Food Chemistry 453 (2024) 139686 Available online 16 May 2024 0308-8146/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Impact of the biomass pretreatment and simulated gastrointestinal digestion on the digestibility and antioxidant activity of microalgae Chlorella vulgaris and Tetraselmis chuii Samuel Paterson a,1, Marta Majchrzak a,1, Denisa Alexandru a, Serena Di Bella b, Samuel Fernández-Tomé b, Elena Arranz b,c, Miguel Angel de la Fuente a, Pilar Gómez-Cortés a, Blanca Hernández-Ledesma a,* a Department of Bioactivity and Food Analysis, Institute of Food Science Research (CIAL, CSIC-UAM, CEI UAM+CSIC), Nicolás Cabrera 9, 28049 Madrid, Spain b Department of Nutrition and Food Science, Faculty of Pharmacy, Complutense University of Madrid (UCM), Plaza Ramón y Cajal s/n, 28040 Madrid, Spain c Departmental Section of Food Science. Faculty of Science, Autonomous University of Madrid (UAM) and Institute of Food Science Research (CIAL, CSIC-UAM, CEI UAM+CSIC), Nicolás Cabrera 9, 28049 Madrid, Spain A R T I C L E I N F O Keywords: Microalgae Simulated gastrointestinal digestion Antioxidant activity Phenolic compounds Bioaccessibility A B S T R A C T Chlorella vulgaris and Tetraselmis chuii are two microalgae species already marketed because of their richness in high-value and health-beneficial compounds. Previous studies have demonstrated the biological properties of compounds isolated from both microalgae, although data are not yet available on the impact that pre-treatment and gastrointestinal digestion could exert on these properties. The aim of the present study was to analyze the impact of the biomass pre-treatment (freeze/thaw cycles plus ultrasounds) and simulated gastrointestinal digestion in the bioaccessibility and in vitro antioxidant activity (ABTS, ORAC, Q-FRAP, Q-DPPH) of the released digests. The cell wall from microalgae were susceptible to the pre-treatment and the action of saliva and gastric enzymes, releasing bioactive peptides and phenolic compounds that contributed to the potent antioxidant ac tivity of digests through their radical scavenging and iron reduction capacities. Our findings suggest the potential of these microalgae against oxidative stress-associated diseases at both, intestinal and systemic level. 1. Introduction Today, the combination of a growing world population, the increasing incidence of chronic and non-communicable diseases (NCDs), simultaneously with consumer interest in sustainable and health- promoting products, has led global health organizations such as the Food and Agriculture Organization (FAO) and the World Health Orga nization (WHO) to recommend an eating pattern more focused on the consumption of functional foods with low environmental impact (Craig et al., 2021). All these together, has started a continuing worldwide search of alternative and sustainable sources of bioactive compounds. Among these sources, microalgae have generated great interest due to their sustainable nature and their richness in high value nutrients and bioactive compounds (Siegrist & Hartmann, 2023; Thavamani, Sferra, & Sankararaman, 2020). Chlorella vulgaris and Tetraselmis chuii are two of the most studied microalgae species whose consumption has been already approved by the European Food Safety Authority (EFSA) and the United States Department of Agriculture (USDA). Both microalgae have a high nutri tional density in terms of protein quality, and content of essential amino acids, polyunsaturated fatty acids, polysaccharides, vitamins, and min erals (Barghchi et al., 2023; Paterson, Fernández-Tomé, Galvez, & Hernández-Ledesma, 2023). Moreover, multiple bioactive properties such as anti-inflammatory, antioxidant, immunomodulatory, antimi crobial, antidiabetic, and antiatherogenic actions have been attributed to these two species (Barghchi et al., 2023; Kokkali et al., 2020; Nunes, Fernandes, Vasco, Sousa, & Raymundo, 2020), giving them a great biological and pharmacological potential (Hamouda et al., 2022; * Corresponding author. E-mail addresses: samuel.paterson@csic.es (S. Paterson), martamaj11@gmail.com (M. Majchrzak), denisa.alexandru@alumnos.upm.es (D. Alexandru), seredibe@ ucm.es (S. Di Bella), sfernandeztome@ucm.es (S. Fernández-Tomé), elena.arranz@uam.es (E. Arranz), ma.delafuente@csic.es (M.A. de la Fuente), p.g.cortes@csic.es (P. Gómez-Cortés), b.hernandez@csic.es (B. Hernández-Ledesma). 1 All authors have equally contributed to the study Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem https://doi.org/10.1016/j.foodchem.2024.139686 Received 22 March 2024; Received in revised form 29 April 2024; Accepted 13 May 2024 mailto:samuel.paterson@csic.es mailto:martamaj11@gmail.com mailto:denisa.alexandru@alumnos.upm.es mailto:seredibe@ucm.es mailto:seredibe@ucm.es mailto:sfernandeztome@ucm.es mailto:elena.arranz@uam.es mailto:ma.delafuente@csic.es mailto:p.g.cortes@csic.es mailto:b.hernandez@csic.es www.sciencedirect.com/science/journal/03088146 https://www.elsevier.com/locate/foodchem https://doi.org/10.1016/j.foodchem.2024.139686 https://doi.org/10.1016/j.foodchem.2024.139686 https://doi.org/10.1016/j.foodchem.2024.139686 http://crossmark.crossref.org/dialog/?doi=10.1016/j.foodchem.2024.139686&domain=pdf http://creativecommons.org/licenses/by-nc-nd/4.0/ http://creativecommons.org/licenses/by-nc-nd/4.0/ Food Chemistry 453 (2024) 139686 2 Panahi, Darvishi, Jowzi, Beiraghdar, & Sahebkar, 2016; Taroncher, Rodríguez-Carrasco, Barba, & Ruiz, 2023). The agri-food industry is increasingly interested in the emerging market of microalgae, as a sustainable alternative in the prevention, control, and treatment of NCDs (Barkia, Saari, & Manning, 2019; Camacho, Macedo, & Malcata, 