Impact of Enzyme- and Ultrasound-Assisted Extraction Methods on

Article pubs.acs.org/JAFC

Impact of Enzyme- and Ultrasound-Assisted Extraction Methods on Biological Properties of Red, Brown, and Green Seaweeds from the Central West Coast of Portugal Dina Rodrigues,*,† Sérgio Sousa,§ Aline Silva,§ Manuela Amorim,§ Leonel Pereira,# Teresa A. P. Rocha-Santos,†, ⊥ Ana M. P. Gomes,§ Armando C. Duarte,† and Ana Cristina Freitas†,

⊥

†

CESAM − Centre for Environmental and Marine Studies and Department of Chemistry, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal § CBQF − Centro de Biotecnologia e Quı ́mica Fina − Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino Almeida, 4200-072 Porto, Portugal # MARE − Marine and Environmental Sciences Centre / IMARFaculdade de Ciências e Tecnologia, Universidade de Coimbra, 3004-517 Coimbra, Portugal ⊥ ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa, Viseu, Portugal ABSTRACT: Seaweeds are an excellent source of bioactive compounds, and therefore the use of sustainable and food compatible extraction methods such as enzyme-assisted (EAE) and ultrasound-assisted extraction were applied on Sargassum muticum, Osmundea pinnatifida, and Codium tomentosum. Extracts were evaluated for proximate characterization and biological properties. Higher extraction yields were observed for C. tomentosum EAE (48−62%; p < 0.05 for Cellulase and Viscozyme), followed by O. pinnatif ida (49−55%; p < 0.05 except Alcalase) and S. muticum (26−31%; p < 0.05). S. muticum extracts presented the highest nitrogen (25 ± 2 mg/glyoph extract) and total phenolics (261 ± 37 μgcatechol equiv/glyoph extract) contents, whereas higher sugars (78 ± 14 mgglucose equiv/glyoph extract) including sulfated polysaccharide (44 ± 8 mgNa2SO4 acid/glyoph extract) contents characterized O. pinnatif ida extracts. A higher effect on hydroxyl-radical scavenging activity (35−50%) was observed for all extracts, whereas S. muticum Alcalase and C. tomentosum Cellulase extracts exhibited higher prebiotic activity than fructooligosaccharides. O. pinnatif ida and C. tomentosum EAE showed inhibitory potential against α-glucosidase (38−49%). KEYWORDS: Sargassum muticum, Osmundea pinnatifida, Codium tomentosum, water-based extraction, biological properties

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INTRODUCTION Seaweeds of great taxonomic diversity are still an untapped source of diverse chemical compounds of interest including nutrients such as polysaccharides, proteins, minerals, vitamins, and dietary fibers.1 The ingestion of such seaweed compounds has the advantages of being of natural origin and affecting positively the health of individuals or even reducing the risk of certain pathologies.2 The biological importance of seaweeds can be found in recent reviews;3,4 some of them have demonstrated important biological properties such as antioxidant, antibacterial, anticoagulant, and antitumor activities, all of which represent added value for these alternative food species.3,4 The extraction and isolation of compounds of interest from seaweeds, able to be ingested or used for food purposes, need to rely upon compatible methods with economically viable yields, yet such objectives do include several challenges. Seaweeds are known to have a chemically and structurally heterogeneous rigid wall, which poses limitations to efficient extraction of the intracellular and wall compounds.5 Several extraction approaches with seaweeds have been tested in recent years. Water-based extraction is food compatible, nonexpensive, and environmentally friendly but has low selectivity with low extraction efficiency.6,7 Extraction based on several organic solvents has been attempted with higher selectivity and efficiency, but some are nonfood compatible such as hexane, © 2015 American Chemical Society

