Marine organisms, including macro-algae, are a valuable source of structurally diverse bioactive metabolites with various biological activities due to their living mode in highly competitive and aggressive surroundings, which are very different in many aspects from terrestrial environment, a situation that demands the production of quite specific and potent bioactive molecules. As a result, the importance of these organisms as a natural resource of bioactive compounds, which may lead to the development of new drugs and functional foods or nutraceuticals, is growing rapidly [1
Macro-algae have been consumed in Asian countries since ancient times and their dietary intake has been shown to decrease blood pressure in humans [2
]. However, the angiotensin I-converting enzyme (ACE) inhibitory activity from macro-algae has not been extensively studied [3
]. ACE belongs to the class of zinc metal proteases that catalyzes the conversion of angiotensin I to a potent vasoconstrictor angiotensin II and also promotes the degradation of the vasodilator bradykinin [4
]. Therefore, this multifunctional enzyme plays a key role in the control of blood pressure, since the inhibition of its activity leads to a decrease in the angiotensin II concentration and an increase in the bradykinin level, and consequently in the reduction of hypertension that is one of the major causes of chronic diseases and a high risk factor of cardiovascular diseases worldwide [5
]. A wide variety of synthetic drugs have been extensively used in treatment of hypertension and most of them have an ACE-inhibitory activity, however, these drugs can cause certain adverse side effects [6
]. Therefore, searching for ACE-inhibitors from natural resources, such as marine organisms including macro-algae [3
], has shown a growing interest in the field of nutraceutical, pharmaceutical and functional foods industries. The most commonly studied natural ACE-inhibitors were protein hydrolysates and peptides, however, others molecules that affect the ACE activity include the phorotannins group that are the predominant polyphenols in brown algae, being particularly abundant in Fucaceae [10
]. In addition to its strong ACE-inhibition, phlorotannins have been recently demonstrated to possess numerous health benefits, including potent antioxidant effects (for a review see [14
]). Since functional food products with multi-bioactivities are receiving wider attention, the potential antioxidant of macro-algal protein hydrolysates/peptides with ACE-inhibitory activity has also been investigated [15
]. In fact, it is well known that natural antioxidants are powerful substances that play an important role against various diseases (atherosclerosis, cancer, chronic inflammation, cardiovascular disorders, hypertension) and ageing process, directly related to oxidative stress [18
There is substantial scientific evidence that enzymatic hydrolysis of food protein sources is an efficient method to recover potent bioactive peptides that may present lower side effects [1
]. Furthermore, enzymatic proteolysis can also release other bioactive compounds such as phorotannins bound to proteins, as reported for some brown macro-algae [20
The Azores Islands (Portugal) being isolated in the middle of Atlantic Ocean and surrounded by waters with low pollution levels [21
] is a very promising location to look for new marine ingredients with medicine-like effects in treating or preventing chronic diseases. Traditionally, the Azorean population has gathered seaweeds either as food or for chemicals extraction. The brown seaweed Fucus spiralis
, which is abundant in the Azorean intertidal zone, is a local delicacy, particularly the frond tips (the receptacles) that are picked and eaten fresh [22
]. Previous studies on its nutritional and/or pharmacological value from our research group have reported that F. spiralis
is a good source of valuable biochemical compounds [23
] and its methanol extract could also be a source of powerful ACE-inhibitory phlorotannins with potential impact on human health [9
]. F. spiralis
from other origins have been also demonstrated to possess high antioxidant properties, mainly linked to its phlorotannin content [24
This is, for the best of our knowledge, the first study of F. spiralis enzymatic protein hydrolysate as source of compounds with multi-bioactivities for potential use in food and pharmaceutical industries and it was aimed to: (i) optimize the protein extraction process to obtain higher yield of F. spiralis protein hydrolysate, (ii) fractionate the cellulase–bromelain protein hydrolysate by ultrafiltration membranes into different molecular weight fractions (<1, 1–3 and ≥3 kDa), (iii) evaluate the ultrafiltrate fractions for multifunctional properties in vitro, namely the ACE-inhibitory activity and the antioxidant properties using different assays, such as the scavenging of 2,2-diphenyl-1-picrylhydrazyl radical, the ferrous ion-chelating and the ferric reducing antioxidant power, (iv) determine the amino acids profile, recovery yield, protein, peptide and total phenolic contents of the ultrafiltrate fractions, and (v) evaluate the in vitro digestibility of F. spiralis crude protein. The findings of the current study can also contribute to the increasing database of medicinal macro-algae.