2019). However, despite the great op portunities that offers the growth and exploitation of these microalgae, their use remains limited due to challenges associated with the scarce existing evidence on the bioactivity, digestibility, and bioavailability of their bioactive compounds, especially in terms of the comparability and reproducibility between research findings (Paterson, Gómez-Cortés, de la Fuente, & Hernández-Ledesma, 2023; Vieira et al., 2021). Moreover, in the case of microalgae C. vulgaris and T. chuii, their complex cell wall represents an additional challenge for human digestion, as it hinders bioaccessibility and nutrient release (de Carvalho et al., 2020; Schwenzfeier, Wierenga, & Gruppen, 2011). To overcome this limita tion, pre-treatment of biomass has become an essential step as it helps to expose different molecules to the action of digestive enzymes by elimi nating or disorganizing the microalga cell walls (de Carvalho et al., 2020; Krakowska, et al., 2018). Several methods, including ultrasounds (US), freeze-thaw cycles, enzymatic and chemical disruption, have dis played promise for pre-treating microalgal biomass (Sousa, Pereira, Vicente, Dias, & Geada, 2023). The antioxidant activity of microalgae C. vulgaris and T. chuii has been reported, although the existing evidence have been referred to specific, isolated, and purified compounds from their biomasses (Cunha, Coscueta, Nova, Silva, & Pintado, 2022; El-fayoumy, Shanab, Gaballa, Tantawy, & Shalaby, 2021; Gil-Cardoso et al., 2022). However, to our knowledge, there is a lack of evidence on the antioxidant properties of whole biomass of these two species after being subjected to in vitro gastrointestinal digestion or on the impact that pre-treatment of the biomass might have on the microalgae susceptibility to digestion and on the consequent release of antioxidant compounds. Therefore, the main objective of the present work was to evaluate the impact that both, biomass pre-treatment and simulated gastrointestinal digestion, exerts on the antioxidant activity of C. vulgaris and T. chuii to better address and understand the full potential of these microalgae as sustainable sources of health protective compounds. 2. Materials and Methods 2.1. Samples and reagents Commercial C. vulgaris and T. chuii microalgae biomass were sup plied by AlgaEnergy S.A. (Madrid, Spain). Pepsin (EC 232–629-3; 2424.6 U/mg), pancreatin (232–468-9; 7.2 U/mg), bile, 2,2′-azino-bis- (3-ethylbenzothiazolin-6-ammonium sulfonate) (ABTS), fluorescein disodium (FL), 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2′-azobis(2-amidinopropane) dichlorohydrochloride (AAPH), potassium persulfate (K2S2O8), monosodium phosphate (NaH2PO4), sodium carbonate (Na2CO3), disodium phosphate (Na2HPO4), sulfuric acid (H2SO4), monopotassium phosphate (KH2PO4), anhydrous sodium sulfate (Na2SO4), calcium chloride (CaCl2), sodium hydroxide (NaOH), trifluoroacetic acid (TFA), chlorohydric acid (HCl), gallic acid, and acetonitrile (ACN) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chloroform, methanol and hexane were pur chased from Osgonsa (Madrid, Spain). 2.2. Characterization of microalgae biomass The experimental and methodological Data Flow Chart of the study is shown in Fig. 1. The protein content of microalgae biomass was deter mined by Kjeldahl (method 981.10, AOAC International) (Bradstreet, 1954), employing a block digester (J.P. Selecta, Barcelona, Spain), and a Buchi Kjeldahl K-314 distillation unit (BÜCHI Labortechnik AG, Flawil, Switzerland). Analysis was carried out in duplicate and a conversion factor of 5.95 was used (López et al., 2010). The amino acid (AA) content of the microalgae biomass was deter mined in duplicate by cation exchange chromatography (Methods, 2023 - AOAC International), along with a Biochrom 30 series Amino Acid Analyser (Biochrom, Cambridge, UK). The carbohydrate content of the microalgae biomass was determined in triplicate using the sulfuric acid ultraviolet absorption method (SA-UV method) based on Ehnert, See hase, Müller-Renno, Hannig, and Ziegler (2021), using glucose as a standard (0–0.2 mg/mL, R2 value = 0.9954, Fig. S1). The microalgae lipid extraction procedure was performed in triplicate using chloroform and methanol (2:1, v/v) and following Figueiredo, da Costa, Silva, Domingues, and Domingues (2019) procedure. 2.3. Pretreatment of microalgae biomass Microalgae biomass was used intact (non-treated biomass) and subjected to a pre-treatment process (pre-treated biomass) consisting in cycles of freezing (− 20 ◦C, 15 h) and thawing (room temperature, 9 h) over a period of 5 consecutive days, followed by US treatment. The US treatment was performed using the Fisherbrand™ Sonicator 505 (Branson, Danbury, USA) with 70% of amplitude (20 kHz, 500 W) using Fig. 1. Experimental and methodological Data Flow Chart. S. Paterson et al. Food Chemistry 453 (2024) 139686 3 a 0.63 cm probe and a total of 6 cycles, each lasting 20 s, with a resting period of 1 min between cycles. 2.4. Microscopy of microalgae biomass To investigate the effect of the pre-treatment on the microalgae cell wall integrity, microscopy images of the non-treated and pre-treated microalgae biomass were taken following the protocol described by Alonso-Español et al. (2023) using the inverted microscope for cell culture (Leica DM IL, Wetzlar, Hessen, Germany) for the electronic im ages and the JEOL 6400 JSM microscope (JEOL, Tokyo, Japan) with a thermionic cathode electron gun attached to a tungsten filament and a secondary electron detector at 25 kV (KV, 3.5 nm - 10 nm) of image resolution for the scanning electron microscopy (SEM) images. 