Contents lists available at ScienceDirect Algal Research journal homepage: www.elsevier.com/locate/algal Potential of microalga Isochrysis galbana: Bioactivity and bioaccessibility Bonfanti C.a,b, Cardoso C.b,c,⁎, Afonso C.b,c,⁎, Matos J.b,d, Garcia T.a,b, Tanni S.a, Bandarra N.M.b,c a Botucatu Medical School, UNESP - Universidade Estadual Paulista, Botucatu Campus, Botucatu, São Paulo, Brazil bDivision of Aquaculture and Upgrading (DivAV), Portuguese Institute for the Sea and Atmosphere (IPMA, IP), Rua Alfredo Magalhães Ramalho, 6, 1495-006 Lisbon, Portugal c CIIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal d Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 16, 1749-016 Lisbon, Portugal A R T I C L E I N F O Keywords: Isochrysis galbana Lipid composition ω3 PUFA Anti-inflammatory activity Bioaccessibility A B S T R A C T The lipid composition and anti-inflammatory activity of the microalga Isochrysis galbana were studied. Moreover, the influence of bioaccessibility on composition and bioactivity was evaluated through the application of an in vitro model of the human digestion. The fatty acid (FA) profile was characterized by abundance of poly- unsaturated FA (PUFA) and, within PUFA, ω3 PUFA were the most abundant. High contents of myristic, oleic, linoleic, α-linolenic, and stearidonic acids as well as docosahexaenoic acid (DHA) were determined. A low level of hydrolysis of triacylglycerols (TAGs) and polar lipids was observed during digestion. Total lipid bioaccessi- bility and specific FA bioaccessibility were low (between 7 and 15%). The highest bioaccessibility percentages were determined for palmitic, oleic, and linoleic acids as well as total ω6 PUFA and the lowest bioaccessible percentages were calculated for myristic and stearidonic acids, DHA, and total ω3 PUFA. Chemical affinity phenomena could be an explanation for these results. Regarding anti-inflammatory activity, it was only detected in the lipid extract of I. galbana prior to digestion (79 ± 7% of cyclooxygenase-2, COX-2, inhibition). No activity was found in the bioaccessible fraction extract. Apparently, the COX-2 inhibitory compounds were not rendered bioaccessible. 1. Introduction Microalgae are an important aquatic resource [1]. Moreover, mi- croalgae are a promising and valuable natural source of bioactive compounds, such as ω3 polyunsaturated fatty acids (ω3 PUFA). Due to its potential, microalgal biomass can be incorporated as a functional ingredient in order to enhance the nutritional value of foods and, thus, to positively affect human health. In particular, the microalga Isochrysis galbana is potentially pro- mising to the food industry due to its significantly high lipid content (20–30% w/dw), representing a rich source of ω3 PUFAs, namely ei- cosapentaenoic acid (EPA, 20:5 ω3) and docosahexaenoic acid (DHA, 22:6 ω3) [2]. Accordingly, it can be regarded as an additional source of essential oils to fisheries, thus covering the needs of increasing human population. It also supplies sterols, tocopherols, colouring pigments, and other bioactive substances [2,3]. EPA and DHA are associated with decreased morbidity and mor- tality from cardiovascular and other diseases as well as with foetal development [4]. Reviewed evidence also pointed to benefits for the development of the neural system in children [5] and prevention of mild cognitive decline in elderly [6]. Furthermore, EPA has been claimed to enhance anti-inflammatory properties of high-density lipo- protein, among other effects [7]. On the other hand, the reports of anti-inflammatory effects on rats due to I. galbana [8] may correspond to the action of bioactive sub- stances in I. galbana, including EPA and other than EPA. These sub- stances may be peptides, carotenoids, or sulphated polysaccharides [9]. In the case of the microalga Chlorella, evidence linking extracts of this organism to anti-inflammatory outcomes has been stronger [1,10]. More specifically, for the microalga Porphyridium cruentum, a sul- phoglycolipidic fraction has been shown to display an anti-in- flammatory effect [11]. A fatty acid (FA) analysis showed that this fraction contained large amounts of palmitic acid, 16:0 (26.1%) and EPA (16.6%) as well as noticeable amounts of the 16:1 ω9 (10.5%) [11]. These results show that microalgae have anti-inflammatory po- tential, which is stimulating the development of innovative nu- traceuticals. However, in future applications as nutraceuticals, it must be taken into account that the absorbable quantity of a compound in the gas- trointestinal (GI) tract is not accurately predicted by its total content in https://doi.org/10.1016/j.algal.2017.11.035 Received 16 September 2017; Received in revised form 27 November 2017; Accepted 27 November 2017 ⁎ Corresponding authors at: Division of Aquaculture and Upgrading (DivAV), Portuguese Institute for the Sea and Atmosphere (IPMA, IP), Rua Alfredo Magalhães Ramalho, 6, 1495-006 Lisbon, Portugal. E-mail addresses: carlos.cardoso@ipma.pt (C. Cardoso), cafonso@ipma.pt (C. Afonso). Algal Research 29 (2018) 242–248 Available online 11 December 2017 2211-9264/ © 2017 Elsevier B.V. All rights reserved. T http://www.sciencedirect.com/science/journal/22119264 https://www.elsevier.com/locate/algal https://doi.org/10.1016/j.algal.2017.11.035 https://doi.org/10.1016/j.algal.2017.11.035 mailto:carlos.cardoso@ipma.pt mailto:cafonso@ipma.pt https://doi.org/10.1016/j.algal.2017.11.035 http://crossmark.crossref.org/dialog/?doi=10.1016/j.algal.2017.11.035&domain=pdf the microalgae. Bioaccessibility corresponds to the share of the initial content that is rendered free from the microalgal structure into the GI tract [12]. Thus, determining bioaccessibility may contribute to the assessment of the effective nutraceutical potential of any given micro- algal biomass. A bioaccessibility study requires the utilization of an adequate in vitro digestion model that reliably simulates human diges- tion. Different techniques have been developed [13] and optimized [12,13], being the static model with digestive compartment distinction and complete digestive juices, including enzymes in all steps, one of the best models. The objective of this study was to determine the lipid composition, anti-inflammatory activity, and bioaccessibility effects on these aspects for the microalgae I. galbana, paving the way for a deeper knowledge of the biochemical processes involved in any anti-inflammatory effects and a more realistic assessment of the nutraceutical potential. 2. Material and methods 2.1. Sample collection and preparation Samples of Isochrysis galbana were supplied by Necton (Necton, Companhia Portuguesa de Culturas Marinhas, SA, Olhão, Portugal). This material was already freeze-dried and was analysed without fur- ther processing. 2.2. Proximate composition The moisture and ash contents were determined according to Association of Official Analytical Chemists (AOAC) methods [14]. The protein level was quantified according to the Dumas method [15]. Crude lipid content was determined following the Bligh and Dyer ex- traction method [16]. Carbohydrate content was determined by dif- ference. 2.3. Fatty acid profile Fatty acid methyl esters (FAME's) were prepared by acid-catalysed transesterification using the methodology described by Bandarra et al. [17]. Samples were injected into a Varian Star 3800 CP gas chroma- tograph (Walnut Creek, CA, USA), equipped with an auto sampler with a flame ionisation detector at 250 °C. FAME's were identified by com- paring their retention time with those of Sigma–Aldrich standards (PUFA-3, Menhaden oil, and PUFA-1, Marine source from Supelco Analytical). The limit of detection (LOD) is 1 mg/100 g. Results were calculated in mg/100 g of edible part using the peak area ratio (% of total fatty acids) and the lipid conversion factors, which were, in turn, calculated according to Weihrauch et al. [18]. 2.4. Lipid class determination The relative weight of each lipid class was determined by analytical thin-layer chromatography (TLC) using a previously described method [19]. An eluent mixture of hexane:diethyl ether:acetic acid (50:50:2 by volume) and a plate coated with 0.25 mm silica gel G were used. Lipid class identification was done by comparison with standards (Sigma Chemical Co., St. Louis, MO, USA). Specifically, glyceryltrioleate (triacylglycerol, TAG), glyceryl 1,3-dipalmitate (diacylglycerol, DAG), DL-α-monoolein (monoacylglycerol, MAG), oleic acid (free fatty acid, FFA), and L-α-phosphatidylcholine (phospholipid, PL, polar lipid) were used. The relative percentage of each lipid class was determined using a GS-800 densitometer and version 4.5.2 of Quantity One 1-D Analysis software from Bio-Rad (Hercules, CA, USA). 2.5. Anti-inflammatory activity To extract lipids from freeze-dried I. galbana microalgae and respective bioaccessible fraction, the Bligh and Dyer method [16] and the methodology described in Section 2.7. were used, respectively. Extracted lipids were directly dissolved in 100% dimethyl sulfoxide (DMSO) (see below Section 2.5.2). 2.5.1. Aqueous extract preparation for in vitro anti-inflammatory activity An aqueous extract of the freeze-dried I. galbana microalgae was prepared with the purpose of attaining a fraction with anti-in- flammatory properties to be tested in vitro. Accordingly, approximately 200 mg of freeze-dried I. galbana mi- croalgae was weighed and homogenized with 2 ml of Milli-Q water using a model Polytron PT 6100 homogenizer (Kinematica, Luzern, Switzerland) at a velocity of 30,000 rpm during 1 min. Afterwards, the mixture was subjected to a thermal treatment (at 80 °C for 1 h). Both the microalgae and bioaccessible extraction mixtures were centrifuged (3000 ×g at 4 °C during 10 min) and the respective supernatant was evaporated using vacuum rotary evaporator with the water bath tem- perature at 65 °C and inert gas (nitrogen) stream. 