Potential of a Renin Inhibitory Peptide from the Red Seaweed

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Potential of a Renin Inhibitory Peptide from the Red Seaweed Palmaria palmata as a Functional Food Ingredient Following Confirmation and Characterization of a Hypotensive Effect in Spontaneously Hypertensive Rats Ciaran Fitzgerald,*,† Rotimi E. Aluko,‡ Mohammad Hossain,† Dilip K. Rai,† and Maria Hayes† †

Food Biosciences Department, Teagasc Food Research Centre, Ashtown, Dublin 15, Ireland Department of Human Nutritional Sciences, University of Manitoba, Winnipeg R3T 2N2, Canada



ABSTRACT: This work examined the resistance of the renin inhibitory, tridecapeptide IRLIIVLMPILMA derived previously from a Palmaria palmata papain hydrolysate, during gastrointestinal (GI) transit. Following simulated GI digestion, breakdown products were identified using mass spectrometry analysis and the known renin and angiotensin I converting enzyme inhibitory dipeptide IR was identified. In vivo animal studies using spontaneously hypertensive rats (SHRs) were used to confirm the antihypertensive effects of both the tridecapeptide IRLIIVLMPILMA and the seaweed protein hydrolysate from which this peptide was isolated. After 24 h, the SHR group fed the P. palmata protein hydrolysate recorded a drop of 34 mm Hg in systolic blood pressure (SBP) from 187 (±0.25) to 153 (± 0.64) mm Hg SBP, while the group fed the tridecapeptide IRLIIVLMPLIMA presented a drop of 33 mm Hg in blood pressure from 187 (±0.95) to 154 (±0.94) mm Hg SBP compared to the SBP recorded at time zero. The results of this study indicate that the seaweed protein derived hydrolysate has potential for use as antihypertensive agents and that the tridecapeptide is cleaved and activated to the dipeptide IR when it travels through the GI tract. Both the hydrolysate and peptide reduced SHR blood pressure when administered orally over a 24 h period. KEYWORDS: in vitro simulated gastrointestinal digestion, spontaneously hypertensive rats, renin inhibitory peptides, red macroalga



recently been reported from sources such as peas11 and hemp seed.12 Previous work carried out by this group isolated a renin inhibitory tridecapeptide with the amino acid sequence IRLIIVLMPILMA from a protein hydrolysate generated using the food-grade enzyme papain from the red seaweed Palmaria palmata.13 This peptide, IRLIIVLMPILMA, had a renin inhibitory IC50 value of 3.34 mM compared to the positive control used. The aim of this work was to assess if the tridecapeptide IRLIIVLMPILMA could survive gastrointestinal (GI) digestion and therefore could be used as a viable functional food ingredient that positively affects hypertension. In silico cleavage analysis coupled with a simulated GI digestion method were used to assess the resistance of this peptide in vivo using spontaneously hypertensive rats (SHRs). The peptide hydrolysate from which the tridecapeptide IRLIIVLMPILMA was isolated along with the chemically synthesized peptide was administered separately via oral gavage to SHRs. The effect on the blood pressure of the animals was observed over 24 h.

INTRODUCTION Therapeutic peptides of natural origin are used widely in the pharmaceutical industry.1 Peptide therapies range from anticancer and antimicrobial applications to peptide use in the treatment of the symptoms of Alzheimer’s disease.1 Cardiovascular disease (CVD) is the biggest natural cause of death in Western society, and there is a focus by pharmaceutical and functional food manufacturers on the prevention of its onset.2 High blood pressure or hypertension is the main contributory factor to CVD.2 To fight hypertension, different components of the renin angiotensin aldosterone system (RAAS) can be positively affected. High blood pressure is ordinarily treated with the use of drugs such as captopril and enalopril that are proven and effective antihypertensive agents.3 These drugs work by inhibiting angiotensin converting enzyme (ACE-I), the second rate-limiting enzyme of the renin angiotensin system (RAS) that forms part of the RAAS.3 However, inhibition of this enzyme can lead to side effects such as development of a dry cough and angioneurotic edema due to the disruption of kinin metabolism.4 The enzyme renin is the initial rate-limiting enzyme in the RAAS, and even though its importance within this system is known since 18985 and its substrate specificity described since 1957, commercialization of viable renin inhibitory drugs is a relatively recent occurrence.6 Inhibition of renin has advantages over ACE-I inhibition as it is not associated with the negative side effects previously mentioned.4 Peptides that inhibit ACE-I were discovered from a range of natural sources including soy,7 cereal,8 fish,9 and macroalgae.10 Similar to the trend observed with synthetic renin inhibitors, naturally occurring renin inhibitors have only © 2014 American Chemical Society