2.5. Simulated gastrointestinal digestion The simulated gastrointestinal digestion of microalgae biomass was based on the INFOGEST static in vitro protocol (Brodkorb et al., 2019), with some modifications. A pool of saliva was made from 7 healthy volunteers and the simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) were preheated at 37 ◦C and prepared according to the INFOGEST protocol. Pepsin from porcine gastric mucosa was mixed with ultrapure water to achieve a final concentration of 33 mg/mL. Pancreatin from porcine pancreas was prepared by dissolving it in SIF to a final concentration of 111.1 mg/mL. Finally, bovine bile was mixed with SIF to a final concentration of 12.9 mg/mL. The digestion involved the exposure of the microalgae biomass to three successive digestive phases: oral, gastric, and intestinal. In the oral phase, 4.5 mL of saliva were mixed with 0.5 mL of ultrapure water and added to the microalgae samples (1 g). The mixture was incubated at 37 ◦C with 120 rpm of agitation for 2 min in the Environmental Shaker – Incubator ES 20/60 (Biosan Medical-biological Research & Technolo gies, Warren, MI, USA). For the gastric phase, the oral bolus was mixed with 4.8 mL of SGF and adjusted to pH 3.0. Pepsin (300 μL, 1/100 w/w), CaCl2 (3 μL) and ultrapure water were added to reach a final volume of 12 mL, and the mixture was incubated at 37 ◦C with agitation for 2 h. In the intestinal phase, 5.1 mL of SIF were added to the gastric chyme and the pH was adjusted to 7.0. The bile (1.5 mL, 1:50 w/w), pancreatin (3 mL, 1:3 w/w), CaCl2 (24 μL) and ultrapure water were added to reach a final volume of 24 mL. The resultant homogenate was incubated at 37 ◦C with agitation for 2 h. At the end of this phase, the enzymes were inactivated at 95 ◦C for 15 min. The intestinal chyle was centrifuged at 2000 g in an EppendorfTM Centrifuge 5804R (Hamburg, Germany), at 4 ◦C for 30 min. Two fractions were obtained, the supernatant corre sponding to the absorbable fraction (AF) and the pellet corresponding to the non-absorbable fraction (NAF). Five digestions of each sample were conducted as well as control digestions with milli-Q water. Samples were lyophilized and stored at − 20 ◦C until further analysis. 2.6. Characterization of absorbable and non-absorbable fractions 2.6.1. Measurement of bioaccessibility Bioaccessibility was measured using weight changes, before and after in vitro digestion, and calculated by eq. [1] (Jin et al., 2020). Bioaccessibility (g/g) = 1– non − absorbable fraction weigth (g) microalgae biomass weigth (g) (1) 2.6.2. Determination of the protein content and profile The protein content of the AFs and NAFs was determined in triplicate using the bicinchoninic acid (BCA) method, following the protocol described by Paterson, Fernández-Tomé, et al. (2023) and the in structions of the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scien tific). Bovine serum albumin (BSA, 25–1000 μg/mL) was used as standard. Electrophoresis was carried out in a Criterion™ Cell electrophoresis tank (Bio-Rad, Hercules, CA, USA) using 1× XT MES buffer prepared by mixing 50 mL of 20× XTMES with 950 mL of Milli-Q water as it has been previously described by Paterson, Gómez-Cortés, et al. (2023). The samples were diluted to a concentration of 2 mg of protein/mL (50 μg protein/well) in sample buffer. The molecular weight marker was Pre cision Plus Protein™ Dual Xtra Prestained Protein Standards (Bio-Rad). Gels were stained with Bio-Safe Coomassie G-250 Stain reagent and subsequently washed to obtain and analyze images with the Versadoc Imaging System gel reader and the Image Lab v 6.1 software (Bio-Rad). 2.6.3. Total polyphenols by fast blue assay by QUENCHER method The total polyphenols content (Q-Fast Blue BB) was performed using the QUENCHER procedure (QUick, Easy, New, CHEap, and Reproduc ible), in which samples are submitted to direct contact with the reagents of each assay, following the methodology described by (Garcia-Herrera et al., 2022). Analysis was carried out in triplicate and results were calculated using a standard calibration curve (gallic acid, GA, concen tration 0.52–133.33 μg/mL) and expressed in mg of GA equivalent (GAE)/g of sample. 2.7. Determination of the antioxidant activity 2.7.1. ABTS radical scavenging activity The ABTS radical neutralization activity was assessed using the methodology outlined by Re et al. (1999). Trolox, ranging from 25 to 200 μM, was employed as the standard. Absorbance values were read using the Multiskan™ FC plate reader (Thermo Scientific) and then plotted against the quantity of Trolox (in μmoL) or the sample (in g). The ratio of the sample’s slope to the slope of Trolox was calculated to determine the Trolox equivalent antioxidant capacity (TEAC), expressed as μmol Trolox equivalent (TE)/g of sample. 2.7.2. Oxygen radical antioxidant capacity The assessment of the Oxygen Radical Antioxidant Capacity (ORAC) was carried out in accordance with the procedure outlined by Hernán dez-Ledesma, Dávalos, Bartolomé and Amigo, 2005. Fluorescence measurements were recorded at 485 nm excitation and 520 nm emission wavelengths at 2-min intervals over a 120-min duration using the FLUOstar OPTIMA plate reader (BMG Labtech, Offenburg, Germany). The resulting ORAC values were expressed as μmol TE/g of sample. 2.7.3. Ferric reducing antioxidant power (FRAP) assay by QUENCHER method Determination of the antioxidant activity by the ferric reducing antioxidant power (FRAP) by QUENCHER methodology (Q-FRAP) assay was performed following the methodology of Del Pino-García, García- Lomillo, Rivero-Pérez, González-Sanjosé and Muñiz (2015) and Garcia- Herrera et al. (2022). Absorbance was read at 595 nm in triplicate, using a Synergy HTX (Agilent Biotek, Santa Clara, CA, USA) multi-mode reader. A calibration curve with Trolox was used as standard (3.125–250 μg/mL), and the results were calculated as mg of TE/g of sample. 