2.5.2. Cyclooxygenase (COX-2) inhibition assay The prepared extracts were dissolved in 100% DMSO to prepare a stock with a concentration of 10 mg/ml. The extract was tested at 1 mg/ml and 100 μg/ml using a commercial cyclooxygenase (COX-2) inhibitory screening assay kit, Cayman test kit-560,131 (Cayman Chemical Company, Ann Arbor, MI, USA). The COX-2 inhibitor screening assay directly measures the amount of Prostaglandin F2α generated from arachidonic acid (AA, 20:4 ω6) in the cyclooxygenase reaction. A volume of 10 μl each of test extract or DMSO was used. The reaction was initiated by addition of 10 μl 10 mM AA and each reaction tube was incubated at 37 °C for 2 min. The reaction was terminated by addition of 50 μl 1 N HCl and saturated stannous chloride. Assays were performed using 100 units of human recombinant COX-2. An aliquot was removed and the prostanoid produced was quantified spectro- photometrically (412 nm) via enzyme immunoassay (ELISA) after 18 h incubation, washing, addition of Ellman's reagent, and further 90 min incubation. Interference by solutions and digestive enzymes used in the bioaccessibility method was taken into account by subtracting COX-2 inhibition of the bioaccessibility blank from the COX-2 inhibition measured with the bioaccessible fraction samples. 2.6. In vitro digestion model An in vitro digestion model was chosen for the determination of bioaccessibility in freeze-dried I. galbana microalgae. The model was comprised of three sections, which enable the simulation of digestion in three different parts of the GI tract: mouth, stomach, and small intes- tine. The solutions and enzymes used in this model followed Afonso et al. [12]. Briefly, approximately 1.5 g freeze-dried I. galbana was weighed taking into account the assumptions defined by Versantvoort et al. [20]. The sample was mixed with 4 ml of artificial saliva at a pH 6.8 ± 0.2 for 5 min, then 8 ml of artificial gastric juice (pH 1.3 ± 0.02 at 37 ± 2 °C) was added, and pH was lowered to 2.0 ± 0.1. The mixing lasted 2 h in a “head-over-heels” movement (37 rpm at 37 ± 2 °C). Finally, 8 ml of artificial duodenal juice (pH 8.1 ± 0.2 at 37 ± 2 °C), 4 ml of bile (pH 8.2 ± 0.2 at 37 ± 2 °C), and 1.33 ml of HCO3 – solution (1 M) was added. The pH of the mixture was set at 6.5 ± 0.5 and agitation for 2 h was identical to gastric conditions. The mixture generated in the in vitro model was subjected to centrifugation at 2750 ×g for 5 min, thus yielding a non- digested portion and the bioaccessible fraction. While chemicals were supplied by Merck (Darmstadt, Germany), enzymes were attained from Sigma (St. Louis, MO, USA). 2.6.1. Calculation of bioaccessibility The percentage (%) of each I. galbana microalgae constituent (C) in the bioaccessible fraction was estimated as follows: C. Bonfanti et al. Algal Research 29 (2018) 242–248 243 = ×C%C bioaccessible [ ] 100/[S]bioaccessible Being: [C] = Concentration of constituent. [S] = [C] in the initial sample (before digestion). 2.7. Lipid extraction in the bioaccessible and undigested fractions For bioaccessible samples, the Bligh and Dyer method [16] was slightly modified since the lipid fraction was rendered available by the digestion procedure. Four milliliters chloroform was added to the bioaccessible fraction followed by 1 min homogenisation in a vortex and a centrifugation at 2000 ×g for 5 min. Then, the upper phase was removed. In the next step, 4 ml chloroform and 2 ml of water were added followed by 1 min homogenisation in a vortex and a cen- trifugation at 2000 ×g for 5 min at a temperature of 4 °C. After the removal of the upper phase, the previous operation was repeated. The organic phase was then filtered through a filter containing anhydrous sodium sulphate and then evaporated in a rotary evaporator. The lipid samples were weighed, solubilised in chloroform and stored at −20 °C until further analysis. For the undigested fraction, the procedure was just as described by Bligh and Dyer [16]. 2.8. Statistical analysis To test the normality and the homogeneity of variance of data, the Kolmogorov-Smirnov's test and Levene's F-test, respectively, were used. Data, which corroborate these assumptions, were analysed by one-way Anova distribution using the Tukey HSD to determine the difference in the content of constituents between the initial and the bioaccessible fraction of samples or between fatty acids. When normality and/or homogeneity of variance were not verified (initial 18:0 content and ω3/ ω6 vs bioaccessible 18:0 content and ω3/ω6), data were tested non- parametrically with Kruskal-Wallis test and Mann-Whitney's U test. For all statistical tests the significance level (α) was 0.05. All data analysis was performed using STATISTICA 6 (Stat-sof, Inc. USA, 2003). 