Influence of Extractive Solvents on Lipid and Fatty Acids Content of Edible Freshwater Algal and Seaweed Products, the Green Microalga Chlorella kessleri and the Cyanobacterium Spirulina platensis

Total lipid contents of green (Chlorella pyrenoidosa, C), red (Porphyra tenera, N; Palmaria palmata, D), and brown (Laminaria japonica, K; Eisenia bicyclis, A; Undaria pinnatifida, W, WI; Hizikia fusiformis, H) commercial edible algal and cyanobacterial (Spirulina platensis, S) products, and autotrophically cultivated samples of the green microalga Chlorella kessleri (CK) and the cyanobacterium Spirulina platensis (SP) were determined using a solvent mixture of methanol/chloroform/water (1:2:1, v/v/v, solvent I) and n-hexane (solvent II). Total lipid contents ranged from 0.64% (II) to 18.02% (I) by dry weight and the highest total lipid content was observed in the autotrophically cultivated cyanobacterium Spirulina platensis. Solvent mixture I was found to be more effective than solvent II. Fatty acids were determined by gas chromatography of their methyl esters (% of total FAMEs). Generally, the predominant fatty acids (all results for extractions with solvent mixture I) were saturated palmitic acid (C16:0; 24.64%–65.49%), monounsaturated oleic acid (C18:1(n-9); 2.79%–26.45%), polyunsaturated linoleic acid (C18:2(n-6); 0.71%–36.38%), α-linolenic acid (C18:3(n-3); 0.00%–21.29%), γ-linolenic acid (C18:3(n-6); 1.94%–17.36%), and arachidonic acid (C20:4(n-6); 0.00%–15.37%). The highest content of ω-3 fatty acids (21.29%) was determined in Chlorella pyrenoidosa using solvent I, while conversely, the highest content of ω-6 fatty acids (41.42%) was observed in Chlorella kessleri using the same solvent.


Introduction
Many recent studies have focused on the chemical composition of seaweeds, their positive contributions to human health and their possible usage as foodstuffs. Seaweed consumption has a long tradition in Asian countries and has increased in European countries in recent years, therefore approximately 20 species of edible algae are now available on the European market. Nowadays, freshwater algae and seaweeds have been extensively studied as good sources of many bioactive substances such as fatty acids, sterols, proteins, amino acids, minerals, polysaccharides or selected halogenated compounds, with extensive health benefit activities [1][2][3][4][5][6]. Freshwater algae and seaweeds, like fruits and vegetables, exhibit antibacterial, anti-inflammatory, anticancer, antiviral, anticoagulant, and other interesting properties [7][8][9]. There are many possibilities for their usage, especially in medicine, pharmacy and the food industry. For instance, seaweeds have been utilized industrially as a source of agar, carrageenans and alginates [1,10,11] and freshwater algae and seaweeds have been evaluated as nutraceutical foods [12,13].
Lipids, including their fatty acids (FAs), are essential human nutrients that can be classified as saturated (SFAs), monounsaturated (MUFAs), and polyunsaturated FAs (PUFAs), according to the absence or presence of unsaturated bonds. Humans are able to synthesize SFAs and MUFAs, but unfortunately, PUFAs with the first double bond on the third or sixth carbon atom (essential fatty acids-EFAs) are essential because they cannot be synthesized by humans [4,14].
The contemporary Western human diet is known for an increased intake of SFAs and ω-6 FAs that results in an imbalance of ω-3 and ω-6 FAs [4,23]. Seaweeds contain a higher proportion of ω-3 FAs, which are components of all cell membranes and are precursors of biochemical and physiological reactions in the body, acting against atherosclerosis, hypertension, inflammatory diseases, cystic fibrosis, rheumatoid arthritis and helping prevent mental illnesses [2,4,24].
The chemical composition of seaweed is affected by many factors such as the seaweed species, location and time of harvest, intensity of light, water chemistry at the location, and the part of plants used [2,25]. Studies of the lipid profile in seaweeds have investigated the seasonal variation [26], effect of growth conditions [27,28], and differences among diverse seaweed tissues [29]. Most studies which were focused on the total lipid contents and on the FA profiles have concerned fresh seaweeds [26,28,29]. However, the effect of algae and seaweed processing, i.e., drying, packaging, transporting, and subsequent storage, on the lipid content and FAs composition is rarely studied.
The present paper evaluates and compares the lipid profiles of nine commercially available seaweed microalgal and cyanobacterial products, and autotrophically cultivated samples of the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. In addition, yield of lipids and FA profiles of seaweeds extracted using different solvents were compared. The new information presented herein should be useful to support more abundant consumption of dry seaweed products as a source of ω-3 and ω-6 FAs, and also for better revaluating the real contribution of freshwater algal and seaweed products to PUFA enrichment of the human food chain.

