2.1 Materials

Sulphuric acid, hydrochloric acid, potassium hydroxide (KOH), dimethyl sulphoxide (DMSO), acetonitrile, ethanol, methanol, hexane, ethyl acetate, ferrozine, copper sulphate pentahydrate, Folin-Ciocalteu (F-C), sodium carbonate (Na₂CO₃), EDTA, glycerol, lutein and additional reagents were purchased from VWR International (Leuven, Belgium). Butylated hydroxytoluene (BHT), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), 1,1-diphenyl-2-picrylhydrazyl (DPPH), acetylthiocholine iodide (ATChI), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), pyrocatechol violet, galanthamine, β-carotene, zeaxanthin, canthaxanthin, astaxanthin, and fucoxanthin were purchased from Sigma-Aldrich (Steinheim, Germany). Neoxanthin and violaxanthin standards were kindly provided by Dr Monya Costa and Dr João Silva (ALGAE Research Group, CCMAR).

2.2 Microalgal biomass production

Tetraselmis chuii was supplied by Laboratório Experimental de Organismos Aquáticos (LEOA), University of Algarve, Portugal. Microalgal cells were grown in 100-L plastic bags in sterilized sea water (37 ppm), supplemented with modified ALGAL medium [26], under a photon flux density of 80 μmol/m²/s at room temperature. The biomass obtained was harvested by centrifugation (Avanti J-25, Beckman Coulter, CA, USA) at 4000 g for 10 min, at room temperature (RT), and stored at -20 °C until further analysis.

2.3 Extraction

Briefly, 73 g of T. chuii wet biomass was dispersed in ethanol (300 mL) and stirred at reflux temperature for 90 min. Subsequently, the mixture was immediately centrifuged (Beckman Coulter Avanti J-25) at 4000 g for 10 min (ET-1). The insoluble matter (i.e., residual biomass) underwent further extraction using ethanol twice, at reflux temperature, for 60 min (ET-2; 150 mL) and 30 min (ET-3; 150 mL). The extracts were afterwards pooled, centrifuged and filtered (Whatman n° 4). Ethanol and water were removed from the mixture using a rotatory evaporator, under reduced pressure, to obtain a crude extract.

2.4 Saponification of crude extract

The UM was separated from the crude lipid extract of T. chuii by saponification [27] with few modifications. Briefly, 40 mL of absolute ethanol and 10 mL of aqueous KOH (6 g/10 mL) solutions were added to crude extract (1.0 g), and the resulting mixture was stirred at reflux temperature for 90 min. Solvent was then evaporated to 2/3 of its volume, transferred into a separating funnel, and distilled water (250 mL) added. The UM was extracted with a mixture of hexane and ethyl acetate (85:15; v/v) 3 times, and the combined solvent layers were washed with distilled water until neutral pH. The solvent was dried over anhydrous sodium sulphate and filtered, and the solvent removed using a rotary evaporator; the UM was stored at -20 °C for further analysis.

2.5 Evaluation of biological activities in the UM fraction

2.6 Determination of carotenoid composition of the UM fraction

Carotenoid composition was determined in an HPLC (Knauer Advanced Scientific Instruments, Berlin, Germany) system comprising of a Knauer pump model 1000, diode-array-detector (DAD) model 2600 with a vacuum degasser, using a Luna 5u C18 100A (5 μm, 250 x 4.6 mm, Phenomenex®) column and ClarityChrom software. Carotenoids were eluted with a gradient mobile phase composed of acetonitrile (0-15 min), acetonitrile : ethyl acetate (50:50; 15-35 min) and acetonitrile (35-45 min), at a flow rate of 1 mL/min with an injection volume of 20 μL as described in our previous report [31]. Carotenoids were identified and quantified using individual calibration curves for each standard (β-carotene, lutein, violaxanthin, zeaxanthin, neoxanthin, canthaxanthin, astaxanthin, and fucoxanthin).

2.7 Determination of total phenolic contents (TPC)

TPC of the UM was determined by the Folin-Ciocalteu (F-C) assay according to Velioglu et al., [32]. Briefly, 5 μL of UM fraction (at 10 mg/mL) were mixed with 10-fold diluted F-C reagent in distilled water (100 μL), followed by incubation at RT for 5 min. Upon incubation, 100 μL of Na₂CO₃ (75 g/L, w/v) were added, and the solution was further incubated at RT for 90 min. The absorbance of the solution was measured at 725 nm using a microplate reader (Biotek Synergy 4). Results were expressed as gallic acid equivalents (GAE), in milligrams per gram of extract (dry weight, DW), using a calibration curve with gallic acid standard solutions at concentrations ranging from 0.002 to 2 mg/mL (r² = 0.999).