3. Results and discussion 3.1. Microalgal characterization 3.1.1. Proximate composition The proximate composition of studied I. galbana species is displayed in Table 1. Moisture content was low (7.7 ± 0.0%, w/w) owing to the fact that studied microalgae was freeze-dried. The dry matter of this microorganism was mainly composed of protein and lipids, 42.3 ± 1.2%, w/w, and 25.3 ± 0.2%, w/w, respectively. None- theless, ash and carbohydrate fractions were also a significant share of the biomass. The observed proximate composition was similar to that reported by other authors for I. galbana [21,22]. Indeed, the biomass of this mi- croorganism is very rich in lipids, even in comparison with other lipid- rich microalgae, such as Diacronema vlkianum [22]. 3.1.2. Lipid composition The distribution of lipid classes in the lipid fraction of I. galbana microalgae is presented in Table 2 and the fatty acid composition of I. galbana microalgae is shown in Table 1. There were two main lipid classes in the studied I. galbana biomass: triacylglycerols (TAGs) and free fatty acids (FFAs). Polar lipids re- presented a relatively small share of 10.2 ± 0.7% of total lipids. For lipid-rich biomass (lipid content exceeding 5%, w/w), TAGs are usually the most abundant class in fish and other organisms [23], but these empirical relations are not necessarily applicable in microalgae [24]. These authors found a high share (exceeding 80%) of polar lipids in Isochrysis sp. However, there are other authors who have reported a high percentage of TAGs [25]. The large share of FFAs may be due to different causes: effects of culture medium composition on microalgal metabolism [21]; lipid breakdown in stressed or dying cells [24]; and hydrolytic degradation of the biomass, which may have occurred during frozen storage (9 months at −20 °C) [26]. Studies on I. galbana [21] have reported a wide variability in the proportion of polar lipids and neutral lipids as a result of changes in the cultivation conditions and time of harvest, that is, a dependence on the different phases of the growth cycle. A high proportion of TAGs has been correlated with the absence of cellular division at the onset of stationary phase in aged cultures of microalgae [27]. Regarding the FA profile, two main aspects may be underlined: on the one hand, PUFAs were the main FA group (46.2 ± 0.4% of total FAs), followed by saturated FAs (SFA, 27.8 ± 0.2%), and mono- unsaturated FAs (MUFA, 23.0 ± 0.2%); on the other hand, within the PUFA group, ω3 PUFAs were clearly more abundant than ω6 PUFAs, thereby yielding an ω3/ω6 ratio of nearly 3. Other aspects are also worth mentioning at a more detailed level: myristic acid (14:0) was the most abundant SFA (14.1 ± 0.1%); oleic acid (18:1 ω9) represented more than half total MUFA (14.7 ± 0.1%); almost all ω6 PUFAs were linoleic acid (18:2 ω6); and ω3 PUFAs were mainly composed of similar proportions, in the 9–11% range, of α-li- nolenic acid (18:3 ω3), stearidonic acid (18:4 ω3), and DHA. EPA content was very low, barely exceeding 1% of total FAs. Table 1 Proximate composition (% wet weight) and fatty acid profile (in % of total fatty acids and in mg/100 g wet weight) of the studied microalgae I. galbana before and after digestion (bioaccessible fraction). Microalgae before digestion Microalgae after digestion Proximate composition (% wet weight) (Bioaccessible fraction) Moisture 7.7 ± 0.0 Lipid 25.3 ± 0.2 Protein 42.3 ± 1.2 Carbohydrate 9.9 ± 1.2 Ash 14.8 ± 0.1 Microalgae before digestion Microalgae after digestion Fatty acid (% total fatty acids) (mg/100 g wet weight) (% total fatty acids) (mg/100 g wet weight) 14:0 14.1 ± 0.1A 3311 ± 30 11.2 ± 0.7B 16.9 ± 1.0 16:0 10.6 ± 0.0A 2489 ± 6 12.9 ± 0.9A 19.4 ± 1.2 18:0 0.5 ± 0.0A 127 ± 1 3.0 ± 0.6B 4.6 ± 0.9 Σ SFAa 27.8 ± 0.2A 6513 ± 40 29.4 ± 1.2A 44.2 ± 1.4 16:1 ω7 5.0 ± 0.0A 1174 ± 7 4.9 ± 0.1A 7.4 ± 0.1 18:1 ω9 14.7 ± 0.1A 3452 ± 15 17.4 ± 0.4B 26.2 ± 0.9 20:1 ω11 0.9 ± 0.0A 214 ± 8 1.0 ± 0.0A 1.5 ± 0.1 Σ MUFAb 23.0 ± 0.2A 5389 ± 37 25.1 ± 0.3B 37.8 ± 0.9 18:2 ω6 9.6 ± 0.0A 2245 ± 5 11.4 ± 0.2B 17.2 ± 0.4 20:4 ω6 0.3 ± 0.0A 69 ± 3 1.0 ± 0.0B 1.5 ± 0.1 18:3 ω3 10.9 ± 0.0A 2557 ± 5 10.5 ± 0.1B 15.8 ± 0.3 18:4 ω3 11.3 ± 0.0A 2644 ± 9 10.3 ± 0.3B 15.5 ± 0.6 20:4 ω3 0.2 ± 0.0A 37 ± 0 0.2 ± 0.0A 0.2 ± 0.0 20:5 ω3 1.2 ± 0.0A 276 ± 1 1.1 ± 0.1A 1.7 ± 0.1 22:5 ω3 0.2 ± 0.0A 42 ± 3 0.3 ± 0.1A 0.4 ± 0.1 22:6 ω3 9.2 ± 0.2A 2146 ± 42 8.0 ± 0.3B 12.1 ± 0.7 Σ PUFAc 46.2 ± 0.4A 10,829 ± 85 45.5 ± 0.9A 68.7 ± 2.1 Σ ω3 33.6 ± 0.3A 7867 ± 61 30.7 ± 0.8B 46.2 ± 1.6 Σ ω6 12.1 ± 0.1A 2829 ± 22 14.3 ± 0.3B 21.6 ± 0.6 Σ ω3/Σ ω6 2.8 ± 0.0A 2.8 ± 0.0 2.1 ± 0.1B 2.1 ± 0.1 Values are presented as average ± standard deviation. Different uppercase letters within a row correspond to statistical differences (p < 0.05) between microalgae before and after digestion (bioaccessible fraction). a SFA, saturated fatty acid. b MUFA, monounsaturated fatty acid. c PUFA, polyunsaturated fatty acid. C. Bonfanti et al. Algal Research 29 (2018) 242–248 244 The FA profile of I. galbana microalgae can vary widely as a function of cultivation conditions and growth phase at harvest time [28]. For instance, an inverse relation it has been observed between EPA and DHA levels and