Total Lipid Contents
Total lipid contents of analyzed samples were determined in extracts obtained by different solvents (Table 1). Evidently, extraction using a 1:2:1 mixture of methanol/chloroform/water (solvent I) resulted in higher contents of lipids among all determined samples, ranging from 1.32% (P. palmata) to 18.02% (S. platensis), whereas the extraction with hexane (solvent II) was less effective in relation to lipid contents, that ranged from 0.64% (P. palmata) to 13.41% (S. platensis) for the same algal samples as in the previous analysis.
Moreover, the total lipid content seemed to be influenced by the cultivation or technology process. Here, the results showed much higher total lipid contents in autotrophically cultivated microalgae compared to freshwater edible products in pill form using both solvent systems. In detail, autotrophically cultivated cyanobacterium S. platensis contained 18.02% lipids (I), in comparison with 10.23% in the spirulina product (I). Similarly, 18.01% was measured in the autotrophically cultivated green microalga C. kessleri compared to 3.70% in the green microalga C. pyrenoidosa product after extraction with solvent I. Further, the content of total lipids obtained from the green microalga C. pyrenoidosa with solvent I (3.70%) was lower than established by Ortega-Calvo et al. in dry C. vulgaris algal product (8.6%) after extraction by dichloromethane/methanol (2:1) [19]. Similarly, D'Oca et al. presented results of lipid contents in C. pyrenoidosa in the range from 1.55% to 20.74%, depending on the different solvents and extraction methods used [18]. A similar result (14.3%) for total lipid content in the cyanobacterium S. platensis after extraction by a mixture of chloroform/methanol (2:1) was published by Babadzhanov et al. [17]. On the other hand, Ortega-Calvo et al. reported much lower contents of lipids in the cyanobacterial food products of S. platensis, S. maxima, and Spirulina sp. (6.4%-7.5% in dry weight) using a mixture of dichloromethane/methanol (2:1) [19].
It was obvious that freshwater green microalgae and cyanobacteria contained higher concentrations of lipids than seaweed products. This could be caused by the specific metabolism and growth conditions of these algae.
In accordance with our findings, significant discrepancies in the efficiency of various solvent mixtures used for lipid extraction were reported in several studies [15,[17][18][19][20]. That could be caused by the presence of different lipid components in the algal biomass. Generally, a higher amount of polar lipid compounds in algal biomass results in worse lipid extraction yields by nonpolar solvents and vice versa [18].
Significant differences in the FA composition of the nine algal products and the autotrophically cultivated S. platensis and C. kessleri were established. Variations in FA contents are ordinarily attributable both to environmental and genetic differences [20,32]. Nevertheless, the extractive solvents had an influence on the number and amount of identified FAs. The impact of the solvents used on the total number of identified FAs and different amounts of determined SFAs, MUFAs and PUFAs are presented in Figures 1-4.    From Figure 1 can be concluded that 9 to 22 or 7 to 21 FAs were identified, respectively, depending on whether solvent I or II was used. The solvent I was found more effective than solvent II in relation to a number of identified FAs for almost all samples, except for autotrophically cultivated S. platensis, where the same efficiency for both solvents was established.
The different efficiency of the two solvents used for the extraction of algal lipids in relation to the proportions of the different FAs is evident too from the obtained results shown in Figures 2-4. Higher amounts of SFAs ( Figure 2) were obtained from all samples except for Spirulina genus using solvent II. The solvent I was more effective for PUFAs for almost in all samples, except for both Spirulina genus samples and the product from the brown seaweed H. fusiformis ( Figure 3). Further, solvent II was more effective for MUFA extraction in both samples of the green freshwater algae C. kessleri and C. pyrenoidosa and in the three brown seaweed products L. japonica, and U. pinnatifida (W, WI). Finally, in samples of autotrophically cultivated S. platensis and E. bicyclis, no difference between the two solvents used was observed ( Figure 4).