MATERIALS AND METHODS

Chemicals. The tridecapeptide IRLIIVLMPILMA was synthesized by GL Biochem Ltd. (Shanghai, China). All enzymes used for the simulated gastric digestion of the tridecapeptide including pepsin from Received: Revised: Accepted: Published: 8352

February 25, 2014 July 23, 2014 July 25, 2014 July 25, 2014 dx.doi.org/10.1021/jf500983n | J. Agric. Food Chem. 2014, 62, 8352−8356

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porcine gastric mucosa, amylase, pancreatin, and porcine bile extract were obtained from Sigma−Aldrich (Dublin, Ireland). All reagents used in the formation of the gastric fluids including potassium chloride, monobasic potassium phosphate, sodium bicarbonate, sodium chloride, and magnesium chloride hexahydrate were also sourced from Sigma−Aldrich (Germany). In Silico Analysis of the Renin Inhibitory Peptide IRLIIVLMPILMA. The program Expasy Peptide Cutter (http://web.expasy.org/ peptide_cutter/) was used to predict where the main proteases of the GI tract could cleave the renin inhibitory peptide IRLIIVLMPILMA. The online tool ToxinPred (http://crdd.osdd.net/raghava/ toxinpred/index.html) was used to predict the toxicity of the peptide and its fragments after hydrolysis.14 Simulated Gastric Digestion of the Peptide IRLIIVLMPILMA. The simulated gastric digestion method was carried out according to the method described by the European Cooperation in Science and Technology (COST) funded group INFOGEST (http://www.costinfogest.eu/). This method was devised under COST action FA1005 in an attempt to promote harmonization of currently used digestion models. Simulation of the gastric process was carried out in three phases as shown in Figure 1. Initially, an oral phase containing 1 g of

(SDF) (Table 1) was added to the SGF along with pancreatin at a concentration of 100 U/mL trypsin activity. Porcine bile extract was also added at a concentration of 2.4 mg/mL of the total volume. The mixture was adjusted to pH 7 using 1 M NaOH and left to stir in a water bath at 37 °C for 2 h before being deactivated using heating at 90 °C for 20 min and subsequently freeze-dried. Removal of Polyethylene Glycols from P. palmata Protein Digested Samples Using a Titanium Dioxide Cleanup Procedure. Digested samples were cleaned-up for further analysis using Pierce graphite spin columns available as part of the Pierce titanium dioxide phosphopeptide enrichment and cleanup kit (Thermo Fischer Scientific, Waltham, MA, USA). The graphite spin columns were used according to the manufacturer’s instructions. Briefly, the column was prepared by placing it in a 1.5 mL Eppendorf tube followed by centrifugation at 2000g for 1 min to remove storage buffer. The column was then rinsed twice with 100 μL of NH4OH and subsequently centrifuged at 2000g. The graphite was activated by adding 100 μL of acetonitrile followed by centrifugation at 2000g for 1 min before adding 100 μL of 1% trifluoroacetic acid (TFA) in water (1:99 v/v) followed again by centrifugation of the column at 2000g for 1 min. After column preparation, 50 mg of the hydrolysate was added and allowed to bind to the column for 10 min with periodical vortexing. The column was then centrifuged at 1000g for 3 min, and the flow through was discarded. The column was subsequently placed in a new tube and washed with 200 μL of 1% TFA followed by centrifugation at 2000g for 1 min. The column was then placed in a new tube and spun at 2000g for 1 min following addition of 100 μL of 0.1% formic acid (FA) in a 50:50 acetonitrile/water v/v ratio. This step was repeated three times, and the resulting elution was freezedried. Tandem Mass Spectrometry Analysis of the Hydrolysate. The enriched digested fraction was further analyzed using an electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer coupled to a nanoultra performance liquid chromatography (nano-UPLC) system (Waters Corporation, Milford, MA, USA) used in positive ionization mode. The sample was dissolved in water/ acetonitrile (80:20, v/v). The redissolved hydrolysate was injected onto a nano-UPLC Acquity column BEH130 C18 (100 μm × 100 mm, 1.7 μm particle size) preceded by a Symmetry C18 (180 μm × 200 mm, 5 μm particle size) trapping column. Mobile phases consisted of solvent A, which contained 0.1% FA in water, and solvent B, which contained 0.1% FA in acetonitrile. Trapping of the peptide/peptide fragments was achieved using a loading time of 3 min at a flow rate of 5 μL/min with 98% solvent A and 2% solvent B and then elution onto the analytical column at 300 nL/min. Chromatographic conditions consisted of 95% solvent A and 5% solvent B isocratically for 2 min, followed by a linear gradient from 85% to 20% solvent A over 63 min. Mass spectrometry (MS) data were acquired in MSe mode with collision energy for a full mass scan of 6 eV and a collision energy ramp of 15−35 eV for a mass range m/z 155 to m/z 1600. The QTOF was calibrated externally using MS/MS fragment ions of Glufibrinopeptide (Glu-fib). In Vivo Determination of the Antihypertensive Effect of the P. palmata Protein Hydrolysate and the Tridecapeptide IRLIIVLMPILMA in Spontaneously Hypertensive Rats. All rat experiments were performed according to protocols approved by the University of Manitoba Animal Care Protocol and Management