growth temperature in Isochrysis, as well as in other microalgae [29]. In particular, EPA contents may experience wide variations between batches with different cultivation conditions [28]. Some traits experience smaller changes, such as the abundance of myristic acid [21,22,28]. High levels of stearidonic acid, such as those observed in current study, have also been measured by other authors [21]. Some aspects, such as the high oleic and linoleic acids levels and low EPA level, are not readily found in the literature [21,22,28]. Moreover, taking into account human EPA + DHA requirements and considering that I. galbana may be used in food applications, the amount of freeze-dried microalga needed to meet the recommended daily intake of EPA + DHA (500 mg/day) according to the American Heart Association may be calculated [30]. On the basis of the absolute FA composition in Table 1, an amount of 20.6 g of the studied freeze- dried I. galbana can be estimated, which is a low amount and may en- able the incorporation of this microalga in foods. In addition, it should be stressed that given the fact that high EPA + DHA levels before di- gestion (especially DHA in I. galbana) do not ensure a high intestinal absorption of these lipids, a study on microalgal lipid digestion is warranted (see below). 3.2. Microalgal lipid digestion 3.2.1. Lipid hydrolysis The level of lipid hydrolysis achieved in the bioaccessible fraction can be assessed by the distribution of lipid classes, as displayed in Table 2. There was a significant level of TAGs remaining after digestion (26.3 ± 4.5% of total lipids). FFAs increased with respect to the initial sample (prior to digestion), but by only 15%, reaching 54.0 ± 5.7%. Polar lipids were slightly reduced from 10.2 ± 0.7% to 7.5 ± 0.4% of total lipid. Accordingly, these results indicated a low hydrolysis of TAGs and polar lipids. This contrasts with other studies claiming a high level of lipid hydrolysis with no TAGs detected in the bioaccessible fraction [12,23,31,32]. However, all these studies were on fish and not based on microalgae. Unfortunately, there are very few studies on microalgal lipid bioaccessibility [13,33]. The latter authors also reported low li- polysis levels in another species of microalga, Nannochloropsis oculata, at least, in the absence of prior treatment of the microalgal biomass. Regarding polar lipids, though it has been claimed that these amphi- philic lipids modify the surface structure of oil droplets in such a way that access to lipids by pancreatic lipases is enhanced [34], no extensive microalgal polar lipid lipolysis was detected. Nevertheless, it should be noted that for some polar lipids, such as phosphatidylcholine, total li- polysis is not necessary for bioaccessibility [35]. 3.2.2. Bioaccessible fatty acids The FA profile of the bioaccessible fraction of the studied micro- algae I. galbana is presented in Table 1 and, on the basis of the com- parison between the FA profile before and after digestion (bioaccessible fraction), the bioaccessibility percentages were calculated for the total lipids and each FA, being shown in Fig. 1. There were some differences between the FA profile before diges- tion (Table 1) and in the bioaccessible fraction. However, the overall proportion of PUFA and SFA was constant. Within the SFA group, the palmitic acid content surpassed that of myristic acid, which was re- duced in comparison to the FA content in the microalga prior to di- gestion, thereby suggesting differences in the degree of bioaccessibility of this SFA. Regarding MUFA, the relative abundance of oleic acid in the bioaccessible fraction was higher than in the initial sample, Table 2 Lipid class distribution (% of total lipid) before and after digestion of the studied microalgae I. galbana and in other studies. Microalgae Initial vs Bioaccessible Lipid Classes Source TAG⁎ FFA⁎⁎ Polar Lipids Other I. galbana Initial 34.5 ± 0.3a 39.8 ± 1.0a 10.2 ± 0.7a 15.5 ± 1.4a Current study Bioaccessible 26.3 ± 4.5a 54.0 ± 5.7b 7.5 ± 0.4b 12.2 ± 1.6a Current study I. galbana Initial 26–61 – 39–74 – [21] Isochrysis sp. Initial 2.8 < 0.2 83.0 14.1 [24] I. galbana Initial > 50 – < 40 – [25] N. oculata Bioaccessible – < 55 – – [33] Current study values are presented as average ± standard deviation. For the current study data, different lowercase letters within a column correspond to statistical differences (p < 0.05). ⁎ TAG, triacylglycerol. ⁎⁎ FFA, free fatty acid. A B a bcd cd f ef de ef ef bc bcd b ab b bcd Fig. 1. Bioaccessibility (%) of total lipid and each fatty acid in the studied microalgae I. galbana. A: Total lipid and saturated (SFA) and monounsaturated fatty acids (MUFA); B: Polyunsaturated fatty acids (PUFA). Different lowercase letters correspond to statistical differences between fatty acids (p < 0.05). C. Bonfanti et al. Algal Research 29 (2018) 242–248 245 exceeding 17% of the total FAs. A similar increase was also determined for the two main ω6 PUFAs, linoleic