3. Materials and Methods
3.1. Chemicals and Reagents
Methanol (MeOH) and acetonitrile (ACN) HPLC grade were purchased from Fluka Chemika (Steinheim, Switzerland). Ammonium sulphate, sodium carbonate (Na2CO3), sodium phosphate, sodium tetraborate decahydrate, iron (II) chloride, sodium hydroxide (NaOH), ethanol, phenol and 85% phosphoric acid were from E. Merck (Darmstadt, Germany). Acetyl chloride and isobutanol were purchased from Alltech Associates (Deerfield, IL, USA). Amino acids standard mixture and sequanal grade 6 N HCl were purchased from Pierce Chemicals (Rockford, IL, USA). Sodium chloride (NaCl), ethyl acetate, heptafluorobutyric anhydride (HFB-IBA), sodium caseinate, 2-mercaptoethanol, brilliant Blue G-250, albumin from bovine serum (BSA), potassium ferricyanide, iron (III) chloride, ferrozine, trichloroacetic acid (TCA), trizma base, zinc chloride, hippuric acid (HA), hippuryl-l-histidyl-l-leucine (HHL), Folin–Ciocalteu reagent (FCR), phloroglucinol, butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydrochloric acid (HCl), glutathione, O-phthaldialdehyde (OPA), β-mercaptoethanol, sodium dodecyl sulphate, angiotensin I-converting enzyme (ACE) from porcine kidney, bromelain (B4882), cellulase from Aspergillus niger (C1184), chymotrypsin (C9381), peptidase (P7500) and trypsin (T8003) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ultrafiltration membrane system and membranes were purchased from Millipore Co (Bedford, MA, USA).
3.2. Collection and Preparation of F. spiralis Sample
F. spiralis Linnaeus (Ochrophyta, Phaeophyceae) sample was collected in January 2013 from the littoral of São Miguel Island of Azores Archipelago (37°40′ N and 25°31′ W), Portugal, and a voucher specimen was prepared (voucher number AZB, SMG-13-04) and deposited in the Herbarium AZB—Ruy Telles Palhinha of the Department of Biology at the University of Azores. Within 24 h of collection, F. spiralis sample was first washed in seawater followed by distilled water to remove encrusting material, epiphytes and salts, and then air-dried and stored in an air-tight container in a freezer (−80 °C) for not more than 6 months until further analysis. Prior to the analytical procedures, the sample was defrosted and dried at 40–45 °C for 48 h (avoiding overheating that could lead to oxidation), and then was grounded into a fine powder of 0.5 mm particle size, re-dried at 40 °C and stored in the dark under N2 in a desiccator at a refrigerated temperature of 4–5 °C.
3.3. Extraction of Protein from F. spiralis
The protein of F. spiralis
was extracted according to the method described by Wong et al. [70
] with slight modifications. Five grams of sample powder were suspended in deionized water (1:10 w
) to induce cell lysis by osmotic shock that facilitated subsequent protein extraction. The suspension was gently stirred overnight at 35 °C, which was found to be the optimal temperature for macro-algae protein solubility. After incubation, the suspension was centrifuged at 10,000× g
and 4 °C for 20 min. The supernatant was collected and the pellet was re-suspended in deionized water with 0.5% 2-mercaptoethanol (v
) being the pH adjusted to 12.0 with 1 M NaOH. The solution was gently stirred at room temperature for 2 h before centrifugation under the same conditions as above. The second supernatant was collected and combined with the first one. The combined supernatant was stirred at 4 °C and the pH was adjusted to 7.0 before precipitation with ammonium sulphate. The extraction method mentioned above was repeated three times on the residue. The combined supernatant was precipitated by slowly adding ammonium sulphate, with stirring, until 85% saturation (60 g/100 mL) and allowed to stand for 30 min. Then the solution (precipitated protein) was removed by centrifugation at 10,000× g
, 4 °C for 20 min. The pellet obtained was dialyzed against distilled water until the total dissolved solutes of dialysate, measured by its conductivity, was similar to that of the distilled water. The retentate containing the protein concentrate of F. spiralis
was lyophilized in a freeze-drier and stored at −20 °C until required. The protein content was determined by Bradford method [71
] using BSA as the calibration standard.
