Introduction

The growing need to produce safe and healthy food in a global context of limiting resources and climate changes suggests the opportunity to exploit natural and sustainable sources of nutrients and functional ingredients for the food sector. Microalgae are a heterogeneous group of microorganisms at the base of the aquatic food chain, rich sources of nutrients and bioactive molecules. Their mass cultivation has the advantage of a high sustainability, as they absorb CO2 from the atmosphere, withstand extreme environmental conditions, have a high productivity and do not compete with terrestrial crops for agricultural land [1, 2].

Among the several thousands of microalgal species available in nature, only a limited number are known and much less are commercially exploited. With a biorefinery approach, microalgal biomass may have multiple industrial and biotechnological applications, from biofuel production to the extraction of high-value products for the pharmaceutical, nutraceutical, functional food and cosmeceutical markets [3]. Nonetheless, the content of high-value proteins, lipids, carbohydrates, vitamins, pigments and other health-promoting bioactive substances, together with their safety requisites, makes some microalgal species suitable for direct consumption, although nowadays their use in human nutrition is limited to the few species allowed by novel food regulations [4,5,6].

Up to now, most of the microalgal species commercially exploited find their main market in aquaculture as feed for larval and juvenile molluscs, crustaceans and fish and for the zooplankton needed in the sea-farm food chain [7]. A deep knowledge of the chemical composition of microalgae grown under controlled and standardized conditions is the first step to understand and promote the potentiality of the whole biomass as a nutritional supplement or ingredient in the food sector. The golden-brown flagellate Isochrysis galbana, the diatom Phaeodactylum tricornutum, the green alga Tetraselmis suecica and the Eustigmatophyta Nannochloropsis gaditana are marine microalgae finding a common application in aquaculture as feed for farmed aquatic species [8,9,10]. More recently, these species are also receiving interest as potential feedstock for biofuel and as sources of high-value molecules for the nutraceutical, pharmaceutical and cosmetic industries [11,12,13]. The unicellular red alga Porphyridium cruentum raises biotechnological interest as a source of valuable polyunsaturated fatty acids, pigments and other high-value compounds for cosmetic and pharmaceutic formulations [14, 15].

The main nutritional interest of microalgae raises especially from their valuable lipid profile due to the high polyunsaturated fatty acid (PUFA) content, although they are also reported to be good sources of proteins, carbohydrates, pigments and antioxidants [16,17,18,19,20].

This study was designed to gather a comprehensive characterization of the nutritional profile, minerals and trace elements of I. galbana, P. tricornutum, T. suecica, N. gaditana and P. cruentum biomass produced at an industrial plant in outdoor photobioreactors with the aim to evaluate their potential value and suitability to food applications.

Materials and methods

Microalgal biomass

The commercial microalgal biomass from Porphyridium cruentum (Phylum Rhodophyta, Class Porphyridiophyceae), Isochrysis galbana (Phylum Haptophyta, Class Coccolythophyceae), Phaeodactylum tricornutum (Phylum Bacillariophyta, Class Bacillariophyceae), Tetraselmis suecica (Phylum Chlorophyta, Class Chlorodendrophyceae) and Nannochloropsis gaditana (Phylum Ochrophyta, Class Eustigmatophyceae) was produced at the industrial plant of Archimede Ricerche Srl (Camporosso, Imperia, Italy; http://www.archimedericerche.com). Microalgae were grown in Green Wall Panel photobioreactors (3000–10,000 L), provided with automated controls to maintain optimal growth conditions. Growth occurred on natural sunlight in enriched artificial seawater, the F/2 medium, containing a mixture of nutrients, microelements and vitamins [21]. The supply of food-grade carbon dioxide as the carbon source was automatically regulated by a pH-control system. Before the onset of the stationary phase, cultures were stopped and cells harvested by centrifugation (Westfalia Separator, Dűsseldorf, Germany). Biomass was provided to our laboratory as homogeneous freeze-dried samples packed under vacuum (3–7% moisture values). Samples were stored at − 80 °C at our laboratory until analyses.