Figure 1. Schematic representation of the simulated, in vitro gastric digestion protocol used. The simulated digestion was carried out in three stages: (1) oral stage; (2) gastric stage; (3) duodenal stage. the synthesized peptide was mixed with 1 mL of simulated salivary fluid (SSF) (Table 1) and 150 U/mL amylase. This mixture was left to stir in a water bath (Stuart shaking water bath, Staffordshire, UK) at 37 °C for 2 min. The second phase of the simulation was the gastric phase. The oral phase was mixed with 2 mL of the prepared simulated gastric fluid (SGF) (Table 1) along with pepsin at a concentration of 1000 U/mL SGF. The pH was adjusted to pH 3 using 1 M hydrochloric acid (HCl) and left to stir in a water bath (Stuart shaking water bath) at 37 °C for 2 h. The final stage of the digestion was the duodenal phase. For this phase, 4 mL of the simulated duodenal fluid

Table 1. Concentrations of Electrolytes Used to Create the Simulated Saliva Fluid, Simulated Gastric Fluid, and Simulated Duodenal Fluid simulated saliva fluid

simulated gastric fluid

simulated duodenal fluid

compd

conc (mM)

compd

conc (mM)

compd

conc (mM)

KCl KH2PO4 NaHCO3 NaCl MgCl2(H2O)6

12.50 14.02 7.99 4.10 0.29

KCl KH2PO4 NaHCO3 NaCl MgCl2(H2O)6

35.08 0.63 25.98 41.00 0.59

KCl KH2PO4 NaHCO3 NaCl MgCl2(H2O)6

6.91 0.56 43.00 32.85 0.32

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Review Committee in accordance to the Canadian Council on Animal Care regulations. Adult (20 week old) SHRs weighing between 360 and 400 g were kept under a 12 h day/night cycle at 21 °C and fed a standard chow diet and water ad libutum. The SHRs were split into four groups of four rats each and administered the following treatment: (a) P. palmata hydrolysate dissolved in phosphate buffer saline (PBS, pH 7.2) at 50 mg/kg body weight, (b) the synthesized tridecapeptide IRLIIVLMPILMA dissolved in PBS at 50 mg/kg body weight, (c) the positive control captopril dissolved in PBS at 10 mg/kg body weight, and (d) PBS only. Each group received a 1 mL dose of each treatment via oral gavage. Before blood pressure was determined, rats were first anesthetized in a chamber at 40 °C with 4% isofluorane for 4 min to avoid stress-related blood pressure effects. The systolic blood pressure (SBP) of the SHRs was measured at 0, 2, 4, 6, 8, and 24 h by the tail cuff method while the rats were in an unconscious state using the Mouse Rat Tail Cuff Blood Pressure System (IITC Life Sciences, Woodland Hills, CA, USA). The change in SBP mm Hg (ΔSBP) was determined by subtracting the SBP at time n (where n is equal to 2, 4, 6, 8, or 24 h) from the SBP at time 0. Statistical Analysis. The student’s t test was performed using GraphPad Prism version 5.04 for Windows, GraphPad Software (La Jolla, CA, USA) and was considered significantly different if P < 0.05.