3.4. In Vitro F. spiralis Protein Digestibility Evaluation
The in vitro digestibility of F. spiralis
protein concentrate suspension was determined using a freshly prepared multiproteolytic enzyme solution (trypsin, chymotrypsin and peptidase), according to Paiva et al. [72
], in order to reproduce the actual digestion environment in vivo. Fifty milliliters of aqueous protein suspension (6.25 mg protein/mL) in glass distilled water were adjusted to pH 8.0, while stirring in a 37 °C water bath. The multi-enzyme solution [1.6 mg/mL trypsin (10,000 BAEE units/mg protein), 3.1 mg/mL chymotrypsin (80 units/mg solid) and 1.3 mg peptidase/mL (50–100 units/g solid)] was maintained in an ice bath and adjusted to pH 8.0. Five milliliters of the multi-enzyme solution were then added to the protein suspension and the pH change in the mixture, caused by the enzymatic digestion, was measured after exactly 10 min. Sodium caseinate was used as control and the in vitro protein digestibility of the algae was expressed as a relative percentage to that of the sodium caseinate normalized at 100%.
3.5. Preparation of F. spiralis Protein Hydrolysate (FSPH)
The enzymatic reaction of the F. spiralis protein concentrate was carried out using two enzymes, cellulase followed by bromelain. One gram of protein concentrate sample was suspended and homogenized in 10 mL of ice-cold distilled water and each enzyme (substrate/enzyme ratio to 100:1, w/w) was individually added to the homogenate. For the enzymatic reaction was used cellulase at 50 °C and pH 4.5 (adjusted with 0.1 M HCl) followed by bromelain at 37 °C and pH 7.0 (adjusted with 0.5 M NaOH). The hydrolytic reaction was carried out for 20 h and after this period, the digestion solution was boiled for 10 min in order to inactivate the enzymes. The protein hydrolysate solution was centrifuged at 3000× g for 15 min, filtered through 0.45 μm filters, lyophilized in a freeze-drier and stored at −20 °C for further analysis.
3.6. Fractionation of FSPH by Ultrafiltration
The digested FSPH with cellulase and bromelain enzymes was further fractionated in a cell dialyzer system through three different ultrafiltration membranes with molecular weight cut-off (MWCO) of 1 and 3 kDa to obtain ultrafiltrates (Fr1 < 1 kDa, 1 kDa ≤ Fr2 < 3 kDa and Fr3 ≥ 3 kDa). All the pooled FSPH fractions were lyophilized in a freeze-drier and assayed for ACE-inhibitory and antioxidant activities. The recovery yield of the ultrafiltration fractions was calculated using the gravimetric method.
3.7. Protein and Peptide Contents Analysis of FSPH Fractions
The protein content was determined by Bradford method [71
] using BSA as the calibration standard and its peptide content was measured by OPA method [73
] using glutathione as the calibration standard, according to Ghanbari et al. [46
3.8. Amino Acids Composition of FSPH Fractions
The amino acids composition of FSPH fractions was determined according to Paiva et al. [3
]. The dried FSPH fractions (0.5 mg) were placed in a small reaction vials, exposed to a stream of dry nitrogen, then capped and submitted to an acid hydrolysis at 100 °C for 24 h with 100 µL 6 N HCl containing 0.1% phenol for tyrosine protection. After cooling until room temperature, the samples were evaporated under a stream of dry nitrogen and then derivatized slowly by adding the mixture of acetyl chloride:isobutanol (1.25:5 mL, v
, the reagent was obtained by adding the acetyl chloride to isobutanol precooled to −20 °C) and heating at 100 °C for 60 min. Next, the sample vials were opened and the mixtures exposed to a stream of dry nitrogen to remove excess reagent. After cooling in an ice bath, the samples were supplemented with 200 µL of acetonitrile and 50 µL of the derivatization reagent HFB-IBA, and heated again at 100 °C for 15 min. After evaporation of the excess reagent at 115 °C under a stream of dry nitrogen and cooling to room temperature, samples were dissolved in 300 µL of ethyl acetate and an aliquot (1 µL) was used for GC analysis. The GC analysis was performed using a Bruker GC model 450-GC gas chromatograph equipped with a split/splitless injector and a flame ionization detector (FID) using a wall-coated open tubular (WCOT) fused silica AT-Amino acid capillary column (25 m × 0.53 mm i.d., 1.2 µm film thickness) from Heliflex/Alltech (San Jose, CA, USA). The temperature started at 60 °C for 3 min, programmed at a rate of 4 °C/min to 210 °C and then held at this temperature for further 20 min. The injector and detector temperatures were held constants at 260 °C and 280 °C, respectively. Helium was the carrier gas at a flow rate of 28 cm/s.