Proximate analysis

Moisture, ash, and crude protein (Kjeldahl N x 4.78) were determined according to AOAC methods [22]. Moisture and ash contents were determined gravimetrically by oven drying at 105 °C, up to a constant weight, and incineration in a muffle furnace at 550 °C, respectively. Nonprotein nitrogen (NPN) was determined by the Kjeldahl method after protein precipitation with 10% (w/v) trichloroacetic acid. Total carbohydrates were determined spectrophotometrically by the phenol–sulphuric acid method using glucose for the calibration curve [23]. Total lipids were determined according to a chloroform:methanol:water extraction protocol specific for microalgae developed at our laboratory and partly derived by previous methods [24, 25]. Briefly, 1–2 g dry algal powder was mixed with 10 mL methanol containing 500 ppm tert-butylhydroquinone (TBHQ) in a centrifuge tube and vortex-mixed for 30 s (Reax 2000, Heidolph, Schwabach, Germany). Chloroform (10 mL) was added and the mixture was vortexed again (30 s) and sonicated (37 kHz, 10 min, room temperature) in an ultrasonic bath (Ultrasonic S60 H, Elma Schmidbauer GmbH, Singen, Germany). After centrifugation (1300×g, 15 min at 5 °C), the extract was recovered and the residual biomass was extracted and sonicated repeating the procedure once more. The two extracts were kept separated, each added 5 mL of 1% w/v sodium chloride in water, vortexed and centrifuged (1300×g, 10 min, 5 °C). After centrifugation, the upper phases in each tube were discarded, while the lower phases were combined and filtered through a Whatman no. 2 filter paper funnel containing anhydrous sodium sulfate to remove any water residue. Solvent in the lipid extract was removed by rotary evaporation (R-210 Rotavapor®, Bűchi, Switzerland) at 40 °C and the lipid content was determined gravimetrically. Results are expressed as weight percentages on a dry matter basis.

Minerals and trace elements

Mineral (Ca, Mg, Na, K, P) and trace element (Fe, Zn, Cu, Mn) contents were quantified by Inductively Coupled Plasma Optical Emission Spectrometry (Optima 8000™ ICP-OES, Perkin-Elmer, Waltham, MA, USA) after liquid ashing (6 mL HNO3 + 1 mL H2O2) of the samples in a microwave digestion system (1200 Mega, Milestone Srl, Italy). Standard Reference Materials, Cabbage (IAEA-359, International Atomic Energy Agency Reference Materials Group) and Haricots vert (BCR 383, Community Bureau of Reference, Brussels), were analyzed as a check on the accuracy of the analysis.

Mercury

Total mercury levels were evaluated by Thermal Decomposition–Amalgamation–Atomic Absorption Spectrophotometry (DMA-80 Direct Mercury Analyzer, Milestone Srl, Italy) as previously described [26].

Fatty acids

Fatty acids were methylated using boron trifluoride in methanol as esterification reagent [27]. The esterified fatty acids were quantified by GC–MS-FID (7890A Series-Agilent Technologies Santa Clara, CA, USA). Separations were accomplished on a Mega-wax column (30 m × 0.32 mm i.d., 0.25 μm film thickness). The Certified Reference Material Supelco® 37 Component FAME Mix C4-C24 (Supelco, Bellefonte PA, USA) was analyzed as a control of the accuracy of the analysis. Fatty acids were identified comparing retention times with known authentic standards and using the NIST08 Mass Spectral Library (National Institute of Standards and Technology, Gaithersburg, MD, USA). Some minor fatty acids were identified only by matching their mass spectrum with the mass spectral database. Relative quantities are expressed as weight percent of total fatty acids. For nutritional evaluation purposes, percent of total fatty acids data have been converted to amounts per 100 g biomass according to Exler and Kinsella [28].

Statistical analysis

Data are reported as the average ± standard deviation of triplicate analyses on microalgal biomass. Analysis of variance (One-Way ANOVA) and Multiple-Range LSD test were applied to find out significant differences (P ≤ 0.05) among species. The software used for statistical analyses was Statgraphics® Centurion, Version XV (StatPoint Technologies Inc., Warrenton, VA, USA).