2.8 GC-MS analysis of UM

Identification and quantification of the rest of the components present in the UM fraction was determined by GC-MS (Agilent 6890 Network GC System, 5973 Inert Mass Selective Detector), equipped with a DB5-MS capillary column (25 m × 0.25 mm internal diameter, 0.25 μm film thickness) using helium as carrier gas, using total ion mode and the MS spectra of the National Institute of Standards and Technology (NIST) library as reference.

2.9 Statistical analysis

Results were expressed as mean ± SEM, and analysed by ANOVA to reveal differences and interactions between treatments or using the Tukey HSD test when parametricity of data did not prevail, using SigmaPlot software for Windows (Version 11.0, Systat Software, Inc.). Differences at p < 0.05 were considered significant. All measurements were carried out in triplicate.

3.1 Evaluation of bioactive properties of UM

Upon its separation from the soap layer, UM was assayed for the presence of antioxidants and metal chelators in order to determine the biomass upgrade potential of this fraction. UM displayed radical scavenging activity (RSA) towards the DPPH radical of 90.7 ± 1.3% at 10 mg/mL (Table 1), which was similar to that of the positive control (BHT) (89.2 ± 0.4 at 1 mg/mL). The obtained scavenging activity is very promising, considering that UM is a complex mixture of compounds (e.g. hydrocarbons, sterols, carotenoids and phenolic compounds). Molecules preventing or delaying the formation of free radicals are essential for the stabilization of food products during storage [37]. They can also play a vital role in the development of cosmeceuticals products for the health care and cosmetic industries [38]. In addition, previous reports have also proposed that a combination of phenolic compounds and carotenoids can synergistically prevent the oxidation of low density lipoproteins (LDL) [39]. Such property might contribute to the future design of functional foods or ingredients based on their antioxidant activity and their synergistic interactions. In addition, the demand by the consumer for natural sources of antioxidants has been growing, as common synthetic antioxidants (e.g. butylated hydroxytoluene, BHT, and butylated hydroxyanisole, BHA) have important drawbacks such as toxicity and carcinogenicity [40]. The high unsaponifiable content may also recommend the exploitation of microalgae biomass as a low cost renewable source of phytosterols for industrial processing in the fields of cosmetics and nutraceuticals.

The metal chelating capacity of the UM fraction was slightly lower for iron (ICA) than for copper (CCA). ICA ranged between 24.9 ± 6.2 and 88.9 ± 2.7%, whereas the highest concentration tested (10 mg/mL) was able to chelate all copper from the solution (Table 1). Conversely, UM had lower ICA and CCA than the positive control at 1 mg/mL. Most crude extracts contain different components and some of them may not be responsible for the activity of interest and thus decrease the effect observed per unit mass or unit volume. Therefore, taken together, these results suggest that UM contains compounds able to chelate iron and copper effectively, which can be used to upgrade the overall biomass value. Interestingly, metal chelators can act as antioxidants, not only by preventing chain initiation through radical scavenging, but also by binding to metal ions, which can operate as catalysts of radical generation. Indeed, there has been increasing interest in recent years in the use of biodegradable metal chelating agents to replace traditional non-biodegradable agents. For example, ethylenediaminetetraacetic (EDTA) and diethylenetriaminepentaacetic (DTPA) acids have been largely used in several industrial, agricultural, domestic (e.g. chelator in detergents) and biotechnological applications [41]. However, these aminopolycarboxylates are hardly biodegradable, leading to the accumulation of considerable amounts in industrial effluents as well as in aquatic systems, which poses serious environmental concerns [42]. Thus, non-toxic metal chelators present in unsaponifiable matter separated from microalgal biomass could be used for industrial applications not only in the food industry, but also in the formulation of nutraceuticals and cosmetics [42,43].

3.2 Chemical characterization of UM

Analysis and chemical characterization of the bioactive compounds present in the unsaponifiable matter (UM) is a crucial approach in developing an efficient microalgae-based biorefinery platform. In this sense, the UM-fraction was characterized in terms of total phenolic contents (TPC), which was found to be equal to 13.6 mg gallic acid equivalents (GAE)/g DW. Therefore, the saponification method could be an efficient procedure to separate phenolic compounds from the lipids present in the microalgal biomass. Phenolic compounds often display antioxidant activity by donating a hydrogen atom of the phenolic hydroxyl groups, chelating metals, and scavenging free radicals and inhibiting oxidoreductases (e.g. lipoxygenase) [44].