2.7.4. DPPH assay by QUENCHER method The antioxidant capacity by DPPH method was determined as pre viously reported by Del Pino-García et al. (2015) and Garcia-Herrera et al. (2022) using QUENCHER methodology (Q-DPPH). Absorbance was measured at 517 nm in microplates using a Synergy HTX (Agilent Biotek) multi-mode reader. Results were calculated using a standard calibration curve with Trolox (12.5–200 μg/mL) and expressed as mg TE/g of sample. 2.8. Statistical analysis The results obtained were analyzed using the statistical analysis S. Paterson et al. Food Chemistry 453 (2024) 139686 4 software GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). To assess the significance of the differences observed in the calculation of bioaccessibility, a One Sample t-test was employed. For protein con tent, phenolic content and antioxidant activity data, a one-way ANOVA followed by a Tukey test was performed. Differences were considered statistically significant at P < 0.05. 3. Results and discussion 3.1. Characterization of microalgae biomass The results of the protein, carbohydrate, lipid, and amino acid composition of microalgae biomass are shown in Table 1. T. chuii and C. vulgaris had a protein content of 32.19 ± 0.29% and 61.27 ± 0.46%, respectively. Previous studies using Kjeldahl protocol had reported values of 35–40% for T. chuii and 60.0% for C. vulgaris (Muys et al., 2019; Pereira et al., 2019). The protein content was also determined by the BCA assay, obtaining values of 22.26 ± 1.78% and 33.33 ± 2.77% in non-treated T. chuii and C. vulgaris biomasses, respectively. The differ ences between protein values determined by Kjeldahl and BCA assays could be due to the factor used to convert nitrogen into protein content that could be overestimated for these microalga species. In addition, the potential influence of the matrix composition on the results obtained by the BCA assay, as it was previously reported by Hueso, Fontecha and Gómez-Cortés (2022) could contribute on the observed differences. The protein content of pre-treated C. vulgaris biomass was 41.81 ± 3.06%, indicating that the pre-treatment favored the release of proteins that were measured by the BCA assay. However, no changes were observed in the protein content of pre-treated T. chuii biomass, thus, further exploration would be encouraged to understand the protein behavior of this microalga under pre-treatment conditions. The AA composition has been reported as an essential parameter to evaluate the protein quality of any food source (Pereira et al., 2019). Table 1 shows the AA content (g/100 g protein and g/100 g biomass) of T. chuii and C. vulgaris. All AA were analyzed except tryptophan (Trp), as it was degraded by the methodology conditions. Both species had a high AA content, being leucine (Leu) the major essential AA (EAA) in both microalgae. Among non-essential AA (NEAA), glutamic acid (Glu) and aspartic acid (Asp) were the predominant in both species. Comparing the results obtained for both species with the recommendation of the FAO/ WHO/United Nations University (UNU) report (2007), the requirements established for each AA were covered (Millward, Layman, Tomé, & Schaafsma, 2008). The carbohydrate content of T. chuii and C. vulgaris biomasses was 7.58 ± 0.28% and 6.64 ± 0.65%, respectively (Table 1). However, previous findings have reported higher amounts of carbohydrates for T. chuii (8–9% of dry matter, d.m.) and C. vulgaris (16.0% d.m.) (Chia, Lombardi, & da Graça Gama Melão, M., & Parrish, C.C., 2015; Renaud, Van Thinh, & Parry, 1999) under controlled growth conditions. More over, the lipid content determined for T. chuii was 34.57 ± 2.89%, similar to that (32.0%) reported by Banskota, Sperker, Stefanova, McGinn, and O’Leary (2019). However, lower values (11.7–14.0% of d. m.) were reported by Rahman et al. (2017). In the case of C. vulgaris, its lipid content of 10.20 ± 2.00% was in the range reported in the litera ture for this microalga specie (Spínola, Costa, & Prates, 2023). These results obtained after the biomass characterization highlight the great nutritional and biological interest of T. chuii and C. vulgaris. However, the differences found in comparison with literature data support the important influence exerted by factors such as the strain and the culti vation conditions on the nutritional value of microalgae (Ferreira et al., 2021). Thus, the macronutrient content and distribution in T. chuii and C. vulgaris have been demonstrated to depend on the nutrient Table 1 General composition and amino acid profile of microalgae Tetraselmis chuii and Chlorella vulgaris. Tetraselmis chuii g/100 g biomass Chlorella vulgaris g/100 g biomass Lipid 34.57 ± 2.89 10.20 ± 2.00 Carbohydrate 7.58 ± 0.28 6.64 ± 0.65 Proteina 32.19 ± 0.29 61.27 ± 0.46 Amino acid g/100 g protein g/100 g biomass g/100 g protein g/100 g biomass FAO recommendation (g/100 g protein) Essential Lysine (Lys) 3.12 ± 0.43 1.01 ± 0.14 3.92 ± 0.26 2.44 ± 0.16 5.20 Tryptophane (Trp) n.d. n.d. 0.70 Phenylalanine (Phe) 3.53 ± 0.55 1.14 ± 0.18 2.82 ± 0.17 1.75 ± 0.10 4.60b Tyrosine (Tyr) 2.01 ± 0.29 0.65 ± 0.09 2.18 ± 0.11 1.36 ± 0.07 Methionine (Met) 1.33 ± 0.16 0.43 ± 0.05 1.45 ± 0.09 0.90 ± 0.05 2.60c Cysteine (Cys) 1.21 ± 0.07 0.39 ± 0.02 0.72 ± 0.00 0.45 ± 0.00 Threonine (Thr) 3.03 ± 0.41 0.98 ± 0.13 2.58 ± 0.12 1.60 ± 0.07 2.70 Leucine (Leu) 4.87 ± 0.47 1.57 ± 0.15 4.51 ± 0.21 2.80 ± 0.13 6.30 Isoleucine (Ile) 2.15 ± 0.19 0.69 ± 0.06 1.79 ± 0.02 1.11 ± 0.01 3.10 Valine (Val) 3.30 ± 0.40 1.06 ± 0.13 2.77 ± 0.14 1.72 ± 0.09 4.20 Non-essential Aspartic acid + Asparagine (Asx) 6.46 ± 0.92 2.08 ± 0.30 5.15 ± 0.37 3.20 ± 0.23 Glutamic acid + Glutamine (Glx) 8.59 ± 0.97 2.77 ± 0.31 6.32 ± 0.43 3.92 ± 0.27 Serine (Ser) 3.03 ± 0.42 0.98 ± 0.14 2.35 ± 0.15 1.46 ± 0.09 Histidine (Hys) 0.78 ± 0.11 0.25 ± 0.03 1.13 ± 0.07 0.70 ± 0.04 Arginine (Arg) 3.53 ± 0.35 1.14 ± 0.11 3.13 ± 0.20 1.94 ± 0.12 Alanine (Ala) 4.43 ± 0.57 1.43 ± 0.18 4.44 ± 0.29 2.76 ± 0.18 Proline (Pro) 3.31 ± 0.48 1.07 ± 0.16 2.56 ± 0.17 1.59 ± 0.10 Glycine (Gly) 3.40 ± 0.47 1.09 ± 0.15 2.75 ± 0.19 1.71 ± 0.12 EAA 24.55 7.92 22.74 14.13 NEAA 33.53 10.81 27.83 17.28 TAA 58.08 18.73 50.57 31.41 EAA*100/TAA (%) 42.27 44.97 40.0 EAA*100/NEAA (%) 73.22 81.71 60.0 HAA*100/TAA (%) 45.01 45.96 AAA*100/TAA (%) 9.54 9.89 a : Protein measured by Kjeldahl method; b: Phe + Tyr; c: Met + Cys; EAA: essential amino acids; NEAA: non-essential amino acids; TAA: total amino acids; HAA: hydrophobic amino acids (Ala + Val + Ile + Leu + Tyr + Phe + Trp + Met + Pro + Cys); AAA: aromatic amino acids (Phe + Trp + Tyr). S. Paterson et al. Food Chemistry 453 (2024) 139686 5 availability, salt stress, light intensity, temperature, and metabolism, among other factors. Consequently, the high adaptability and mallea bility of these microalgae make the modulation of the environmental growing parameters an affordable way to produce nutritionally rich microalgae (Andreeva et al., 2021; Jahromi, Koochi, Kavoosi, & Shah savar, 2022; Muys et al., 2019). 3.2. Pre-treatment of microalgae biomass Mechanically, with the use of freeze-thaw cycles, ice crystals are formed from intracellular water which made cells expand and occa sionally break. In the case of US, the waves transferred through the medium created local pressure changes and bubbles that collapse generating a cavitation phenomenon that is responsible for the ruptures and loss of integrity caused on the cell wall surface (Mercer & Armenta, 2011). Notably, the combination of freeze-thaw cycles and US has demonstrated to be particularly effective in the disruption of other microalgae cell walls (Geada et al., 2019). Microscopy images (Figs. 2 and 3) showed disorganization and loosening of the microalgae cell wall. Pre-treated T. chuii microscope images had a chaotic background full of particles in which most of the microalgae cells were more asymmetrical and have lost their original shape (Fig. 2A and C). More over, its corresponding SEM images (Fig. 2B and D) revealed a disrupted, frayed and almost disintegrated cell wall that could facilitate the release of compounds, and enhance their bioaccessibility after the simulated gastrointestinal digestion. In contrast, pre-treated C. vulgaris micro scopic images (Fig. 3A and C) had a clearer background in which only some microalgae cells have lost their original shape and most remain similar to the intact biomass. In addition, pre-treated C. vulgaris SEM image (Fig. 3D) mainly showed disorganization instead of disintegration of the cell wall, indicating that the perforation of the cell wall was not fully achieved after the freeze-thaw cycles and US pre-treatment. As Savchenko et al. (2017) reported, C. vulgaris has a thicker and more difficult to break cell wall than T. chuii, thus the pre-treatment of C. vulgaris becomes more critical in the favouring and release of nutri ents and intracellular compounds into the extracellular medium. Therefore, different, or stronger disintegration mechanisms should be considered. In the Postma et al. (2017) work, SEM images of bead- milling pre-treated C. vulgaris revealed that before disintegration, the cells have uniform spherical shape, but appeared to be cracked upon bead impact after which the cell content was released, leaving an empty cell wall envelope. 3.3. Behavior of microalgae under simulated gastrointestinal digestion Bioaccessibility is defined as the fraction of a food/component that is released from the food matrix in the gastrointestinal tract that becomes available for absorption (Canelli et al., 2020). In our study, bio accessibility values for non-treated T. chuii and C. vulgaris, calculated using the formula indicated in section 2.6.1, were 0.51 and 0.50 g/g of biomass, respectively. Kose, Ozen, Elibol, and Oncel (2017) reported a bioaccessibility value of 0.40 g/g of biomass for C. vulgaris. Although close to our results, these differences could be due to the conditions of the in vitro digestion method used. Interestingly, the values determined in pre-treated samples were different between species. In the case of T. chuii, the pre-treatment increased its bioaccessibility up to 0.55 g/g of Fig. 2. Microscopy images of the non-treated and pre-treated Tetraselmis chuii biomass. Electronic microscopy images of (A) non-treated and (C) pre-treated biomass (0.4 × 50); Scanning Electron Microscopy (SEM) images of (B) non-treated and (D) pre-treated biomass. S. Paterson et al. Food Chemistry 453 (2024) 139686 6 biomass while the bioaccessibility of C. vulgaris was not affected. Sørensen et al. (2023) reported that bead-milling cell wall disruption of T. chuii facilitated efficient release of total phenolic compounds, chlo rophyll A, chlorophyll B and total carotenoids after gastrointestinal digestion, supporting the idea that the pre-treatment before the gastrointestinal digestion increased the bioaccessibility of the micro algae biomass, as confirmed in our study. However, the greater wall thickness of C. vulgaris could explain the absence of changes in the bioaccessibility of the resulting digests from pre-treated