and mean length (cm) were determined (**Table 1**).

Average 40 specimens of mussels (from each season and each farm) were used for a proximate, fatty acid and fat-soluble vitamins, cholesterol and pigments analysis. All shucked mussels were cut into small pieces and homogenized at 800 rpm for 5 min, using a Moulinex blender.


**Table 1.** Biometric characteristics of mussel samples (mean ± SD).

#### **2.2. Standards and reagents**

marine environments. Nowadays, mussels are harvested commercially and are of considerable significance for aquaculture worldwide. Farmed marine mussels from the Mytilidae family, especially genera *Mytilus*, are important for the human diet in the provision of high levels of proteins, omega-3 polyunsaturated fatty acids (PUFAs), fat-soluble vitamins, and carbohydrates. In recent years, the functional properties of mussel lipids have been investigated and few dietary supplements, based on lipid extracts of mussels, have been presented at the market [1, 2]. Due to these facts, the importance of marine mussels as a source for bioactive substances with anti-inflammatory, antimicrobial, and lowering cholesterol level agents, is increasing rapidly. In addition, mussels have recently become one of the most commercially important species from the Bulgarian Black Sea [3]. The assessment of the proximate composition and the lipid qualities may facilitate consumer acceptance and predict the market feasibility of aquaculture mussels in our region. However, the information about the nutritional qualities of mussels from the Bulgarian Black Sea waters, based on their chemical composition, fat-soluble pigments, cholesterol and PUFA contents is very limited. In this article, we studied the seasonal changes of mussel primary metabolites as proteins, lipids, and carbohydrates with a focus on lipid bioac-

tive components such as fatty acids, cholesterol, fat-soluble vitamins (A, E and D<sup>3</sup>

of the Bulgarian Black Sea coast.

**2. Materials and methods**

**2.1. Sample collection**

182 Biological Resources of Water

(astaxanthin, beta-carotene) in farmed mussels (*Mytilus galloprovincialis*) from the Northern part

All mollusk samples were purchased from two mussel farms in spring (March 2015), summer (July 2015), and autumn (October 2015). The mussel farms are located in one of the most ecologically non-polluted areas along the Northern part of the Bulgarian Black Sea coast (Kavarna). The samples were immediately frozen at −20°C and stored in a fridge. The biometric characteristics as mean weight (g) and mean length (cm) were determined (**Table 1**).

Average 40 specimens of mussels (from each season and each farm) were used for a proximate, fatty acid and fat-soluble vitamins, cholesterol and pigments analysis. All shucked mussels were cut into small pieces and homogenized at 800 rpm for 5 min, using a Moulinex blender.

Mean weight 11.0 ± 0.5 12.0 ± 0.5 13.0 ± 0.5 Mean length 4.5 ± 0.3 5.5 ± 0.5 6.0 ± 0.5

Habitat Demersal Food habits Herbivorous

**Table 1.** Biometric characteristics of mussel samples (mean ± SD).

n, number of specimens; SD, standard deviation.

**Spring (n = 45) Summer (n = 43) Autumn (n = 46)**

) and pigments

Fatty Acid Methyl Esters (FAME) Mix standard (SUPELCO FAME, Mix C4-C24), and nonadecanoic acid and methyl ester nonadecanoic acid standards were purchased from Sigma– AldrichTM. Pure solid substances of all-trans-retinol, cholecalciferol, alpha-tocopherol, astaxanthin, beta-carotene, and total-cholesterol are HPLC-grade reagents, purchased from Sigma-AldrichTM. All used chemicals were of analytical, HPLC, and GC grade (Scharlau, Scharlau Sourcing Group, Spain).

#### **2.3. Proximate composition analysis**

The homogenized mussel tissues (2.000 ± 0.005 g) were dried at 105 ± 2°C in an air oven for 16–18 hours to a constant weight [4]. The moisture was calculated as weight loss. The crude protein content was determined by the Kjeldahl method [5]. The total lipids (TLs) were estimated according to Bligh and Dyer procedure [6] and the results were presented as g per 100 g wet weight (g 100 g−1 ww). The carbohydrates were determined according to [7]. The method was based on the treatment of the mussels' tissue with a methanolic KOH solution, followed by acid hydrolysis of starch to glucose. The glucose quantity was determined through the oxidation with a bivalent copper from a copper reagent and was then converted into starch. The energy values were calculated by multiplying fat, protein, and carbohydrate with appropriate coefficients (4.0 kcal/g for proteins and carbohydrates and 9.0 kcal/g for lipids) [8].