diethyl ether, benzene, and acetonitrile, for which solvent residues and environmental pollution are further drawbacks.8,10 Enzyme-assisted extraction (EAE) has emerged to considerable interest because its hydrolytic action on seaweeds could weaken or disrupt cell wall structure and also break down complex interior storage compounds, releasing intracellular compounds such as polysaccharides, peptides, or amino acids.9 Algal cell walls are typically fibrous composites of microfibrillar polysaccharides embedded in a matrix of polysaccharides and proteoglycans.10 The extraction efficiency of compounds from seaweeds is limited due to the presence of these complex cell walls with mixtures of sulfated and branched polysaccharides associated with proteins and various bound ions such as calcium and potassium.11 EAE has shown potential to improve extraction yield, to release secondary plant metabolites, and to maintain bioactive properties of the extracts.8 Several studies have reported a higher extractability of bioactive compounds from several brown algae and considered EAE an economical and sustainable method based on solvent-free process with low costs, high extraction rates, and high yields.7,12 UltrasoundReceived: Revised: Accepted: Published: 3177

September 2, 2014 March 9, 2015 March 10, 2015 March 10, 2015 DOI: 10.1021/jf504220e J. Agric. Food Chem. 2015, 63, 3177−3188

Article

Journal of Agricultural and Food Chemistry Table 1. Summary of Optimum Hydrolysis Conditions, Characteristics, and Enzyme Sources optimum conditionsa enzyme

pH

temperature (°C)

Alcalase Flavourzyme Cellulase Viscozyme L

8.0 7.0 4.5 4.5

50 50 50 50

characteristics

brand/source

endopeptidase endoprotease and exopeptidase cellulase multienzyme complex of carbohydrases: arabanase, cellulase, β-glucanase, hemicellulase, and xylanase

Sigma-Aldrich/Bacillus lichenifarmis Sigma-Aldrich/Aspergillus oryzae Sigma-Aldrich/Aspergillus niger Sigma-Aldrich/Aspergillus sp.

a Information based on Wang et al.13 and Heo et al.;25 pH values of S. muticum, O. pinnatif ida, and C. tomentosum water solutions before extraction were 5.6, 4.3, and 6.6, respectively.

and antidiabetic potential of water-based enzyme-assisted extracts for these studied species.

assisted extraction (UAE), an alternative energy input-assisted extraction method, is based on sound wave migration, which generates cavitations that grow and collapse, leading to disruption of cells and their walls.13 UAE and EAE have been reported as alternative approaches with great potential to extract bioactive substances from marine algae.14 Considering the limitations of water-based extraction (low selectivity and low extraction yields), yet wishing to target efficient food compatible methods, the main objective of this study was to obtain extracts rich in bioactive compounds using alternative water-based approaches such as EAE and UAE on representative red, brown, and green species of the Central West Portuguese coast, some of which are less studied such as Osmundea pinnatifida and Codium tomentosum. Hence, this study was based on three steps: (i) performance of different extraction modes and evaluation of respective yield, (ii) chemical characterization of each extract obtained from each of the three different seaweeds, and (iii) evaluation of the biological potential of the extracts in terms of antioxidant, antidiabetic, and prebiotic activities. Seaweeds such as Sargassum species (Phaeophyceae, brown algae) have been found to be good sources of dietary fiber and of phenolic compounds and carotenoids with antioxidant activity, all of which play important roles in the prevention of intestinal and neurodegenerative diseases, respectively.15−17 Several other compounds able to scavenge and/or neutralize oxidative compounds (antioxidant activity) have been isolated from other seaweeds including phenolic compounds, sulfated polysaccharides, or proteins.18,19 Prebiotics are defined as nondigestible compounds mainly of carbohydrate nature that selectively enhance the activity and viability of beneficial bacteria in the intestine by providing these with fermentable substrates that lead to the production of short-chain fatty acids.20 They have been associated with several health benefits in the large intestine such as modulation of beneficial gut bacteria, hypocholesterolemic effect, reduction of cancer and obesity risks, or increase of the bioavailability and uptake of some minerals (Ca and Mg).20 Inhibitory compounds of αglucosidase or α-amylase with antidiabetic activity are of much interest because by acting as competitive inhibitors of the enzymes they inhibit the hydrolysis of oligo-, tri-, and disaccharides to glucose and other monosaccharides in the small intestine and thereby delay postprandial glucose absorption, lowering blood glucose.21 Inhibition of the carbohydrate-hydrolyzing enzymes has been reported for different extracts of red and green seaweeds.22 To our knowledge this is the first study applying water-based extraction through combined EAE and UAE technologies in Sargassum muticum, O. pinnatif ida, and C. tomentosum seaweeds, and there are no papers regarding the prebiotic