tridecapeptide IRLIIVLMPILMA following in silico analysis using the online tool Expasy Peptide Cutter. It shows that the peptide is cleaved at position 3, 6, and 11 by pepsin at both pH 1.3 and pH > 2. More importantly, however, in terms of renin inhibitory activity, the enzyme trypsin cleaves the peptide at position 2 at the carboxy end of the amino acid arginine releasing the dipeptide, amino acid sequence IR. Previous work showed that this dipeptide inhibited renin, and ACE-I activities at concentrations of 1 mg/mL previously when isolated from a pea peptide hydrolysate.11 To confirm the results obtained using in silico analysis, the tridecapeptide was subjected to the in vitro simulated gastric digestion outlined in Figure 1. In addition, the sample was analyzed using UPLC-MS in order to identify the breakdown products and the dipeptide IR. Figures 2 and 3 show the UPLC



RESULTS AND DISCUSSION The feasibility of functional food or pharmaceutical application of bioactive peptides depends on overcoming several challenges including lack of oral bioavailability. In addition, bioactive peptide action is often hampered by proteolytic attack. In this work, the resistance of a renin inhibitory tridecapeptide with the amino acid sequence IRLIIVLMPILMA was assessed using a combination of in silico analysis followed by a simulated GI digestion procedure and in vivo administration to SHRs. Table 2 shows the resultant peptide fragments obtained from the

Figure 3. Nano-ESI-MS spectra data showing singly protonated molecular ions for (a) peptide IRLIIVLMPILMA at m/z 1495 and doubly protonated at m/z 748, (b) hydrolyzed peptide fragment LIIVLMPILMA at m/z 1226, and (c) dipeptide IR at m/z 288.

Table 2. Cleavage Points of the Peptide IRLIIVLMPILMA by the Main Enzymes of the Gastrointestinal Tract Using the Online Tool Expasy Peptide Cutter name of enzyme

no. of cleavages

positions of cleavage sites

pepsin (pH 1.3) pepsin (pH > 2) trypsin

3 3 1

3, 6, 11 3, 6, 11 2

chromatograms obtained following MS analysis of the tridecapeptide following in vitro simulated GI digestion according to the INFOGEST procedure and the corresponding nano-ESI-MS spectra data. The base peak intensity chromato-

Figure 2. (a) Base peak intensity (BPI) chromatogram of the hydrolyzed analytes. (b) Extracted ion chromatograms (XIC) for [M + 2H]2+ ions at m/z 748.43. (c) [M + H]+ ions at m/z 1226. (d) [M + H]+ ions at m/z 288. 8354

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Figure 4. (a) Short-term (24 h) changes in systolic blood pressure (SBP) of spontaneously hypertensive rats (SHRs) after oral administration of saline, captopril, P. palmata protein hydrolysate, and the synthesized peptide IRLIIVLMPILMA. (b) SBP of SHR rats over 24 h after oral administration of saline, captopril, P. palmata protein hydrolysate, and the synthesized peptide IRLIIVLMPILMA. Values are mean ± SEM (n = 3). *P < 0.05 compared to the control (saline).

The MS data obtained confirms the in silico cleavage analysis results that predicted that the renin inhibitory dipeptide IR remains intact during normal digestive processes. Previous studies examined the ACE-I inhibitory activity in pea and whey protein digests using in vitro digestion but chose to analyze the resulting hydrolysates with SDS-PAGE and HPLC.15 In this study, ESI-Q-TOF mass spectrometry was used in tandem with UPLC due to the high sensitivity of peptide detection that ESIQ-TOF offers along with the rapid and high degree of separation offered by UPLC.16 The results shown in Figure 4 present the short-term changes in SBP observed over a 24 h period of SHRs fed a diet of the seaweed protein papain hydrolysate, the synthesized tridecapeptide, a positive control (captopril), and a saline solution (negative control). Figure 4a displays changes in SBP over a 24 h period whereas Figure 4b shows the actual SBP over the same time period following administration of the