Results and discussion

Proximate composition

The macronutrient profile of algal biomass showed wide differences among species (Table 1) in accordance with their different taxonomic position. Crude protein, total lipid, total carbohydrate, energy and ash contents differed significantly (P ≤ 0.05) among species. The crude protein content ranged from 19.6 to 33.2 g 100 g−1 dw, with P. cruentum showing the lowest and N. gaditana the highest value (Table 1). Intermediate levels (26–29 g 100 g−1dw) were shown by I. galbana, P. tricornutum and T. suecica. Such values confirm these microalgae as a protein-rich biomass, although values found in the literature are variable and highly dependent on species, culturing conditions and growth stages [18, 19, 29]. In addition, the analytical method used and the N-to-protein conversion factor adopted may highly affect the final value. We adopted 4.78 as a N-to protein conversion factor according to Lourenço et al. [30] as this factor, in place of the conventional 6.25, takes into account the relevant presence of nonprotein nitrogen (NPN) in microalgae. This nitrogen fraction, mainly represented by nucleic acids, free amino acids and inorganic nitrogen, is present at variable levels in microalgae, depending on the species and growth phase. The NPN levels observed in our study, ranging from about 1 g 100 g−1 dw (P. cruentum and N. gaditana) to 3.2 g 100 g−1 dw (I. galbana), indicate a not negligible and highly variable contribution of this fraction to total nitrogen, accounting for a minimum of 15% (N. gaditana) to over 50% (I. galbana) of total nitrogen (Table 1). These values are in accordance with the NPN ranges reported in the literature [30, 31].

Table 1 Chemical composition and energy content of biomass from the five microalgal species objects of this study
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Microalgal biomass from the five species under study was characterized by sharply different lipid levels (Table 1). The lowest total lipid content was shown by P. cruentum (5.69 g 100 g−1 dw), whereas the highest levels were attained by N. gaditana (27.89 g 100 g−1 dw) and I. galbana (31.1 g 100 g−1 dw) (Table 1). These values are in accordance with the literature, reporting P. cruentum as a species accumulating carbohydrates and Nannochloropsis sp. and Isochrysis sp. as oleaginous species receiving interest as sources of n-3 fatty acids in aquaculture and as potential feedstock for biofuel production [18, 32,33,34]. Although the lipid content of microalgae is species specific, its level is also highly dependent on culture conditions and time of harvest. This fact explains the variable levels found in the literature.

Carbohydrates are nutrients highly represented in microalgae, both as intracellular storage carbohydrates and cell wall structural polysaccharides. Total carbohydrate contents (Table 1) were comparable in the three species N. gaditana, P. tricornutum and I. galbana (15.9–17.7 g 100 g−1 dw), whereas T. suecica (24.0 g 100 g−1 dw) and P. cruentum (42.2 g 100 g−1 dw) showed significantly higher values (P ≤ 0.05). These data are in general accordance with literature values [18, 32, 35, 36]. The high carbohydrate content of P. cruentum is peculiar of this unicellular red alga, encapsulated within a sulphated polysaccharide-rich cell wall [18, 32]. The accumulation of substantial amounts of carbohydrates in P. cruentum, under selected conditions, has been object of studies and represents one of the elements raising biotechnological interest towards this species [14, 15]. Sulphated polysaccharides, also released in the growth medium as exopolysaccharides, have multiple interests because of demonstrated antiviral, antioxidant and anti-inflammatory activities valuable in the pharma sector and rheological properties useful for food applications as thickening agents [37, 38]. The second species most abundant in carbohydrates, T. suecica (24.01 g 100 g−1 dw), is known for its richness in intracellular starch so as to be considered a potential feedstock for bioethanol production [12]. In addition, the peculiar cell wall polysaccharide of T. suecica has been reported to contain an interesting monomer, 3-deoxy-d-manno-oct-2-ulosonic acid, with precious pharmacological properties and a high economic value [36]. In Nannochloropsis sp., cellulose is the main component of its tough and highly recalcitrant cell wall, protected by an outer hydrophobic algaenan layer, whereas in P. tricornutum, the cell wall is mainly composed of a sulphated glucuronomannan [39, 40]. Beta-glucans, sulphated polysaccharides, cellulose and other microalgal polysaccharides may also act as dietary fiber within the gastrointestinal tract thus helping regulate energy intake, and serum glucose and lipid levels.