acid and AA, entailing a corre- sponding increase in the total ω6 PUFA level. On the other hand, the stearidonic acid and DHA contents were slightly reduced after diges- tion. The other ω3 PUFAs, as a percentage of total FAs, were left un- changed. Hence, the total ω3 PUFA content in the bioaccessible fraction was lower than in I. galbana biomass before digestion, 30.7 ± 0.8% vs 33.6 ± 0.3% of total FAs. The opposite variations of total ω6 PUFA and total ω3 PUFA entailed a decrease of the ω3/ω6 ratio to only 2.1 ± 0.1 in the bioaccessible fraction of I. galbana. These variations had obvious implications for the bioaccessibility percentages. Both total lipid bioaccessibility and each particular FA bioaccessibility were low and within a narrow range of approximately 7–15%. The highest bioaccessibility percentages were determined for palmitic, oleic, and linoleic acids, as well as total ω6 PUFA, thus, agreeing with the relative enrichment of these FAs in the FA profile of the bioaccessible fraction. On the other end of the spectrum of varia- tion, the lowest bioaccessible percentages were calculated for myristic and stearidonic acids, DHA, and total ω3 PUFA. This also matched the observed variations in the FA profile of the bioaccessible fraction with respect to the initial (before digestion) FA profile. The remaining FAs exhibited intermediate bioaccessibility percentages. First, it should be highlighted that the results show the importance of studying human digestion of microalgal lipids, given the observed significant variations. For instance, ω6 PUFAs were more available for intestinal absorption than ω3 PUFAs. Nevertheless, the deviations with respect to the initial FA profile of I. galbana were not large. For fish (sole), a study on lipid bioaccessibility showed large variations to the ω3 PUFA content prior to digestion [31]. Just as in the current study, ω3 PUFA level was lower in the bioaccessible fraction of sole. Since FA in vitro bioaccessibility in the whole microalgal biomass was not pre- viously researched and investigation into microalgal foods is still a nascent area [36], there are only studies on other matrices, such as fish, as a reference. There are also some studies on microalgal FA apparent digestibility — relating FA contents in the diet and feces, thereby enabling an es- timate of the share of the initial FA content in the microalga that is assimilated by the organism — through in vivo animal experiments with salmon [37]. This study used untreated microalgal biomass (Schi- zochytrium sp.) and found evidence supporting high microalgal digest- ibility for lipids in general and ω3 PUFA in particular. This seems to oppose the low FA bioaccessibility (including ω3 PUFA) in I. galbana, but it should be noticed that salmon feed is extruded — extrusion pressure and temperature may favour cell disruption — and there are differences between microalgae species [38]. This deserves further in- vestigation, since the possibility of using unprocessed microalgal bio- mass directly in animal and human nutrition would be advantageous over extracting microalgal oil (economic costs in extraction, loss of nutrients, and higher risk of oxidation of ω3 PUFA). Specifically, aquaculture is in urgent need of adequate volumes of new ω3 PUFA sources [37], more sustainable in the long term than fish oil and less expensive than microalgal oil. The percentages of lipid and FA bioaccessibility in I. galbana were very low and unprecedented, if compared with other bioaccessibility studies on fish lipids and using a similar in vitro model [12,23,31,32]. In these studies, lipid bioaccessibility was always above 50% — salmon and gilthead seabream — [23,32] or 29% — meagre and sole — [12,31]. Therefore, there seems to be a higher difficulty in digesting microalgal biomass. Indeed, a study on N. oculata [33] showed that neither proteins nor lipids of intact microalgal cells were accessible to the mammalian digestive enzymes used in the applied digestion model. There was only some improvement in lipid digestion after microalgal treatment by pH-shift processing (from pH 7.0 to pH 10.0). A possible explanation was the microalgal cell wall composition of Nanno- chloropsis, consisting predominantly of cellulose, surrounded by an outer layer of algaenan, which may block enzymes from acting on the cell [39]. It is also known that algal cell walls change during growth and if microalgae are subjected to nitrogen stress [40]. However, Iso- chrysis genus lacks a cell wall, only possessing a plasma membrane covering [41]. For this reason, utilization of Isochrysis in nutrition has been advised as easily assimilated biomass. Accordingly, the explana- tion for the low lipid bioaccessibility in I. galbana must lie elsewhere. It is possible that chemical interaction phenomena play a role in these bioaccessibility results. In this case, microalgal lipids would have dis- played higher affinity for the non-bioaccessible fraction