biomass, as its cell wall and food matrix could not be completely disaggregated, being Fig. 3. Microscopy images of the non-treated and pre-treated Chlorella vulgaris biomass. Electronic microscopy images of (A) non-treated and (C) pre-treated biomass (0.4 × 50); Scanning Electron Microscopy (SEM) images of (B) non-treated and (D) pre-treated biomass. Fig. 4. Electrophoresis (SDS-PAGE) gel of Chlorella vulgaris and Tetraselmis chuii (50 μg protein/mL). MW: Protein molecular weight marker (2–250 kDa); NAF: Non- absorbable fraction; AF: Absorbable fraction; ND: Non-digested microalgae biomass. S. Paterson et al. Food Chemistry 453 (2024) 139686 7 needed a stronger disruption method to fully release its nutritional compounds. In any case, further studies should be needed to validate and confirm the observed effects of pre-treatment on the bioaccessibility of both species. The protein profiles of non-digested pretreated C. vulgaris and T. chuii biomasses and their corresponding AFs and NAFs were analyzed through the SDS-PAGE (Fig. 4). Firstly, in the NAF of the digestion blank, bands correspondent to the main gastrointestinal enzymes used, like pepsin (38.3 kDa), trypsin (24 kDa), chymotrypsin (27 kDa), pancreatic α-amylase (55.4 kDa) and pancreatic lipase (52 kDa) appeared after precipitation during centrifugation. Moreover, numerous bands of low molecular weight that could come from the salivary proteome such as proline-rich proteins, immunoglobulins, statherins, cystatins and hista tins were also visible (blank NAF). In the AF from the digestion blank, less intense and defined low molecular weight bands were seen that could correspond to small proteins and peptides contained in the saliva or to peptides released by the autolysis or heterolysis of gastrointestinal enzymes during the digestion process. In the case of the undigested biomasses, C. vulgaris and T. chuii showed bands with molecular weights ranged from 3.2 to 224.1 kDa and 2.8 to 250.0, respectively. This range of protein molecular weights might correspond to chloroplast-related proteins, which were previously detected with bands between 7.80 and 46.0 kDa (Alavijeh, Karimi, Wijffels, van den Berg, & Eppink, 2020; Tejano, Peralta, Yap, Panjaitan, & Chang, 2019). Moreover, Piasecka and Baier (2022) reported bands between 50 and 80 kDa for C. vulgaris that could correspond to the stress response heat-shock proteins (Hsp 70 and Hsp 90), as well as other proteins involved in intracellular movements and growth such as dyneinand and α-tubulin. The high diversity in protein composition could be explained by the fact that microalgae do not accumulate distinct storage proteins as N-source (Yaakob, Mohamed, Al-Gheethi, Ravishankar, & Ambati, 2021). Therefore, most proteins detected would be enzymes involved in photosynthesis or other essential activ ities for survival and growth. For example, previous SDS-PAGE gels of other microalgae like Scenedesmus obliquus, which also belongs in the same Chlorophyta division showed bands between 58 and 62 kDa that corresponded to polypeptides from photosystem I complex (PSI-200) as well as other bands higher than100 kDa that might correspond to the chlorophyll A protein CPIV and P700-chlorophyll A protein 1 from photosystem I (Afify, El Baroty, El Baz, Abd El Baky, & Murad, 2018). Furthermore, in the NAFs of both microalgae, visible at the bottom of the gel, bands similar to those observed in the NAF of digestion blank and corresponding to the digestive enzymes and saliva protein constituents were observed in addition of other low molecular peptides. Finally, in the AFs of both microalgae, medium and small size proteins and peptides (2–47 kDa) appeared. The use of pepsin and trypsin, as well as other enzymes like alcalase, papain, and flavourzyme, for microalgae peptide production has been studied and mentioned before by different authors and microalgae species, thus supporting the partial susceptibility of the microalgae proteins to action of the proteolytic enzymes (Cunha & Pintado, 2022; Y. Li et al., 2021). The protein and phenolics contents of AFs and NAFs obtained from microalgae digests were determined (Table 2). For both microalgae, the protein contents of the NAFs were higher than those determined in the AFs which could be due to the presence of the digestive enzymes and microalgae proteins that remained intact after gastrointestinal digestion and precipitated by centrifugation. The protein contents of the NAFs from pre-treated C. vulgaris were significantly lower than those deter mined for the non-treated biomass. However, previously to the diges tion, the pre-treated C. vulgaris biomass presented higher protein concentration (41.81%) than that presented by the intact biomass (33.33%). This fact could indicate that proteins released under pre- treatment conditions were more susceptible to the digestive process, liberating peptides that remained solubilized in the AF, thus reducing the protein content in the pre-treated NAF in comparison with the NAF obtained from the non-pretreated biomass. The total polyphenol content was determined in the AF and NAF from both microalgae digests (Table 2) by using the highly phenolic selective Fast Blue BB assay. This protocol avoids potential interferences of reducing compounds as occurs with the Folin-Ciocalteu assay (Pico, Pismag, Laudouze, & Martinez, 2020). Fast Blue values determined in the AFs were ranged between 14.30 and 28.43 mg GAE/g fraction. The pre-treatment of the biomass did not result in changes in the phenolic compounds content of the AF from digests of both microalgae. In com parison with the AFs, the phenols content was higher in the NAFs from