FAT SOLUBLE VITAMINS AND FATTY ACIDS COMPOSITION OF BLACK SEA CYSTOSEIRA BARBATA

Brown alga Cystoseira barbata is the most widely distributed seaweed in the Black Sea. There is limited information about fat soluble vitamins content and fatty acids composition of this specie from Bulgarian Black Sea coast. The aim of this study was to determine fat soluble vitamins, pigments, total lipid and fatty acid composition of Cystoseira barbata. Fat soluble vitamins (vitamin E and D), pigments (β-carotene and astaxanthin) and total cholesterol were analyzed simultaneously using HPLC/UV/FL system equipped with RP analytical column. Sample preparation procedure includes alkaline saponification, followed by liquidliquid extraction. Brown seaweed Cystoseira barbata contained high amounts of α-tocopherol and βcarotene. Lipids were extracted by following the method of Bligh and Dyer. The residual lipid fraction was methylated using base-catalyzed transmethylation with methanolic potassium hydroxide. Fatty acid composition was analyzed by GC/MS. Cystoseira barbata was rich in linoleic (C18:2n6) and eicosopentaenoic acid (C20:5n3) although total lipid content was generally low. High levels of αtocopherol correlate with high levels of polyunsaturated fatty acids. As an antioxidant α-tocopherol preserves tissue PUFA from oxidation.


Introduction
Seaweeds have been used since ancient times as food, fodder, fertilizer and as source of medicine. Nowadays seaweeds represent an inexhaustible source of the raw materials used in pharmaceutical, food industries, medicine and cosmetics. They are nutritionally valuable as fresh or dried vegetables, or as ingredients in a wide variety of prepared foods. In particular, seaweeds contain significant quantities of protein, lipids, minerals and vitamins (Manivannan et al., 2008).
Lipids represent only 1-5% of algal dry matter and exhibit an interesting polyunsaturated fatty acid (PUFA) composition particularly omega 3 and omega 6 acids which play an important role in the prevention of cardio vascular diseases, osteoarthritis and diabetes. Brown algae are rich in fatty acids with 20 carbons: eicosapentaenoic acid (EPA, C 20:5 n3) and arachidonic acid (AA, C 20:4 n6) (Banerjee et al., 2009). Marine algae are rich in PUFAs of the n-3 and n-6 series, which are considered essential fatty acids for humans and animals. Some of these FAs (20:3n-6, 20:4n-6, 20:5n-3) have high biological activity and are converted into eicosanoids. In addition, PUFAs are of interest in cosmetics as components of sun lotions and as regenerating and anti-wrinkle products. Because of the huge and renewable biomass and the fact that many of them could easily be cultivated in the sea on a large scale, seaweeds are a potential source of fatty acids for biotechnology and a dietary source of essential fatty acids (Khotimchenko et al., 2002).
Seaweeds are a good source of some water-(B1, B2, B12, C) and fat-soluble (β-carotene with vitamin A activity, vitamin E) vitamins. Seaweed vitamins are important not only due to their biochemical functions and antioxidant activity but also due to other health benefits such as decreasing blood pressure (vitamin C), prevention of cardiovascular diseases (β-carotene), or reducing the risk of cancer (vitamins E and C, carotenoids) (Skrovankova, 2011).
Bulgarian Black Sea coast is rich in algae, regarding biomass and algal biodiversity. According to Guiry and Guiry (2013), taxonomically, the species Cystoseira barbata belongs to the division Ochrophyta, class Phaeophyceae, order Fucales, family Sargassaceae, genus Cystoseira. It represents a group of macroscopic, multicellular algae, widely distributed along the coastal cliffs and rocks, which often form underwater "meadows". Negreanu-Pirjol et al., (2012) reported that lipid soluble substances of Cystoseira barbata possess higher antioxidant activity, compared to other species from the Black Sea. Information available about the lipid composition of this macroalgae is scarce. The main objective of this investigation was to provide knowledge on chemical composition of Cystoseira barbata as an alternative source of fatty acids and fat soluble vitamins.

Data and methodology
Cystoseira barbata was collected in July, 2012 from the region of Varna Bay, Bulgaria. All of the samples were harvested manually from their respective sites and then transported to the laboratory in wet tissue towels in an ice box. They were thoroughly cleaned to remove epiphytes and detritus attached to the fronds. Cleaned samples were frozen and stored at -20°C prior to analysis. The moisture content was determined by oven method at 105°C until constant weight (AOAC, 2000).