Fatty Acid Profiles
FA compositions of cyanobacterial, microalgal and seaweeds products and two samples of autotrophically cultivated green microalga and cyanobacteria obtained by different solvent extractions are presented in Tables 2 and 3 and the results are given in % of total FAMEs.

Saturated Fatty Acids
Pursuant to published data, the most abundant groups of algal lipids among the total FAMEs are SFAs or PUFAs, depending on the algal species [15,33]. The majority of the investigated samples showed the highest proportions of SFAs in their FAMEs distribution regardless of the solvent used. The highest contents of SFAs obtained with the solvents I and II were established in the red seaweeds P. palmata (86.58%/93.26%) and P. tenera (65.56%/76.56%). Conversely, the cultivated freshwater green microalga C. kessleri had the lowest contents of SFAs (28.87%/29.29%).

Monounsaturated Fatty Acids
MUFAs were distributed in less amounts than SFAs and their contents ranged from 6.73% (S. platensis) to 30.38% (L. japonica) in the solvent I extract and from 4.41% (P. tenera) to 33.55% (L. japonica) in solvent II extracts. In general, large differences were found in MUFA contents among the analyzed algal species. The highest contents of MUFAs were determined in brown seaweeds, whilst the lowest contents were detected in the samples of cyanobacteria and red seaweeds, depending on the solvents used.
In keeping with reports on brown seaweeds, MUFAs with higher numbers of carbons were identified as more abundant than in other algal species. The summed contents of oleic acid C18:1(n-9) in L. japonica gave the highest amount (26.65%/30.69%) of total FAMEs, unlike reported data (8.4%) [36]. In the two products from U. pinnatifida (W, WI), higher contents of oleic acid C18:1(n-9) (13.32%/9.35% in W; 11.91%/15.30% in WI) were determined than reported in published data (6.79%-10.2%) [20,35]. The same situation was observed in the last product from the brown seaweed H. fusiformis, where 10.00% and 11.28% were determined, contrary to a published 7.68% values for total FAMEs expressed as a sum of C18:1 [15].

Polyunsaturated Fatty Acids
PUFA contents ranged from 3.04% (P. palmata) to 61.52% (cultivated C. kessleri) using solvent I, whereas solvent II extracts were in the range from 0.00% (P. palmata) to 42.70% (cultivated C. kessleri) of total FAMEs. The extraction of lipids by solvent II seemed to be insufficient for the isolation of PUFAs with higher carbon numbers, as they were not detected in most of the analyzed samples, except for C20:4(n-6) determined in the brown seaweed products.
Generally, α-linolenic (ALA) and linoleic acids (LA) are the primary precursors of ω-3 and ω-6 EFAs, respectively. Both are formed by the gradual desaturation of oleic acid in the endoplasmic reticulum and plantae chloroplasts. Importantly, humans cannot synthesize ALA due to the absence of the ∆ 12 and ∆ 15 desaturases required for the synthesis of ALA from stearic acid (18:0) or PUFAs with the first double bond on the C3 (ω-3) and C6 (ω-6) from the methyl-end. Thus, the level of these PUFAs in the human body depends on their intake from the diet [4]. Generally, ω-3 PUFAs play crucial roles in many biochemical pathways which results in various health benefits, especially cardioprotective effects that result from their considerable anthiatherogenic, antithrombotic, anti-inflammatory, antiarrhytmic, hypolipidemic effects, and other health benefits, based on the complex influence of the concentrations of lipoproteins, fluidity of biological membranes, function of membraned enzymes and receptors, modulation of eicosanoids production, blood pressure regulation, and finally on the metabolism of minerals [4,[38][39][40][41][42].
Fish oil is considered as the main source of essential PUFAs. Nevertheless, fish also cannot synthesize these PUFAs because of the absence of crucial enzymes and the high level of essential PUFAs in fish oil is a direct consequence of the presence of marine microorganisms and algae in the fish trophic chain. The highest content of ω-3 FAs was determined in the green microalga C. pyrenoidosa (21.29%, I) and the highest content of ω-6 FAs was observed in the other cultivated freshwater green microalga C. kessleri sample (41.42%, I).