The high energy content of microalgal biomass (294–465 kcal 100 g−1 dw) was correlated with lipid levels, except that in the case of P. cruentum, where the high energy contribution of carbohydrates counter-balanced its low lipid content. Values within this range are reported in the literature, with the lower levels referred to P. cruentum and the higher ones to the lipid-rich N. gaditana and I. galbana [18, 19, 32, 41]. However, as the microalgal biomass composition may be modulated through nutrient starvation or control of environmental conditions to increase the lipid or carbohydrate content of cells, variable energy values may be attained [35, 42].

Phaeodactylum tricornutum and P. cruentum were the species with the highest ash content (27.9 g and 23.6 g 100 g−1 dw, respectively) compared to the other species (11.5–18.0 g 100 g−1 dw) (Table 1). Comparable values are reported in the literature [32, 43]. However, highly variable ash contents and mineral profiles are reported in the literature, due to the strong influences of culturing and environmental conditions even within the same species.

Mineral composition

The elemental composition of algal biomass is reported in Table 2. Sodium (2.0–4.5 g 100−1 dw) and calcium (0.6–2.9 g 100 g−1 dw) were the most abundant minerals, followed by potassium (1.1–2.4 g 100−1 dw), phosphorus (0.3–1.2 g 100 g−1 dw) and magnesium (0.37–1.4 g 100 g−1 dw). P. tricornutum showed levels of potassium (2.4 g 100 g−1 dw), magnesium (1.4 g 100 g−1 dw) and of trace minerals such as iron (87.7 mg 100 g−1 dw), manganese (14.2 mg 100 g−1 dw) and copper (5.5 mg 100 g−1 dw) much higher than those of the other species (P ≤ 0.05). P. cruentum was the species with the highest Ca:P ratio (6.53), largely surpassing P. tricornutum (2.45), I. galbana and T. suecica (1.84–1.86) and N. gaditana (0.71). As regards safety for human consumption, in the studied species, mercury levels (0.05–0.55 µg 100 g−1 dw) were well below the maximum value of 0.1 mg kg−1 set by the European legislation for food supplements [44]. Taking into consideration the levels of tolerable weekly intake (TWI) established by EFSA for mercury (1.3 and 4.0 μg kg−1 body weight for methyl-mercury and inorganic mercury, respectively), the total mercury levels detected are of no toxicological concern [45].

Table 2 Mineral composition of microalgal biomass from the five microalgal species objects of this study
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Fatty acid profile

The total saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acid compositions of microalgal biomass are reported in Table 3. All microalgae showed a prevalence of PUFA (47.4–59.1% of total fatty acids) over SFA (23.0–35.7% of total fatty acids) and MUFA (6.1–30.5% of total fatty acids).

Table 3 Fatty acid profile of the five microalgal species objects of this study (% of total fatty acids)
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Fatty acid profiles differed widely, both qualitatively and quantitatively, among species. SFA were mainly represented by palmitic acid (C16:0), prevalent in P. cruentum (34.1% of total fatty acids), N. gaditana (21.6% of total fatty acids), T. suecica (18.3% of total fatty acids) and P. tricornutum (14.4% of total fatty acids), and by myristic acid (C14:0), prevalent in I. galbana (16.1% of total fatty acids). MUFA were mainly represented by palmitoleic acid (C16:1 n-7), prevalent in N. gaditana (22.25% of total fatty acids) and P. tricornutum (18.65% of total fatty acids) and by oleic acid (C18:1 n-9) prevalent in T. suecica (19.6% of total fatty acids) and I. galbana (16.5% of total fatty acids). P. cruentum distinguished for the very low MUFA percentage (6.1% of total fatty acids), a feature reported in the literature for this species [18] and consistent with its low lipid content.