GC-MS analysis
Lipids were extracted by following the method of Bligh and Dyer (1959). Algal tissue was extracted first with chloroform: methanol (1:2, v/v) and the residue were extracted thrice with small portions of chloroform: methanol (1:1, v/v). All the extracts were pooled together, filtered and mixed with an equal volume of chloroform and water (1:1, v/v) for phase separation. The lower organic phase was collected and evaporated to dryness in a vacuum, and the total lipids were determined gravimetrically. The residual lipid fraction was methylated by base-catalyzed transmethylation using 2M methanolic potassium hydroxide and n-hexane. After 10 minutes centrifugation (3500 rps), the hexane layer was taken for GC analyses. Gas chromatography was performed by a model FOCUS Gas Chromatograph with autosampler A 2000, equipped with Polaris Q MS detector (Thermo Scientific, USA). The capillary column used was a TR-5 MS (Thermo Scientific, USA) universal column 30m length and 0.25mm i.d., with a wide range of applications for food analysis. Helium was used as a carrier gas at flow rate 1 ml/min. Chromatographic separation was achieved by temperature range: initial temperature -40°C for 4 min followed by 10°C per minute until 235°C and final temperature reach was 280°C for 5 min. The injection volume was 1µl. Peaks were identified according to two parameters: Retention Time (RT) based on available FAME mix standard (SUPELCO F.A.M.E. Mix C4-C24) and mass spectra (ratio m/z) -compared to internal Data Base (Thermo Sciences Mass Library, USA). All analytical determinations were performed in triplicate. Values were expressed as percentage of total fatty acid mass as a mean value and ± standard deviation (SD). All of the chemicals used in the experiments were analytical grade and GC grade (Sharlau).

HPLC analysis
Astaxanthin, β-carotene, α-tocopherol, ergocalciferol and total cholesterol were determined simultaneously using HPLC/UV/FL system equipped with RP analytical column. Sample preparation was performed following the method described by Dobreva, Galunska and Stancheva (2011) with some modifications. Generally, an aliquot of the homogenized tissue (1.500±0.005g) was weighed into a glass tube with a screw cap and 1% of methanolic L-ascorbic acid and 0.3M methanolic potassium hydroxide were added. Six parallel samples of algae tissue were prepared and saponified at 40 o C for 30 min. After cooling the lipids were extracted with n-hexane: dichloromethane. The extracts were pooled together and evaporated under nitrogen. The dry residue was dissolved in methanol: dichloromethane and injected into the HPLC system (Thermo Scientific Spectra SYSTEM). Chromatography was run as a gradient at 1.1 mL/min with a Synergi 4µ Hydro-RP 80A pore 250x4,6 mm reversed-phase column. Solvent A contained methanol-water (93:7), solvent B contained acetonitrile and solvent C -2-propanol. The gradient changed as follows: 0-16.0 min, 100 % solvent A, 20.0-30.0 min, 60% solvent B and 40 % solvent C, 30.0-40.0 min, 50 % of solvent B and 50% solvent C. The gradient was then returned to 100 % of solvent A. Detection of astaxanthin, β-carotene, ergocalciferol and cholesterol was performed by UV detector (for astaxanthin λ=474nm, for βcarotene λ=450nm, for ergocalciferol λ= 265 nm, for cholesterol λ=208 nm). Concentration of αtocopherol was measured by fluorescence at λex = 288 nm and λem = 332 nm. The quantitation was performed by the method of the external calibration, comparing the chromatographic peak areas of the samples with those of the corresponding standards.

Results and discussion
The total lipids content of Cystoseira barbata accounted for 3.35±0.58g per 100 g on a dry weight basis. The fatty acid composition is presented in Table 1. Saturated fatty acids (SFA) were most abundant components with 70.63% of total fatty acids (TFA). The total sum of monounsaturated fatty acids (MUFAs) was 13.08%, whereas total PUFAs were 16.29%.
Algae are a source of vitamins (liposoluble and hydrosoluble) available all along the Black Sea coast, yet not usually consumed fresh or dried. Results obtained from HPLC analysis for various liposoluble constituents in Cystoseira barbata are shown in Table 2. The value of β-carotene in algae in this study exceeds those reported by Gayathri et al., (2004) for fresh carrot (88.33 µg/g) and other vegetables of mass consumption like spinach, cabbage, Swiss chard, averaging 25µg/g. Another interesting carotenoid was detected in Cystoseira barbata algal samples. Astaxanthin content accounted for 1.49±0.04 mg per 100g dry weight. Many studies have associated high dietary intake of carotenoids with lower risks of certain pathologies (Tapiero et al., 2004).
Total cholesterol content was 4.67±1.11 mg per 100g d.w. Although, further analysis of the individual sterol composition is needed, Milkova et al., (1997) reported that Cystoseira barbata from the Black Sea contained in the sterol fraction almost pure fucosterol.

Conclusion
Lipids, fatty acids composition, α-tocopherol, ergocalcipherol, β-caroten, astaxanthin and total cholesterol in brown algae Cystoseira barbata were analyzed in the present study. Palmitic acid (C16:0) was the most abundant fatty acid, followed by EPA (20:5, n-3). The high concentrations of αtocopherol, β-carotene, polyunsaturated fatty acids and the presence of the powerful antioxidant astaxanthin demonstrate possible application of this macroalga as a supplement for use in food and pharmaceutical industry and aquaculture. Further studies concerning proximate analysis, amino acids, carbohydrates, biogenic and toxic elements, as well as individual sterol composition are necessary in order to provide more information for safer and more versatile utilization of these seaweeds.