C. vulgaris digests, although it was lower in the case of NAFs from T. chuii, hence suggesting a different digestibility behavior of poly phenols between both microalgae. The presence of phenolic compounds after simulated gastrointestinal digestion confirmed previous recent findings. Li et al. (2023) showed that during intestinal digestion of Arthrospira sp., the phenolic compounds increased from 3.70 ± 0.27 to 7.93 ± 0.32 mg GAE/g. These authors suggested that the origin behind the well-known antioxidant activity of Arthrospira genus could be attributed to the release of bioactive phenolic compounds during digestion. Food phenolic compounds mainly occur as esters, glycosides and polymers that cannot be absorbed and require hydrolysis by digestive system enzymes or colonic microbiota. The bioaccessibility of poly phenols is affected by many parameters (Wojtunik-Kulesza et al., 2020) and, among these factors, the food matrix is a key element (Grundy, Moughan, & Wilde, 2024). Few studies have identified and quantified individual phenolic compounds in microalgae and these works showed that the phenolic profile of microalgae could be quite different between genus and even when comparing microalgal species from the same genus (Paterson, Fernández-Tomé, et al., 2023). Hence, differences in the type of phenolics compounds between C. vulgaris and T. chuii might be responsible for their different digestibility behavior. Moreover, the different composition in terms of dietary fiber, proteins, lipids, and other sample compounds (Grundy et al., 2024), might have an impact on the liberation and/or degradation of phenols during the gastrointestinal process, hence showing different levels in NAF and AF. When plant cells are broken through mastication or, in our case through a pre-treatment, phenolic compounds might associate with di etary fibers, leading to a modulation of their relative bioaccessibility (Mandalari et al., 2010). Therefore, dietary fiber, proteins, and other components within the food matrix would bond to phenols, ultimately preventing their mixture with gastrointestinal fluids and their release by conventional extractions (Alara, Abdurahman, & Ukaegbu, 2021). As well, the released number of phenolic compounds from the food matrix Table 2 Protein and phenolic compounds content of absorbable (AF) and non-absorbable fraction (NAF) from non-treated and pretreated microalgae Tetraselmis chuii and Chlorella vulgaris digests obtained under simulated gastrointestinal conditions.a, b,c Absorbable fraction Non-absorbable fraction Non- treated Pre- treated Non- treated Pre- treated Tetraselmis chuii Protein (g/100 g fraction) 18.13 ± 1.58a 17.42 ± 1.54a 21.58 ± 2.10b 20.33 ± 1.65b Total phenols (mg GAE/g fraction) 14.30 ± 1.45c 14.83 ± 1.51c 7.44 ± 0.65b 5.24 ± 0.53a Chlorella vulgaris Protein (g/100 g fraction) 23.74 ± 1.75a 22.37 ± 1.94a 34.73 ± 2.80c 26.32 ± 1.71b Total phenols (mg GAE/g fraction) 28.43 ± 1.94a 28.14 ± 1.24a 36.91 ± 2.66c 32.75 ± 2.41b GAE: gallic acid equivalent. a,b,c Means within a row with different superscripts indicate significant dif ferences between samples (P < 0.05). A one-way ANOVA calculation followed by a Tukey test was performed. S. Paterson et al. Food Chemistry 453 (2024) 139686 8 might be altered according to the way it is processed and the interaction of phytochemicals with other food components (Bohin, Vincken, Van Der Hijden, & Gruppen, 2012). All these together could explain the distribution of the total phenolic compounds in the microalgae digests, suggesting that some phenolic compounds were not digested yet while others could be linked to the undigested dietary fiber and proteins of the food-matrix present in the NAFs, especially for polysaccharide-rich cell wall microalgae like C. vulgaris. In addition, the sensitivity to autoxidation is probably overestimated in in vitro digestion models, as oxygen levels are lower in the gastroin testinal tract, and it should be noted that proteolytic enzymes could play a role in the polyphenol bioaccessibility by releasing phenolic com pounds bound to dietary proteins, as observed in the gastric tract for pepsin (Tarko, Duda-Chodak, & Zając, 2013). Lastly, polyphenols may exert anti-inflammatory, antioxidant, anti-cancer, and anti-diabetic ac tivities by positively modulating the gut microbiota, and so the food–gut human axis should be another factor to be considered (Lippolis, Cofano, Caponio, De Nunzio, & Notarnicola, 2023). 3.4. Impact of simulated gastrointestinal digestion on the antioxidant activity of microalgae It has been suggested that a universal and optimized assay protocol for determining the in vitro antioxidant capacity is needed. However, since there is no single method that can realistically measure the total antioxidant activity, current recommendations indicate that both elec tron transfer (ET) and hydrogen atom transfer (HAT) assays should be used to draw a full picture of a biological sample. In our study, the antioxidant activity of AFs and NAFs from simulated digests of both microalgae were determined by ORAC, ABTS, Q-DPPH, and Q-FRAP assays (Figs. 5 and 6). ORAC assay is considered a HAT-based assay whereas ABTS, DPPH and FRAP assay are ET-based protocols (Shahidi & Zhong, 2015). In the case of the antioxidant activity of the AF from C. vulgaris digest, measured by ORAC and ABTS assays, was higher than that determined for NAF (Fig 5A and B). However, the Q-DPPH value of AF was lower than that of NAF and no differences between fractions were observed in non-pretreated samples when Q-FRAP assay was used (Fig 5C and D). On the other hand, T. chuii AFs showed higher antioxi dant activity than its