#### **2.4. Fatty acid analysis**

Fatty acid composition of total lipids at edible mussel tissue was determined by GC of the corresponding methyl esters. The residual lipid fraction was methylated by base-catalyzed transmethylation, using 2 M methanolic KOH and n-hexane according to [9]. To determine the analytical recoveries, the samples of homogenized tissue were spiked with a methanolic solution containing C19:0 (1 mg/ml). Gas chromatography analysis was performed by a FOCUS GC, autosampler A 3000, Polaris Q MS detector (Thermo Scientific, USA). The capillary column was a TR-5 MS (Thermo Scientific, USA), 30 m, 0.25 mm i.d. Helium was used as a carrier gas at flow rate 1 ml/min. Chromatographic separation of fatty acids methyl esters was performed under the following temperature regime: 40°C initial temperature for 4 min, followed by 10°C increase per minute until 235°C were reaches, temperature increase up to 280°C with a stay at this level for 5 min. The sample volume was 1 μl. The injector was a split/splitless injector operated in the split mode (1:10). Peak identification was measured by: retention time (RT) based on fatty acid methyl esters (FAME) mix standard (SUPELCO F.A.M.E. Mix C4-C24), and mass spectra (ratio *m/z*) compared to the internal Data Base (Thermo Sciences Mass Library; Thermo Corporation, Waltham, USA). FAMEs were quantified by the method of external standard. The FA content was expressed as a percentage of total FAs content [10].

#### **2.5. Extraction of fat soluble vitamins, cholesterol, pigments, and HPLC analysis**

The edible tissue of the mussels from the three different farms was used to evaluate its astaxanthin, beta-carotene, and cholesterol content. The extraction and quantity analysis was performed by the method of Dobreva et al. [11]. An aliquot of the homogenized sample (1.000 ± 0.005 g) was weighed into a glass tube with a screw cap, 1% of methanolic L-ascorbic acid and 0.3 M methanolic KOH were added. Six parallel samples of edible tissue from each mussel farm were prepared and subjected to saponification at 50°C for 30 min. The fat-soluble components of interest were extracted with two portions of n-hexane: dichloromethane = 2:1 solution. The combined extracts were evaporated under a nitrogen flow and the dry residue was dissolved in methanol: dichloromethane and injected (20 μl) into the HPLC/UV/FL system. All fat-soluble compounds were analyzed simultaneously by an HPLC system, equipped with an RP analytical column (Synergi Hydro-RP (80 Ǻ, 250 × 4, 6 mm, 4 μm)). Astaxanthin, beta-carotene, and cholesterol were identified by UV detection. The mobile phase composition was ACN:MeOH:iPrOH = 75:20:5 v/v, with the flow rate being 1 mL/min. The qualitative analysis was performed by comparing the retention times of pure substances: at λmax = 208 nm for cholesterol, at λmax = 450 nm for beta-carotene, and λmax = 474 nm for astaxanthin. The quantitation was performed by external calibration, comparing the chromatographic peak areas of the corresponding standards (Astaxanthin, Supelco; Cholecalciferol, Supelco, and Betacarotene, Supelco). The results were expressed as μg per 100 g wet weight (μg·100 g−1ww).

individual FA, FA groups, fat-soluble vitamins, cholesterol and carotenoids, and nutritional

Assessment of Proximate and Bioactive Lipid Composition of Black Sea Mussels...

http://dx.doi.org/10.5772/intechopen.71909

185

The proximate composition of edible mussel tissue varied with the season (**Table 2**). The assessment of the nutritional quality based on the macronutrients content in black mussel was conducted in accordance with Commission Regulation (EC) No. 116/2010 [17]. As water is the main component of mussel tissue, the levels of moisture are ranged between 73.35 and