3.9. Total Phenolic Content (TPC) Determination of FSPH Fractions
TPC was determined according to the method of Waterhouse [74
] with slight modifications. An aliquot of 100 µL of FSPH fraction (2 mg/mL) was mixed with 1500 µL of distilled water and 100 µL of 2N FCR, homogenized in a vortex for 15 s and placed in dark for 3 min. Then, 300 µL of 10% Na2
) was added to the mixture, homogenized and incubated for 5 min at 50 °C. The Abs values were measured at 760 nm. A blank sample was prepared by replacing the sample with Milli-Q water. The phloroglucinol (a basic structural unit of phlorotannins) was used as a standard and the results are expressed as mg of phloroglucinol equivalents (PE) per gram of dried FSPH fraction. A calibration curve was prepared using a concentration range of 50–300 μg/mL.
3.10. ACE-Inhibitory Activity Determination of FSPH Fractions
The determination of ACE-inhibitory activity was performed in vitro by RP-HPLC adapted from the spectrophotometric method described by Cushman and Cheung [75
] with slight modifications [3
]. This method is based on the liberation of hippuric acid from hippuryl-l
-leucine (Hip-His-Leu) catalyzed by ACE. For the assay, 42.5 µL of the sample solution (2 mg/mL) was pre-incubated at 37 °C for 5 min with 10 µL ACE (0.6 mU/mL) enzyme. The mixture was subsequently incubated at the same temperature for 60 min with 20 µL of the substrate (5 mM HHL in 10 µM zinc chloride containing 100 mM sodium trizma base and 300 mM NaCl at pH 8.3). The reaction was terminated by adding 12.5 µL of 5 M HCl. The percentage of ACE-inhibition was determined by an HPLC system from Waters equipped with a 626 pump and 600S controller coupled to a 486 tunable UV detector. An aliquot of 20 µL from the reaction mixture was analyzed on a reverse-phase Ultrasphere ODS analytical column (25 cm × 4.6 mm i.d., 5 µm particle size) (Beckman Coulter, Miami, FL, USA) using an isocratic elution of MeOH: ACN: 0.1% HCl (25:25:50, v
) at a constant flow-rate of 0.6 mL/min and HA and HHL were detected by UV at 228 nm. The average value from three determinations at each concentration was used to calculate the ACE-inhibition (%) rate as follows: % ACE-inhibition = [B − A/B − C] × 100, where A is the absorbance (Abs) of HA generated in the presence of ACE-inhibitor, B the Abs of HA generated without ACE-inhibitor and C the Abs of HA generated without ACE (corresponding to HHL autolysis in the course of enzymatic assay). The IC50
value (mg/mL) was defined as the concentration of inhibitor required to reduce the HA peak by 50% (corresponding to 50% inhibition of ACE activity). The captopril was used as a positive control for ACE-inhibition.