corresponding NAFs in ABTS and Q-FRAP assays (Fig 6B and D), while no statistical differences were observed in Q-DPPH (Fig 6C). What is more, T. chuii NAFs antioxidant activity was unde tectable through the Q-FRAP assay (Fig 6D). Although no data have been reported in the literature on antioxidant compounds released from these microalgae under simulated gastroin testinal conditions, there are previous studies that described the release of peptides by the action of other enzymes. Thus, Liu et al. (2023) re ported antioxidant activity by DPPH, hydroxyl free radical scavenging activity and ORAC assays in A. platensis hydrolyzates prepared by a marine Bacterium pseudoalteromonas sp. JS4–1 extracellular protease. The combination of fermentation with L. plantarum T0A10 and alcalase has also been shown to increase the TEAC values of this microalga specie up to 60% (Verni, Dingeo, Rizzello, & Pontonio, 2021). In addition, isolated C. vulgaris water/ethanol extracts and their combination with a US-assisted extraction have shown to display low in vitro antioxidant activities through the ORAC, ABTS, DPPH and FRAP assays compared to other seaweeds and microalgae like Ascophyllum nodosum, Lith othamnium calcareum and Bifurcaria bifurcata (Agregán et al., 2018; Frazzini et al., 2022), thus highlighting the important role of digestion enzymes over the release of the full antioxidant compounds from microalgae. The changes during the gastro and/or intestinal phases, the effect of the different digestive enzymes and the digestive pH conditions plus the own food-matrix effect could possibly affect the bioaccessibility and bioavailability of the antioxidants present in both AF and NAF (Ket nawa, Reginio, Thuengtung, & Ogawa, 2022).What is more, the differ ences between their antioxidant activities could possibly be due to the degree of solubility of their compounds as the more lipophilic antioxi dant compounds derived from the microalgae matrix, like carotenes, xanthophylls and other pigments could possibly remain in the NAF thus increasing its total antioxidant activity (Rocha, Coelho, Gomes, & Pin tado, 2023). Therefore, in vitro simulated gastrointestinal digestion models could provide information on the biological potency of bioactive components, which will allow us to elucidate their metabolic pathways and bioactivities at target sites. 4. Conclusions Our findings have demonstrated the great nutritional and biological potential of T. chuii and C. vulgaris microalgae. In addition of their nutritional value as source of high-quality proteins, carbohydrates, and lipids, these two microalgae might exert potent antioxidant effects after their gastrointestinal digestion through the release of potential bioactive and bioaccesible peptides, phenolic compounds and other bioactive compounds. The disruption of the microalgae cell wall by a combination of freeze/thaw cycles combined with US confirmed the crucial role that the pre-treatment played to increase the susceptibility of microalgae to the action of digestive enzymes and the release of compounds with po tential ability to protect against oxidative stress at both intestinal and systemic levels. Further studies are needed to fully understand the structural transformations of the two microalgae species during their transit through the gastrointestinal tract, the specific nutrient break down and liberation as well as its correlation with the antioxidant ac tivity observed for the final digests. These studies, that will ultimately help to understand T. chuii and C. vulgaris biological potential as sources Fig. 5. Antioxidant activity by (A) ORAC, (B) TEAC, (C) Q-DPPH and (D) Q- FRAP assays of absorbable (AF) and non-absorbable (NAF) fractions from non- treated and pre-treated (freeze-thaw cycles + ultrasounds) Chlorella vulgaris digests obtained under simulated gastrointestinal conditions. TE: Tro lox equivalent. S. Paterson et al. Food Chemistry 453 (2024) 139686 9 of health beneficial compounds. CRediT authorship contribution statement Samuel Paterson: Writing – original draft, Investigation, Formal analysis. Marta Majchrzak: Investigation, Formal analysis. Denisa Alexandru: Investigation, Formal analysis. Serena Di Bella: Investi gation. Samuel Fernández-Tomé: Writing – review & editing, Writing – original draft, Supervision. Elena Arranz: Writing – review & editing, Writing – original draft, Supervision. Miguel Angel de la Fuente: Writing – review & editing. Pilar Gómez-Cortés: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Blanca Hernández-Ledesma: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability Data will be made available on request. Acknowledgments This research was funded by the Spanish Ministry of Science and Innovation, grant number PID2021-122989OB-I00. E. Arranz and S. Fernández-Tomé thank to the ALIMNOVA UCM Research Group Ref: 951505 (Grant: GFRN32-23). S. Paterson gratefully acknowledges the Autonomous Community of Madrid for his predoctoral contract PIPF- 2022/BIO-24996. S. Di Bella thanks to Universitá degli studi di Palermo (Borsa di studio per Mobilità Internazionale dell’Ateneo A.A. 2023/24 -Traineeship autonomo) Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodchem.2024.139686. References Afify, A. E. M. M. R., El Baroty, G. S., El Baz, F. K., Abd El Baky, H. H., & Murad, S. A. (2018). Scenedesmus obliquus: Antioxidant and antiviral activity of proteins hydrolyzed by three enzymes. Journal of Genetic Engineering and Biotechnology, 16(2), 399–408. https://doi.org/10.1016/j.jgeb.2018.01.002 Agregán, R., Munekata, P., Franco, D., Carballo, J., Barba, F., & Lorenzo, J. (2018). 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