Upon RP-HPLC analysis, three carotenoids, namely β-carotene, lutein and violaxanthin were identified at concentrations between 0.14-2.15 mg/g of the total UM (Table 2). Similar results have been obtained in the eustigmatophyte Nannochloropsis gaditana, where the UM obtained also contained carotenoids, neoxanthin in particular [20]. These pigments are present in most green microalgae, being essential not only for light harvesting, but also to protect the photosynthetic apparatus from the deleterious effects of reactive oxygen species [45]. Carotenoids are known to be important sources of antioxidants, and are associated with various human health benefits, such as prevention of age-related macular degeneration, cataracts, tumoural and cardiovascular conditions, and rheumatoid arthritis and muscular dystrophy ailments [45,46]. Because of this, carotenoids have become increasingly popular as dietary supplements, food colorants and ingredients in cosmetic, nutraceutical and pharmaceutical formulations [47].

The chemical composition of the UM was further analysed by GC-MS and a tentative identification of the compounds present on this fraction was performed (Table 3). The hydrocarbon fraction was composed of saturated, unsaturated and cyclic compounds, with a number of carbons ranging from 12 to 26. Among those, the main component was 1-hexadecene, which corresponded to 17.9% of total hydrocarbons (TH). Other major hydrocarbons, namely octadecane, tetradecane, eicosane, docosane, tetracosane and hexacosane were also detected with relative contents ranging from 4.4 to 14.5% of TH. Some of the minor hydrocarbons were 1-nonadecene, cyclotetracosane, dodecane and 5-eicosane. A combination or mixture of hydrocarbons is well known as mineral oil or liquid paraffin, which is a common ingredient in lipsticks, baby lotions, cold creams, ointments, cosmetics and emollients as well [48]. Rawlings and Lombard [49] reported that mineral oil could improve the skin softness compared to fatty acids, triacylglycerols and wax esters. Hence, the natural biodegradable products in the form of UM of the microalgae can be exploited as ingredients in cosmeceutical industry. Although most of the mineral oils are generally obtained from petroleum products, nowadays there is increasing consumer pressure for the use of natural or marine natural products rather than petroleum-based products.

Besides hydrocarbons, the UM contained others components, including a sterol (campesterol), 2,4-bis(1,1-dimethylethyl) phenol, phytol and phthalate derivatives (Table 3). Sterols, sometimes collectively referred to as “phytosterols”，have been found in various plants and marine organisms, being used in food and feed formulations [50]. Interestingly, these hydroxylated steroids seem to play a major role in preventing coronary heart disease, displaying also a hypocholesterolemic effect [14,51]. Phytosterols like campesterol, stigmasterol and β-sitosterol have been reported to possess antioxidant properties that may be crucial to prevent lipid peroxidation [50,52]. On the other hand, phenol derivatives, such as 2,4-di-tert-butylphenol, have been reported as UV stabilizers, antifungal, cytotoxic, biopreservatives and dietary antioxidants. Moreover, they are often used as intermediates for synthesis and formulation of pharmaceuticals and perfumes [53-55]. Phthalate esters, including diethyl- and mono-(2-ethyl hexyl) phthalates, accounted for 1.9 and 2.4% of the UM, respectively. The GC-MS phthalate fragment pattern of mono-(2-ethyl hexyl) found in T. chuii was identical to that in a previous report. In addition, as phthalates are commonly used as plasticizers to impart flexibility and durability, the UM of T. chuii could therefore be used in the production of bioplastics. These have the advantage of being biodegradable and thus an alternative to those manufactured using fossil-based resources. Moreover, phthalic acid ester has recently been reported as inhibitor of the hydrolytic activity of trypsin [56,57]. Phytol is an acyclic diterpene alcohol and was found to be a major component of UM (Table 3). It is a constituent of chlorophyll, and is used as a precursor for the synthesis of vitamin E. Phytol is considered to be a safe and cost-effective food supplement and has several possible pharmaceutical applications, since it is endowed with antioxidant, antinociceptive, antiallergic and anti-inflammatory activities as well as showed in vitro and in vivo schistosomicidal activity against Schistosoma mansoni [58].