Fatty Acid Profiles of Autotrophically Cultivated Cyanobacteria and Microalga
Autotrophically cultivated S. platensis showed a similar FA composition to the cyanobacterial product of S. platensis, except for a slightly higher content of C16:0 and lower content of C18:3(n-6) in the cultivated alga. In contrast, the other autotrophically cultivated freshwater green alga C. kessleri showed a higher amount of PUFAs than the microalgal product of C. pyrenoidosa, the highest of all analyzed samples. The content of linoleic acid C18:2(n-6), which was the predominant PUFA in cultivated C. kessleri, exceeded the amount of this PUFA in product from C. pyrenoidosa by 51.6%.

PUFAs/SFAs Fatty Acids Ratio
The PUFAs/SFAs fatty acids ratio (hereinafter referred to as ratio) could be used for a rapid evaluation of FA profiles of analyzed samples; the higher value of this ratio means more health benefits. Ratio in the product from S. platensis (0.57/0.66) and in autotrophically cultivated S. platensis (0.46/0.74) are in accordance with the reported ratios in Spirulina sp., S. platensis, and S. maxima (0.25-0.75) [19] and in the cyanobacterium S. platensis (0.54) [17].
Based on the obtained results and data presented in literature [15,[17][18][19][20], it is evident very significant differences exist within the FA profiles in the same species of algae and seaweeds depending on the used solvents and methods of analysis. Further, chemical composition of seaweed and microalgae is affected by many factors (species of seaweed, location and time of harvest, light intensity, water chemistry and the used part of plants); therefore, the results obtained from various analyses may differ [2,25].

Samples and Chemicals
The study was conducted with eight representative species of dried cyanobacterial, microalgal and seaweed products purchased in a special local store in dried form; they were represented by green microalga (Chlorophyta), cyanobacteria (Cyanophyceae), brown seaweeds (Phaeophyta), red seaweeds (Rhodophyta), and two samples of autotrophically cultivated freshwater green microalga Chlorella kessleri (No. 260) and cyanobacterium Spirulina platensis (No. 27) obtained from the Culture Collection of Autotrophic Organisms, Institute of Botany, Academy of Sciences of the Czech Republic, Centre of Phycology (Trebon, Czech Republic). Both autotrophically cultivated species were harvested in exponential growth phase. Characteristics of all the samples are summarized in Table 4. All product samples were pulverized with a mixer (Vorwerk Thermomix TM 31, Wuppertal, Germany) to obtain a homogenous powder with a particle size of 1 mm and they were stored in airtight plastic bags at room temperature (25 °C). Freshwater green microalga Chlorella kessleri and cyanobacterium Spirulina platensis were cultivated autotrophically in a solar photobioreactor as described in the study by Masojídek et al. [43]. For the cultivation of microalgae, BG11 culture medium was used [44]. After the cultivation, the algal biomass was lyophilized (Alpha 1-4 LSC, Christ, Osterode am Harz, Germany) and stored in airtight plastic bags at room temperature (25 °C). All used chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany), except for the standard mixture of 37 FAMEs (FAME Mix, Supelco, Bellefonte, PA, USA), and methyl undecanoate purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA).

Total Lipids Determination
Total lipids of the analyzed samples were extracted using two different solvents. Either a mixture of methanol/chloroform/water (1:2:1, v/v/v) according to the modified method of [45] or n-hexane was used. Specifically, a portion (2 g) of every dried ground sample was weighed into an extraction thimble and subjected to a Soxhlet extraction for 4 h with 100 mL of the solvent mixture. Subsequently, the solvent was removed on a vacuum rotary evaporator (Laborota 4010 Digital, Heidolph, Schwabach, Germany) and the lipid extracts were dried at 105 °C for 2 h (Venticell 111 Komfort, BMT, Brno, Czech Republic). The amount of total lipid contents of all samples was determined gravimetrically [18].