components, which could comprise carbohydrates and other components (Table 1). Furthermore, the low degree of lipolysis and high level of remaining TAGs (Table 2) suggest inhibition of the digestive model lipases by some component(s) in the microalgal biomass. These hypotheses and this lipid bioaccessibility study on microalgae need to be supported by further experimental work. Taking into account the very low bioaccessibility, the differences between bioaccessibility percentages of each FA were not very sig- nificant. Nonetheless, some differences, such as a higher bioaccessi- bility percentage of oleic acid and a lower bioaccessibility percentage of DHA and ω3 PUFA, have been found in fish matrices [23,32]. Chemical structure aspects may underlie these differences. It has been reported that higher level of unsaturation leads to lower bioaccessibility per- centage [23] and DHA and other ω3 PUFA are highly unsaturated. Another consequence of the low lipid bioaccessibility is a lower nutritional value regarding ω3 PUFA content. Effectively, for achieving the recommended daily intake of EPA + DHA according to the American Heart Association, 500 mg/day [30], a much higher quantity of freeze-dried I. galbana, approximately ten times more, must be in- gested than otherwise calculated (20.6 g). Of course, a serving portion of 200 g of freeze-dried microalgae or the incorporation of this quantity in a functional food is unviable. Therefore, it is of paramount im- portance to find options that enhance lipid bioaccessibility in I. galbana. Such options would enable to take advantage of anti-inflammatory substances, EPA [7] and other, present in the I. galbana biomass (see below). 3.3. Microalgal anti-inflammatory activity Anti-inflammatory activity may result from the inhibition of en- zymes — such as cyclooxygenase (COX-2)— involved in the conversion of fatty acids into prostaglandins and leukotrienes, which display an inflammatory action [42,43]. Accordingly, the anti-inflammatory ac- tivity values of the I. galbana biomass measured as a percentage of in- hibition of the enzyme COX-2 were determined and are presented in Table 3. Two different extracts of the initial (prior to digestion) freeze- dried biomass were tested, an aqueous and an oily extract. Further- more, the anti-inflammatory activity of the bioaccessible fraction (oily extract) was also determined. Extract concentration was 5 mg/ml in DMSO. Anti-inflammatory activity was only detected in the oily extract of the initial I. galbana biomass, reaching a value of 79 ± 7% of COX-2 inhibition. For the bioaccessible fraction extract — after eliminating the bioaccessibility blank background interference — and the other (aqu- eous) initial extract, no activity was detected. Table 3 Anti-inflammatory activity (% inhibition of cyclooxygenase-2, COX-2) in the studied microalgae I. galbana before (initial, in aqueous and lipid extract) and after digestion (bioaccessible, in lipid extract). Extract Anti-inflammatory activity (% inhibition of COX-2) Initial Aqueous nda Lipid 79 ± 7b Bioaccessible Lipid nda nd – Not detected. Values are presented as average ± standard deviation. Different lowercase letters within a column correspond to statistical differences (p < 0.05). C. Bonfanti et al. Algal Research 29 (2018) 242–248 246 There are some studies on the anti-inflammatory activity of micro- algae, but methodologies are different, ranging from in vitro assays to in vivo models [44–46]. This makes comparison among studies difficult. Nevertheless, several studies point to the existence of anti-in- flammatory activity in microalgae [44,46]. Whereas some authors found significant activity in aqueous extracts, for instance, in Ar- throspira platensis [44], other authors have reported anti-inflammatory properties of lipid components and precisely in I. galbana [46]. In general, different components may be involved, such as phenolic compounds, carotenoids, phytosterols, alkaloids, polysaccharides or proteins, such as phycocyanin [44]. However, in the current study, li- pids and lipophilic substances seem to be at the root of the anti-in- flammatory activity, since activity was only detected in the lipid ex- tract. Besides EPA, which may have anti-inflammatory properties [7], other lipid fraction substances in I. galbana biomass may contribute to the anti-inflammatory properties. Indeed, it should also be remarked that even though EPA and ω3 PUFA have anti-inflammatory effects [47], their action usually requires in vivo systems, which was not the case in the COX-2 inhibition assay used in current study. A recent study [46] identified glycolipids, such as digalactosyldiacylglycerols, as in- hibitors of the production of pro-inflammatory cytokine in human THP- 1 macrophages. The COX-2 inhibition reported in the current study seems to point to another anti-inflammatory mechanism, thus justifying further study on the nature of the COX-2 inhibitor(s) in the oily fraction of I. galbana. Such inhibitor(s) did not seem to be bioaccessible. If a low bioac- cessibility of the anti-inflammatory compounds in I. galbana is con- firmed, preparation of extracts for nutraceutical applications or mi- croalgal processing through decoction to produce tisane or other alternatives [33] may be advantageous, thereby rendering these com- pounds more bioaccessible. 