gram (Figure 2a) displays the peaks of all hydrolyzed analytes. The extracted chromatogram in Figure 2b displays doubly charged [M + 2H]2+ ions at m/z 748 representing the whole, uncleaved tridecapeptide IRLIIVLMPILMA (theoretical mass = 1494). The corresponding nano-ESI-MS spectra data in Figure 3a shows both the protonated molecular ions for IRLIIVLMPILMA at m/z 1495 and the doubly protonated at m/z 748. Figure 2c displays the extracted chromatogram highlighting the peaks corresponding to the [M + H]+ ions at m/z 1226 representing the cleaved fragment LIIVLMPILMA (theoretical mass = 1225). Figure 3b displays the resultant nano-ESI-MS spectra data hydrolyzed peptide fragment LIIVLMPILMA at m/z 1226. Figure 2d shows the extracted ion chromatogram displaying [M + H]+ ions at m/z 288 the expected observed mass of the peptide IR (theoretical mass = 287) now cleaved from the parent peptide. The consequential nano-ESI-MS spectral data in Figure 3c shows the dipeptide IR at m/z 288. 8355

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(3) Gradman, A. H.; Kad, R. Renin inhibition in hypertension. J. Am. Coll. Cardiol. 2008, 51, 519−528. (4) Gradman, A. H.; Schmieder, R. E.; Lins, R. L.; Nussberger, J.; Chiang, Y.; Bedigian, M. P. Aliskiren, a novel orally effective renin inhibitor, provides dose-dependent antihypertensive efficacy and placebo-like tolerability in hypertensive patients. Circulation 2005, 111, 1012−1018. (5) Tigerstedt, R.; Bergman, P. Niere und Kreislauf. Skand. Arch. Physiol. 1898, 8, 223−271. (6) Skeggs, L. T.; Kahn, J. R.; Lentz, K.; Shumway, N. P. The preparation, purification, and amino acid sequence of a polypeptide renin substrate. J. Exp. Med. 1957, 106, 439−453. (7) Wu, J.; Ding, X. Characterization of inhibition and stability of soy-protein-derived angiotensin I-converting enzyme inhibitory peptides. Food Res. Int. 2002, 35, 367−375. (8) Ma, M.-S.; Bae, I. Y.; Lee, H. G.; Yang, C.-B. Purification and identification of angiotensin I-converting enzyme inhibitory peptide from buckwheat (Fagopyrum esculentum Moench). Food Chem. 2006, 96, 36−42. (9) Kim, S.-K.; Wijesekara, I. Development and biological activities of marine-derived bioactive peptides: A review. J. Funct. Foods 2010, 2, 1−9. (10) Suetsuna, K.; Nakano, T. Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatif ida). J. Nutr. Biochem. 2000, 11, 450−454. (11) Li, H.; Aluko, R. E. Identification and inhibitory properties of multifunctional peptides from pea protein hydrolysate. J. Agric. Food Chem. 2010, 58, 11471−11476. (12) Girgih, A. T.; Udenigwe, C. C.; Li, H.; Adebiyi, A. P.; Aluko, R. E. Kinetics of enzyme inhibition and antihypertensive effects of hemp seed (Cannabis sativa L.) protein hydrolysates. J. Am. Oil Chem. Soc. 2011, 88, 1767−1774. (13) Fitzgerald, C.; Mora-Soler, L.; Gallagher, E.; O’Connor, P.; Prieto, J.; Soler-Vila, A.; Hayes, M. Isolation and characterization of bioactive pro-peptides with in vitro renin inhibitory activities from the macroalga Palmaria palmata. J. Agric. Food Chem. 2012, 60, 7421− 7427. (14) Gasteiger, E.; Hoogland, C.; Gattiker, A.; Wilkins, M. R.; Appel, R. D.; Bairoch, A. Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook; Springer: New York, 2005; pp 571−607. (15) Vermeirssen, V.; van der Bent, A.; Van Camp, J.; van Amerongen, A.; Verstraete, W. A quantitative in silico analysis calculates the angiotensin I converting enzyme (ACE) inhibitory activity in pea and whey protein digests. Biochimie 2004, 86, 231−239. (16) Yang, B.; Dong, W.; Zhang, A.; Sun, H.; Wu, F.; Wang, P.; Wang, X. Ultra performance liquid chromatography coupled with electrospray ionization/quadrupole-time of-flight mass spectrometry for rapid analysis of constituents of Suanzaoren decoction. J. Sep. Sci. 2011, 34, 3208−15. (17) Li, H.; Prairie, N.; Udenigwe, C. C.; Adebiyi, A. P.; Tappia, P. S.; Aukema, H. M.; Jones, P. J.; Aluko, R. E. Blood pressure lowering effect of a pea protein hydrolysate in hypertensive rats and humans. J. Agric. Food. Chem. 2011, 59, 9854−60. (18) Li, C. H.; Matsui, T.; Matsumoto, K.; Yamasaki, R.; Kawasaki, T. Latent production of angiotensin I-converting enzyme inhibitors from buckwheat protein. J. Pept. Sci. 2002, 8, 267−74. (19) Fitzgerald, C.; Gallagher, E.; O’Connor, P.; Prieto, J.; MoraSoler, L.; Grealy, M.; Hayes, M. Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the Zebrafish larvae assay. Peptides 2013, 50c, 119−124.