GC Analysis of FAMEs
FAs were determined by gas chromatography (GC) of their methyl esters (FAMEs) in the lipid extracts obtained by above described method, excluding drying. Briefly, 0.5 M sodium hydroxide in methanol (4 mL) was added to the lipid extract (obtained from 2 g of sample) in a 250 mL flask. The flask was closed and heated for 30 min under nitrogen on a heating block (LTHS 250, Brnenska Druteva, Brno, Czech Republic). Then, freshly prepared 15% boron trifluoride in methanol (5 mL), was added to methylate the samples. After 2 min, heptane (5 mL) and sodium chloride (saturated solvent, 2 mL) were added and the sample was removed from the heating block. Next, heptane (15 mL) and sodium chloride (saturated solvent, 40 mL) were added to extract the FAMEs, the mixture was shaken and phases were separated and subsequently washed with sodium chloride (saturated solvent, 40 mL). The heptane phase was separated and anhydrous sodium sulfate was added. Quantitative determinations of FAMEs were conducted using a Shimadzu GC-2010 gas chromatograph (Shimadzu Corporation, Tokyo, Japan) equipped with a flame ionization detector (FID) and a HP-88 (Agilent Technologies, Englewood, CO, USA) capillary column (100 m × 0.25 mm, 88% cyanopropyl-arylpolysiloxane stationary phase with the thickness of 0.25 μm). The injection volume was 1.0 μL, the temperature of injection port was 250 °C with the split ratio of 1:100 and nitrogen was used as a carrier gas, temperature program was 80 °C/5 min, 200 °C/30 min, 250 °C/15 min. Identification of FAMEs was conducted by comparing their retention times with those of a 37 FAME reference standard. For quantification of FAMEs, methyl undecanoate was used as an internal standard. The FA results are expressed as a percentage of total FAMEs.

Statistics
The results of total lipids were expressed as means with standard deviations (SD) of each sample. Each sample was analyzed in triplicate (n = 3). Statistical differences among the samples were estimated by unpaired t-test and a probability value of p < 0.05 was considered to be statistically significant. Statistical analysis was performed using the StatPlus:mac LE Version 2009 software (AnalystSoft Inc., Atlanta, GA, USA). The analytical FA composition results are expressed as the average of six analyses (n = 6).

Conclusions
This study has examined nine commercially available edible cyanobacterial, microalgal and seaweed products and, moreover, autotrophically cultivated samples of the green microalga Chlorella kessleri and the cyanobacterium Spirulina platensis. Lipid content and FA profiles were determined using two different solvents, a mixture of methanol/chloroform/water (1:2:1, v/v/v, solvent I) and hexane (solvent II). In addition, yields of lipids and FA profiles after the extraction with different solvents were compared and, furthermore, comparison of data obtained from the determination of microalgal and cyanobacterial products and autotrophically cultivated microalga and cyanobacterium was accomplished.
Evidently, edible microalgal and cyanobacterial products contained a higher proportion of lipids than edible seaweed products using both solvent systems, and the highest lipid content was observed in autotrophically cultivated C. kessleri and S. platensis. From the lipid content point of view, the cultivated algae appear to be a better source of lipids than analyzed processed algal products.
The highest content of PUFAs, especially ω-3 FAs, was determined in the microalgal product of the green alga C. pyrenoidosa and two products of the brown seaweed U. pinnatifida (W, WI).
Even though fresh microalgae and unprocessed algae usually contain higher amounts of lipids, the dried edible microalgal product of C. pyrenoidosa examined in this work had a relatively high total lipid content and the highest level of PUFAs, especially ω-3 FAs. This investigation of edible cyanobacterial, microalgal and seaweed products and cultivated algae attested to the presence of health-promoting nutrients, such as PUFAs, especially essential ω-3 FAs, and this fact makes them a useful food supplement.