3.11. Antioxidant Activity Assays on FSPH Fractions
3.11.1. DPPH Free Radical Scavenging Activity (FRSA) Assay
The FRSA of FSPH fractions was determined according to the method of Molyneux [76
] with slight modifications. The FRSA of each FSPH fraction was tested by measuring their ability to quench DPPH. The DPPH, a stable free radical, is reduced changing the purple color of the DPPH radical solution to a bright yellow in the presence of antioxidants that possess hydrogen-donating or chain-breaking properties and the intensity of this can be monitored spectrophotometrically [77
]. An aliquot of 250 µL of FSPH fraction with various concentrations (or BHT) was added to 250 µL of 100 mM DPPH solution. BHT was used as reference sample and a mixture without FSPH sample or BHT was used as the control. The Abs was measured at 517 nm after 30 min in the dark. The FRSA of FSPH fractions was calculated as a percentage of DPPH decoloration using the following equation: % FRSA = (1 − Abssample
) × 100.
3.11.2. Ferrous Ion-Chelating (FIC) Activity Assay
Chelating ability of FSPH fractions was determined according to the modified method of Wang et al. [58
], by measuring the inhibition of the Fe2+
–ferrozine complex formation. An aliquot of 100 µL of each FSPH fraction (concentration 2 mg/mL) was mixed with 135 µL of methanol plus 5 µL of 2 mM FeCl2
. The reaction was initiated by the addition of 10 µL of 5 mM ferrozine. After 10 min at room temperature, the Abs was determined at 562 nm. Methanol instead of ferrozine solution was used as a sample blank, which is used for error correction because of unequal color of the sample solutions. Methanol instead of sample solution was used as a control. Results are expressed as relative iron chelating activity compared with the unchelated (without ferrozine) Fe2+
reaction, and EDTA was used as reference standard. A lower Abs indicated a better FIC ability. The FIC ability was calculated as follows: % FIC ability = [A0
× 100, where A0
was the Abs of the control, A1
was the Abs of the sample or standard and A2
was the Abs of the blank.
3.11.3. Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP of FSPH fractions was determined according to the method of Oyaizu [78
], and evaluated on the basis of their abilities to reduce Fe3+
complex to Fe2+
. An increased Abs value indicates an increased reducing power of the hydrolysate fractions. Each fraction (concentration range 12.5–100 μg/mL) in methanol (0.4 mL) was mixed with 0.4 mL of 300 mM of phosphate buffer (pH 6.6) plus 0.4 mL of potassium ferricyanide (1%, w
) and the mixture was incubated at 50 °C for 20 min. After cooling down, 0.4 mL of TCA (10%, w
) was added, and the mixture was centrifuged at 3000× g
for 10 min. The upper layer (1 mL) was mixed with 1 mL of deionized water plus 0.2 mL of FeCl3
), and the Abs was measured at 700 nm against a blank. The blank solution contained pure methanol instead of the methanolic FSPH fraction. BHT was used for comparison.
3.12. Statistical Analysis
All determinations were performed at least in triplicate and the results were expressed as means ± standard deviations (SD). The statistics analysis was performed using SPSS 17.0 (version 17, SPSS Inc., Chicago, IL, USA) and one-way analysis of variance test (ANOVA) was carried out to assess for any significant differences between the means. Differences between means at the 5% (p < 0.05) level were considered significant. Correlations between the parameters evaluated were obtained using Pearson’s correlation coefficient (r).
Algae remain a relatively untapped source of compounds with ACE-inhibitory and antioxidant activities. To the best of our knowledge, this study revealed, for the first time, that enzymatic hydrolysate fractions with the aforementioned bifunctional properties could be efficiently generated from F. spiralis protein hydrolyzed by the cellulase–bromelain enzymes. The fraction with MW ≥3 kDa, obtained from this hydrolysate using ultrafiltration membranes, showed significantly higher recovery yield, ACE-inhibition and ferric reducing antioxidant power than the other fractions, and also showed strong scavenging of DPPH radical and ferrous ions (Fe2+) chelating activity. The significant bioactivity of this fraction can be attributed to the high concentration of the potent active peptide sequences (even though the fraction contains peptides relatively large in size) and phenolic compounds (high-MW phlorotannins). Although additional research is needed on structural information of the bioactive compounds, overall results from this study indicate that the produced F. spiralis protein hydrolysate fractions can be promising natural sources to develop functional food ingredients for controlling hypertension and/or oxidative stress, the two major causes of cardiovascular diseases. However, in vivo studies are also needed to verify their physiological effects.