Among PUFA, n-3 fatty acids were prevalent in N. gaditana and I. galbana (36.2–36.4% of total fatty acids), T. suecica (34.6% of total fatty acids) and P. tricornutum (32.6% of total fatty acids), whereas n-6 fatty acids, mainly arachidonic (C20:4) and linoleic (C18:2) acids, were prevalent in P. cruentum (43.3% of total fatty acids).

As regards single n-3 fatty acids, P. cruentum, P. tricornutum, and N. gaditana were rich in eicosapentaenoic acid (C20:5, 15.9%, 29.3% and 36.0%, respectively). I. galbana was a good source of stearidonic acid (C18:4, 12.2% of total fatty acids), a precursor of eicosapentaenoic acid, and of its elongation–desaturation product, docosahexaenoic acid (C22:6, 9.0% of total fatty acids), undetectable or present at low levels in the other species. I galbana and T. suecica showed also consistent percentages of α-linolenic acid (C18:3, 12.2% and 15.7% of total fatty acids, respectively).

A high long-chain polyunsaturated fatty acid (LC-PUFA) productivity of Porphyridium, Nannochloropsis, Phaeodactylum, Tetraselmis and Isochrysis spp. is reported in the literature, although values of single fatty acids may largely vary as their synthesis and accumulation depend highly on cultivation conditions and growth stage [11, 41,42,43, 46,47,48,49,50].

As primary producers of n-3 PUFA, essential nutrients for the growth and development of aquatic animals, as well as for humans at any life stages, the nutritional value of marine microalgae is mostly linked to their fatty acid profile [43].

n-3 LC-PUFA are important components of human cells, playing beneficial effects on human health, particularly for their protective effects towards cardiovascular and inflammatory diseases. In addition, n-3 fatty acids have positive effects on the nervous system and brain functions, neurodevelopment and prevention of neurodegenerative diseases [51, 52].

The n-3/n-6 ratio, an indicator of the nutritional value of food, desirable to be high in the human diet for the prevention of cardiovascular and chronic inflammatory diseases and to counter-balance the high intake of n-6 PUFA typical of modern Western diets, was high (2.43–5.01) in all microalgal species except P. cruentum (0.37). The PUFA/SFA ratio, a parameter inversely related to cardiovascular risk when considering human diet [53, 54], was favorably high for all microalgal species (1.64–2.19). The n-3/n-6 and PUFA/SFA ratio values found in the microalgae under study are in line with the literature, underlying the healthy properties of microalgal oils [16, 18, 41].

As the main source of LC-n-3 PUFA in the human diet is marine fatty fish, suffering from overfishing and catch decline, the exploitation of new sustainable sources of these essential nutrients is of great interest.

Dietary considerations

The data here reported suggest some dietary considerations. Microalgal biomass from this study compares favorably to traditional food commodities (eggs, milk, beans, fish, meat, and soybean) for nutrient content [55]. In particular, the concentrations of some mineral elements such as sodium, magnesium, manganese and iron were much higher in microalgae than in most common food items when expressed on a dry matter basis. However, considering that one possible use of microalgal biomass as a food ingredient is in pasta, bakery products or snacks, where the suggested levels of addition generally do not exceed 2–3 g per 100 g of product due to sensory acceptability limits [56,57,58], the high levels of some minerals here found may not represent a concern.

The amounts of nutrients supplied by 3 g of microalgal biomass are reported in Table 4. In nutritional terms, the most significant contribution of microalgae, when added to food products at a 3% level, is in mineral and PUFA. In fact, regardless of the species, the amounts of protein and lipid supplied by 3 g of dry microalgal biomass correspond to less than 2% the daily protein and lipid requirements of a 60-kg adult on a 2000 kcal diet [59]. On the contrary, the same amount of microalga supplies, depending on the species, 9–26% of the recommended dietary allowance (RDA) for Fe, 5–18% of the RDA for Mg, 2–9% for Ca, 1–18% for Cu. The high Ca:P ratio found in most of the microalgae from this study (see Table 2) is a positive attribute suggesting their possible use as mineral supplements to counteract the Ca:P unbalance that characterizes Western diets and that may negatively affect bone health. As regards PUFA, taking into account the adequate intake of 250 mg per day of EPA + DHA recommended for primary cardiovascular prevention of healthy adults [59,60,61], 3 g dry biomass of I. galbana, P. tricornutum or N. gaditana supplies about 34%, 42% and 108% of the recommended EPA + DHA amounts, respectively. Therefore, food enrichment with microalgae, along with fish consumption or fish oil supplementation, would help to set the dietary n-3/n-6 PUFA ratio at the desirable levels and to counter-balance the high intake of n-6 PUFA typical of modern Western diets [62].