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MATERIALS AND METHODS

Chemicals. Ethyl acetate, hydrogen peroxide, boric acid, barium chloride dihydrate, sodium salicylate, sodium carbonate decahydrate, L(+)-ascorbic acid, potassium peroxodisulfate (K2S2O8), Folin− Ciocalteu, and MRS broth were purchased from Scharlau (Sentmenat, Spain). Ethanol, Kjeldahl catalyst, trichloroacetic acid, sodium dihydrogen phosphate 1-hydrate, and ethylenediaminetetraacetic acid disodium salt (EDTA) were purchased from Panreac (Barcelona, Spain). Phenol, 2,2-diphenyl-1-picrylhydrazyl (DPPH), α-glucosidase, Alcalase, Flavourzyme, Cellulase, Viscozyme L, 4-nitrophenyl-α-Dglucopyranoside, acarbose, and catechol were purchased from SigmaAldrich (St. Louis, MO, USA), and ferrous sulfate heptahydrate was purchased from Sigma-Aldrich (Sintra, Portugal). Sulfuric acid was purchased from Fisher Chemical (Leicestershire, UK), and hydrogen chloride and sodium sulfate were obtained from Pronalab (Lisbon, Portugal). 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was purchased from Fluka (Germany), and Lcysteine hydrochloride monohydrate and MRS agar were obtained from Merck (Gernsheim, Germany). D-(+)-Glucose monohydrate and disodium hydrogen phosphate-7-hydrate were purchased from Riedelde Häen (Germany) and riboflavin and L-methionine from Acro̅s Organics (Geel, Belgium); nitroblue tetrazolium (NBT) was purchased from Fisher Scientific (Erembodegem-Aalst, Belgium). Specimens of Seaweeds. Specimens of the red alga (Rhodophyta, Florideophyceae) O. pinnatif ida (Ceramiales) Rhodomelaceae family, the brown alga (Heterokontophyta, Phaeophyceae) S. muticum (Fucales) Sargassaceae family, and the green alga (Chlorophyta, Ulvophyceae) C. tomentosum (Bryopsidales) Codiaceae family were harvested in April 2012 from Buarcos bay (Figueira da Foz, Portugal). The classification of seaweeds was based on AlgaeBase.23 The seaweeds were first washed with running tap water and then with deionized water and then dried in an oven at 60 °C. The dried samples of seaweeds were milled to 0.05) in comparison to HWE. Apparently, cellulase and the multicarbohydrase complex actions of Viscozyme on C. tomentosum cell wall polysaccharides were responsible for higher solid content in lyophilized extracts. Extraction yields with Cellulase and Viscozyme L were of similar magnitude to those observed by Wang et al.9 with red algae Palmaria palmata, which were around 60%. Complex polysaccharides such as alginates, agar, carrageenans, and α- and β-D-mannans are practically nondigested by human gastrointestinal enzymes, whereas microorganism-derived enzymes such as some types of carbohydrases are able to degrade complex polysaccharides, converting water-insoluble seaweeds into water-soluble materials collected in water-based extracts.7 Similar tendencies such as those for C. tomentosum but with lower extraction yield values were observed for S. muticum (Figure 1). Cellulase was responsible for the statistically significant highest extraction yield of 31.3% (p < 0.05). This value is in the range of extract yield (30−40%) reported by Plaza et al.36 with subcritical water extraction (SWE) at 100 and 200 °C and by Hardouin et al.12 with EAE (cellulase) for S. muticum, respectively. Although SWE is known for its speed, high yields, and the use of reduced amount of solvents, EAE with Cellulase may also provide several advantages given the enzyme versatility and robustness; furthermore, similar