4. Conclusions The biomass of the studied I. galbana was rich in protein and lipids. The FA profile was characterized by abundance of PUFA and, within PUFA, ω3 PUFA were the most abundant. High contents of myristic, oleic, linoleic, α-linolenic, and stearidonic acids as well as DHA were determined. Lipid, particularly FA, bioaccessibility was studied through the application of an in vitro model. A low hydrolysis of TAGs and polar lipids during digestion was observed. Moreover, both total lipid bioaccessibility and each particular FA bioaccessibility were low (be- tween 7 and 15%). The highest bioaccessibility percentages were de- termined for palmitic, oleic, and linoleic acids as well as total ω6 PUFA and the lowest bioaccessible percentages were calculated for myristic and stearidonic acids, DHA, and total ω3 PUFA. Chemical affinity phenomena may be at the root of these results. Concerning anti-in- flammatory activity, it was only detected in the oily extract of I. galbana biomass prior to digestion (79 ± 7% of COX-2 inhibition). No activity was detected in the bioaccessible fraction extract. Future work should focus on preparing extracts for nutraceutical applications or microalgal processing through decoction (tisane) for achieving a higher bioacces- sibility of lipid components and anti-inflammatory bioactives. Acknowledgments This work received financial support from CAPES for Carolina Bonfanti (88881.134961/2016-01). This work was also supported by the following Post-Doctoral Grants: Ref.: SFRH/BPD/102689/2014 (“Fundação para a Ciência e a Tecnologia”, FCT) for the author Carlos Cardoso; Ref.: SFRH/BPD/64951/2009 (FCT) and DIVERSIAQUA (MAR2020, Ref.: 16-02-01-FEAM-66) for the author Cláudia Afonso. A doctoral grant awarded by FCT (SFRH/BD/129795/2017) supported the work performed by Joana Matos. The experimental work was funded by the project Algared+ (0055_ALGARED_PLUS_5_E) “Rede Transfronteiriça para o Desenvolvimento de Produtos Inovadores com Microalgas”. Conflict of interest No conflict of interest involving any of the authors. Authors contributions The conception and design of the study was carried out by S Tanni and NM Bandarra, the acquisition of data and its analysis was done by C Bonfanti and T Garcia, and the interpretation of data was done by C Afonso and J Matos. The drafting the article and its critical revision for important intellectual content was performed by C Cardoso. Its final version was approved by NM Bandarra. Statement of informed consent, human/animal rights No conflicts, informed consent, human or animal rights applicable. References [1] J. Matos, C. Cardoso, N.M. Bandarra, C. Afonso, Microalgae as a healthy ingredient for functional food: a review, Food Funct. 8 (2017) 2672–2685. [2] A.P. Batista, L. Gouveia, N.M. Bandarra, J.M. Franco, A. 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http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0210 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0210 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0210 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0215 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0215 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0215 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0215 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0220 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0220 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0220 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0225 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0225 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0225 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0225 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0230 http://refhub.elsevier.com/S2211-9264(17)30888-3/rf0230 Potential of microalga Isochrysis galbana: Bioactivity and bioaccessibility Introduction Material and methods Sample collection and preparation Proximate composition Fatty acid profile Lipid class determination Anti-inflammatory activity Aqueous extract preparation for in vitro anti-inflammatory activity Cyclooxygenase (COX-2) inhibition assay In vitro digestion model Calculation of bioaccessibility Lipid extraction in the bioaccessible and undigested fractions Statistical analysis Results and discussion Microalgal characterization Proximate composition Lipid composition Microalgal lipid digestion Lipid hydrolysis Bioaccessible fatty acids Microalgal anti-inflammatory activity Conclusions Acknowledgments Conflict of interest Authors contributions Statement of informed consent, human/animal rights References