control (saline), the known blood pressure lowering drug captopril (3 mg/kg bw) as a positive control, the P. palmata protein hydrolysate (50 mg/kg bw), and the synthesized peptide IRLIIVLMPILMA (3 mg/kg bw). Interestingly, both the hydrolysate and the tridecapeptide IRLIIVLMPILMA displayed very similar trends in lowering the SBP of the rats over a 24 h period. After 2 h, the group that was fed with captopril recorded a 29 mm Hg drop in SBP from 184 (±0.28) mm Hg to 156 (±1.08) mm Hg . Likewise, the group fed the P. palmata protein hydrolysate recorded a drop of 34 mm Hg in SBP from 187 (±0.25) to 153 (±0.64) mm Hg SBP, while the group fed the tridecapeptide IRLIIVLMPLIMA presented a drop of 33 mm Hg in blood pressure from 187 (±0.95) to 155 (±0.94) mm Hg SBP. After 24 h, the effect of all blood pressure lowering agents was drops of 21, 16, and 17 mmHg SBP, respectively, compared to the SBP recorded at 0 h. Previous papers examined the blood pressure lowering effects of a pea peptide hydrolysate in SHRs.17 Blood pressure was measured using the tail cuff plethysmography method to avoid stressrelated blood pressure effects. However, the pea protein hydrolysates were administered at a concentration of 200 mg/kg, and this dose resulted in a 19 mm Hg drop in SBP of SHRs. Likewise, SHRs treated with an antihypertensive buckwheat hydrolysate recorded an initial decrease in SBP of 27 ± 7.6 mm Hg after 2 h but only when treated with 100 mg/ kg body weight,18 twice the concentration of the P. palmata protein hydrolysate and the tridecapeptide IRLIIVLMPLIMA used in this study. In silico analysis of the peptide using the online tool ToxinPred also showed that the peptide was nontoxic, which indicates that it is suitable for use in functional foods and pharmaceutical agents. In addition, an earlier study carried out by this group found that the P. palmata hydrolysate was nontoxic to zebrafish when administered at a concentration between 1 and 5 mg/mL.19 This study demonstrates that the tridecapeptide IRLIIVLMPLIMA is a renin inhibitory peptide with the ability to impart an antihypertensive effect in vivo confirmed by monitoring changes in SBP in SHRs over a 24 h period. Both the peptide and the P. palmata protein papain hydrolysate may have potential for use as functional ingredients in the growing nutraceutical market to prevent hypertension.



AUTHOR INFORMATION

Corresponding Author

*Phone: +353 (0) 1 8059500; fax: +353 (0)1 8059550; e-mail: ciaran.fi[email protected]. Funding

This work was supported by the Irish Marine Functional Foods Research Initiative, NutraMara programme, which is funded by the Irish Department of Agriculture, Fisheries and Marine and the Marine Institute. It was also supported by the COST action INFOGEST, improving health properties of food by sharing knowledge on the digestive process FA1005. INFOGEST is supported by the EU Framework programme 2011-2015. C.F. is in receipt of the Teagasc Walsh Fellowship. Notes

The authors declare no competing financial interest.



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