RESULTS AND DISCUSSION

Extraction Yield. The extraction yields achieved for each of the different aqueous extractions of the three seaweeds are displayed in Figure 1. Both seaweed species and extraction method were shown to have a significant effect on extraction yield (p < 0.05). In general, in terms of species, higher extraction yields were observed for C. tomentosum (green algae), followed by O. pinnatif ida (red algae) and S. muticum (brown algae) extracts. Interestingly, an inverse correlation between the organic matter of the seaweed and the respective extraction yields was observed; 67, 58, and 55% of organic matter were reported for S. muticum, O. pinnatif ida, and C. tomentosum, respectively.35 According to Wijesinghe and Jeon,11 the amount, diversity, and complexity of polysaccharides in the seaweeds’ cell wall could reduce the extraction efficiency especially with classical extraction methods. In terms of extraction method, EAE extracts were shown to be the most effective. The extraction efficiency of compounds from seaweeds is limited due to the presence of complex cell walls and, therefore, the degradation of their structure is a fundamental step for the release of compounds from seaweeds. Hydrolytic enzymes have been reported to improve the extraction yield in seaweeds,9 an effect that was corroborated with the results reported herein for all three seaweed types studied. 3180

DOI: 10.1021/jf504220e J. Agric. Food Chem. 2015, 63, 3177−3188

Article

Journal of Agricultural and Food Chemistry

Table 2. Contents of Nitrogen, Sugars, Sulfated Sugars, and Total Phenolics in the Different Extracts of S. muticum, O. pinnatif ida, and C. tomentosum Seaweedsa seaweed

a

extraction method

nitrogen (mg/glyoph)

sugars (mgglucose equiv/glyoph extract)

sulfated sugars (MgNa2S4 acid equiv/glyoph extract)

total phenolic content (μgcatechol equiv/glyoph extract)

S. muticum

HWE UAE EAE_CelL EAE_Visc EAE_Alc EAE_Flav

24.6 24.4 22.1 25.1 29.6 25.4

± ± ± ± ± ±

3.58b 0.33b 0.43ab 0.62b 0.01bc 0.74b

40.6 37.8 87.3 29.9 30.7 34.7

± ± ± ± ± ±

2.45b 0. 16b 4.82c 1.95a 0.39a 2.37ab

9.5 10.1 5.9 7.6 7.5 9.7

± ± ± ± ± ±

0.36c 0.27c 0.40a 0.61b 0.50b 0.77c

275.8 235.0 261.9 300.3 289.7 200.5

± ± ± ± ± ±

4.98b 5.57a 29.42ab 11.67b 18.46b 7.40a

O. pinnatif ida


12.7 10.3 11.7 12.5 15.5 19.4

± ± ± ± ± ±

0.50b 0.02a 0.50ab 0.53b l.04c 0.14d

73.9 83.2 102.2 71.3 59.5 76.0

± ± ± ± ± ±

5.15b 6.56b 5.34c 6.48b 3.06ab 5.16b

41.4 42.8 41.4 59.8 38.6 46.0

± ± ± ± ± ±

l.30b l.64b 1.81b l.20d 1.25ab l.97bc

118.6 103.7 112.4 109.2 106.0 123.1

± ± ± ± ± ±

5.98a l.67a 0.27a 2.17a 2.92a 21.89a

C. tomentosum


17.7 19.0 13.1 13.8 22.2 24.1

± ± ± ± ± ±

0.19b 0.54b 0.07a 1.37a 0.36c 0.4c

62.9 69.3 102.93 156.7 40.9 56.8

± ± ± ± ± ±

2.38b 5.73b 5.68c 4.55d 2.17a 0.47ab

11.7 11.7 10.5 12.1 9.9 9.3

± ± ± ± ± ±

0.20c 0.25c 0.29b 0.16c 0.25b 0.33a

107.6 141.1 108.2 125.6 121.8 159.0

± ± ± ± ± ±

3.13a 9.79b 5.94a 8.56ab 7.64ab 11.25b

For each seaweed, different letters indicate significant differences (p < 0.05) between methods of extraction.