Seafood products are considered "low fat" when containing below 3 g of lipids per 100 g wet weight (ww). In the present study, the range of the total lipids (TLs) content is between 1.40 and 2.89 g 100−1 g ww. The highest TL was found in spring mussels, whereas summer specimens presented twice lower values (P < 0.001). TLs that amount below 3 g 100−1 g ww were found in all analyzed seasons; therefore, Black Sea mussels can be classified as "low

The lack of considerable variation in the protein content during the seasons is well illustrated in the results (see **Table 2**). According to [8], the seafood protein content below 15% is considered low. In this study, a significant decrease of the protein content was found in the autumn period as compared to the spring sample (P < 0.001). However, protein levels were significantly above 15% in all samples, and the analyzed mussels can be classified as protein-rich

The observed seasonal pattern in the carbohydrate amounts showed the highest levels in the autumn season and the lowest in the summer period. The accumulated carbohydrates could be utilized under unfavorable conditions and the observed variation in the mussel tissue indicates that the level of mobilized carbohydrate reserves may vary widely and rapidly in response to fluctuation in environmental conditions [18, 19]. It was observed that in warmer seasons, the carbohydrate contents were higher than TL contents in the mussel

**Lipid Protein Carbohydrate Moisture Energy value**

Spring 2.89 ± 0.10 19.92 ± 0.80 2.25 ± 0.08 73.35 ± 1.55 115.00 ± 5.50 Summer 1.40 ± 0.08<sup>a</sup> 18.30 ± 0.50 2.00 ± 0.06 77.15 ± 1.60<sup>a</sup> 94.50 ± 4.50<sup>a</sup> Autumn 2.51 ± 0.12c 17.40 ± 0.55<sup>b</sup> 2.73 ± 0.10b,<sup>c</sup> 76.20 ± 1.40<sup>b</sup> 103.20 ± 5.20<sup>b</sup>

**Table 2.** Proximate composition in molluscs tissue, in g 100 g−1 ww and kCal 100−1 g ww (mean ± SD).

quality indices. The differences were considered significant at p < 0.05.

77.15%, being higher in the summer samples in comparison to other seasons.

**3. Results and discussion**

**3.1. Proximate composition**

fat" food.

a

b

c

regardless of the season.

P < 0.001 (spring vs. summer).

P < 0.001 (spring vs. autumn).

P < 0.001 (summer vs. autumn).

#### **2.6. Nutritional quality indices**

Nutritional qualities were estimated by several indices and ratios of fatty acid composition: the indices of atherogenicity (AI), thrombogenicity (TI), cholesterolemic index (h/H), n-6/n-3 and PUFA/SFA ratios, according to Simopoulos [12]. Ulbricht and Southgate [13] suggest two indices, AI and TI, which might better describe the atherogenic and thrombogenic potential of different unsaturated FA. AI indicates the relationship between the sum of the main saturates and that of the main unsaturated, the former being considered pro-atherogenic (favoring the adhesion of lipids to cells of the immunological and circulatory systems), and the latter being anti-atherogenic (inhibiting the aggregation of plaque and diminishing the levels of esterified FA, cholesterol and phospholipids, thus preventing the appearance of micro- and macrocoronary diseases). TI shows the tendency to form clots in the blood vessels and is defined as the relationship between the pro-thrombogenic (saturated) and the anti-thrombogenic FAs (MUFA, n-6 PUFA, and n-3PUFA). The cholesterolemic index (h/H) presents the functional effects of different PUFAs of the cholesterol metabolism (hypo- and hyper-cholesterolemic effect) and is calculated in accordance with the method, described elsewhere [14]. In addition, the hyperlipidemic and atherogenic potential of mussel lipids, related to cholesterol, SFA, and unsaturated FA content, were determined. To assess the dietary effect of the mussel lipid consumption on serum cholesterol levels, two indices were calculated: cholesterol/SFA index (CSI) and cholesterol index (CI) [15, 16].

#### **2.7. Statistical analysis**

All analytical estimations were performed in triplicate. The results were expressed as a mean and standard deviation (mean ± SD). The obtained data were analyzed using Graph Pad Prism 5.0 software. An unpaired t-test statistical analysis was performed to estimate the differences between the analyzed samples. Thus, the comparison was made for proximate compositions, individual FA, FA groups, fat-soluble vitamins, cholesterol and carotenoids, and nutritional quality indices. The differences were considered significant at p < 0.05.