Table 4 Amounts of selected nutrients supplied by 3 g (dry matter) of microalgal biomass from this study and corresponding percent of recommended dietary allowance (RDA) values
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The dietary pattern changes occurred in the last century in Western industrialized countries have implied not only an increment of the total dietary fat content, but also a qualitative change of fat profile, with a shift towards saturated fat and n-6 PUFA, these latter mainly contributed by vegetable oils rich in linoleic acids, at the expense of n-3 PUFA. The n-6/n-3 PUFA ratio, estimated to be about 1 in the diet of our ancestors, has increased drastically in Western diets, up to a value of 10–20, with several public health implications since a balanced n-6/n-3 ratio is essential for the prevention and treatment of coronary artery disease, hypertension, diabetes, arthritis, osteoporosis, other inflammatory and autoimmune disorders, cancer and mental health [62]. To re-establish a healthy n-6/n-3 ratio, adequate dietary patterns should be promoted, with an increment the consumption of food rich in n-3 PUFA.

Reducing the total fat intake within the recommended range (20–35% of dietary energy) and substituting saturated and trans-fatty acids with mono- and polyunsaturated fatty acids, both n-6 and n-3, result in a favorable modulation of lipid and lipoprotein blood profile with consequent advantages on health and chronic disease risk reduction [63]. In practical terms, the desirable dietary lipid profile change may be actuated through the reduction of red meat, full-fat dairy products and industrially processed product consumption, while incrementing the consumption of lean and poultry meat, seafood products, especially fatty fish, nuts and seeds, rich in omega-3 fatty acids, and olive oil as a source of oleic acid [64]. Within this context, the integration of the diet with marine microalgal oil, rich in LC n-3 PUFA, or with functional products containing microalgae as an ingredient, may represent a valid alternative strategy to help re-equilibrate the dietary lipid profile and provide an additional source of the healthy n-3 fatty acids. Clinical trials of microalgal oil supplementation report beneficial changes of cardio-metabolic risk markers, comparable to those reported with fish oil consumption but with the advantage of lower gastrointestinal complaints at high-dose therapies and no adverse effects [51].

Furthermore, based on the results of our previous study [20], food enrichment with microalgae would provide it with significant amounts of carotenoids, mainly xanthophylls, thus incrementing the antioxidant potential and healthy value of the original food.

Nutrient bioaccessibility and bioavailability, as well as sensory properties and shelf life of food enriched with microalgal biomass, represent relevant issues to be considered in the development of food products enriched with microalgae and deserving specific evaluations for each product and microalgal strain. Other bioactive compounds not covered by this study (vitamins, sterols and phenolic compounds) deserving an in-depth study would be also contributed by microalgae addition to food, thus positively affecting their healthiness and antioxidant potential.

Conclusions

The microalgal biomass of P. cruentum, I. galbana, P. tricornutum, T. suecica and N. gaditana studied was characterized by a good nutritional value and confirmed to be a potentially valuable ingredient for nutritional or nutraceutical purposes.

All species proved to be a good source of PUFA and minerals. P. tricornutum, N. gaditana and I. galbana were the species highest in nutritionally important n-3 LC-PUFA, with positive implications when included in a balanced diet.

The high nutrient density, healthy lipid profile and environmentally sustainable origin make these microalgae a potential food ingredient to be considered in substitution to or at integration of traditional ones and as a viable alternative to fish oil, with the advantage of reducing the dependency on traditional terrestrial and marine finite resources.