Ecklonia cava (brown alga) and reported slightly higher extraction yield values after 6 and 12 h at 30 °C in an ultrasonic bath with 200 W (31.3−34.3%) than after 24 h in a shaking incubator at room temperature (28.7%). Proximate Characterization of the Extracts. In general, and as expected, enzymatic extracts resulting from proteases action presented higher nitrogen content (Table 2); Alcalase and Flavourzyme were responsible for higher content in S. muticum (25−30 mg/glyoph extract; p < 0.05), O. pinnatif ida (15− 19 mg/glyoph extract; p < 0.05), and C. tomentosum (22−24 mg/ glyoph extract; p < 0.05). Indeed, proteases appeared as the most effective enzyme for protein recovery and accessibility. In terms of sugars, EAE extracts obtained from carbohydrase action presented higher content of sugars; Cellulase was responsible for higher content in S. muticum (87 mgglucose equiv/ glyoph extract; p < 0.05) and in O. pinnatif ida (102 mgglucose equiv/ glyoph extract; p < 0.05), whereas higher values were obtained by Viscozyme action in C. tomentosum (157 mgglucose equiv/ glyoph extract; p < 0.05). The different yields of nitrogen and sugar contents in the extracts for the three species (Table 2) did not correlate with the seaweeds proximate composition in terms of total protein and total sugars content. For both parameters, an inverse correlation between their content in the enzymatic extracts and the seaweed composition was observed: a statistically significant higher value of protein content was observed for O. pinnatif ida (23.8 g/100 gdry seaweed) and the lowest for S. muticum (16.9 g/ 100 gdry seaweed); in terms of total sugars S. muticum was characterized by a higher content in sugar (49.3 g/100 gdry seaweed) than O. pinnatif ida and C. tomentosum, with similar contents of 32−33 g/100 gdry seaweed.35 No statistically differences were observed for both nitrogen and sugar contents in the extracts obtained with HWE and UAE for the three seaweeds except for O. pinnatif ida, for which the nitrogen content was higher in the HWE extract than in the UAE counterpart (p < 0.05). These results may indicate that

extraction efficiency of targeted compounds and bioactivity properties is achieved using conventional equipment. No statistically significant differences (p > 0.05) were found between S. muticum extracts obtained from Viscozyme (28.0%) and Flavourzyme (28.1%) and between extracts obtained from UAE (24.0%) and HWE (22.5%) methods. Alcalase action resulted in the lowest extraction yield (25.6%) within the EAE methods applied. In O. pinnatif ida, Cellulase and Flavourzyme were responsible for the statistically significant highest extraction yields (54−55%, p < 0.05), whereas no statistically significant differences (p > 0.05) were found between extracts obtained from Alcalase (49.2%), UAE (49.1%), and HWE (49.9%) treatments. The hydrolytic action of the endoprotease and exopeptidase in the Flavourzyme enzymatic complex from Aspergillus oryzae (Table 1) on peptide bonds of O. pinnatif ida proteins could be responsible, at least in part, for the higher extraction yield given its highest protein content (23.8%) in comparison to S. muticum and C. tomentosum (16.9−18.8%, respectively).35 According to Cian et al.,37 proteolysis promoted by Flavourzyme in Porphyra columbina (red seaweed) residual cake enhanced extraction of peptides. UAE is considered an emerging potential technology being considered an efficient alternative to traditional extraction techniques that has been successfully used in the plant extraction field.38 However, in the present study UAE was not responsible for higher extraction efficiency than HWE for O. pinnatif ida and S. muticum, yet in the case of C. tomentosum it enabled a slightly higher, and statistically significant, value (48.6%) than HWE (44.6%). Because UAE was applied to water extracts after 24 h at 50 °C, an improved yield extract was expected, resulting from ultrasound waves due to the implosion of cavitation bubbles. In addition, it has been reported that UAE should be carefully used because unstable compounds such as carotenoids could be degraded.39 Lee et al.40 evaluated the potential use of UAE in water and methanolic extracts of 3181


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Figure 2. Total antioxidant capacity, DPPH free radical, hydroxyl radical, and superoxide radical scavenging activities of S. muticum, O. pinnatif ida, and C. tomentosum extracts obtained by hot water extraction (HWE), ultrasound-assisted extraction (UAE), and enzyme-assisted extraction (EAE) with Cellulase (Cell), Viscozyme L (Visc), Alcalase (Alc), and Flavourzyme (Flav) at 50 °C. Different letters indicate significant differences (p < 0.05) between extracts for each seaweed.

polysaccharides, was assigned by FTIR-ATR for S. muticum.35 According to Ale and Meyer,41 fucoidans cover several different structural fucose-containing sulfated polysaccharides, comprising many different types of fucose-rich polysaccharides, including sulfated fucogalacturonans found in Sargassum sp., for example. The lower content of sulfated groups in S. muticum in comparison to O. pinnatif ida could be related to the presence of alginates (a nonsulfated polysaccharide), also evidenced by FTIR-ATR analysis. S. muticum, an original species from Japan now found in the Atlantic Ocean and in the Mediterranean Sea, has been reported to possess, when dry, 10−18% alginates.42 A slightly higher content in sulfated groups, in comparison to S. muticum, was observed in C. tomentosum extracts. These could be related to the presence of sulfated xyloarabinogalactans such as sulfated arabinan and sulfated arabinogalactan, which have been identified in C. tomentosum 43 or other sulfated polysaccharides.35 Extracts that resulted from EAE with Cellulase and Viscozyme, as well as from HWE and UAE, were those with higher content in sulfated groups. Total phenolic content varied significantly according to extraction mode and seaweed type (p < 0.05). Higher values for total phenolic content were found for S. muticum extracts, which ranged between 201 and 301 μgcatechol equivalent/glyoph extract (Table 2). Once again, no direct correlation was observed between total phenolic content in each seaweed and those in the respective aqueous extracts. Significant differences in total phenolic content were observed between the three seaweed species, namely, 460, 250, and 169 μgcatechol equivalent/gdry seaweed for C. tomentosum, S. muticum, and O. pinnatif ida, respectively.35 This observation might suggest that none of the extraction modes were sufficiently efficient to extract all phenolics in C. tomentosum and O. pinnatif ida. It has been

the mechanical action resulting from ultrasound waves under the extraction time and temperature conditions used did not promote the release of extra proteins or polysaccharides from the intracellular or wall contents of seaweeds. Variable content in sulfate groups, which was significantly different for each seaweed species and extraction method (p < 0.05), is observable in Table 2. Previous studies with FTIRATR analysis showed that in the brown algae S. muticum, alginates and fucoidans were the main polysaccharides, whereas O. pinnatif ida was mostly an agar producer; sulfate esters of sulfated polysaccharides were observed in the spectrum of C. tomentosum.35 Therefore, the sulfated content in each extract was in part related to the nature and amount of sulfated polysaccharide in each seaweed species as well as the extraction efficiency. High content of sulfate groups was observed in O. pinnatif ida extracts (39−60 μgNa2SO4 acid equiv/glyoph extract) probably from agar, a less sulfated polysaccharide than carrageenans, also characteristic in some red seaweeds. A significantly higher content was obtained with EAE using Viscozyme. According to Domozych,10 red algae possess complex composite cell walls, which are made of cellulose, xylan, or mannan fibrils as well as an extensive matrix of polysaccharides such as agar and carrageenan. For S. muticum extracts, enzymatic activity did not result in higher content of sulfate groups except for extraction performed with Flavourzyme, in accordance with data reported by Hardouin et al.12 for this seaweed. Hot water and UAE extracts were revealed to be more suitable to obtain extracts richer in the sulfate groups. These probably result from fucoidans. The presence of sulfated ester groups (SO), a characteristic component in fucoidan and other sulfate 3182


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quenchers of peroxyl radicals but be only singlet-oxygen scavengers.47 The statistically significant difference between the seaweed extracts (p < 0.05) could be, at least in part, attributed to the higher amount of total phenolic compounds and proteinaceous compounds found in S. muticum (Table 2). The radical scavenging activity of phenolic compounds is, however, dependent on the phenolic structure and number and location of hydroxyl groups,48 and therefore the radical scavenging potential of the extracts is dependent on qualitative and quantitative phenolic combination. Our results are in accordance with those published by Yangthong et al.,49 who reported higher phenolic content and antioxidant activity for brown seaweeds than for red and green seaweeds. Polysaccharides in brown algae such as those with high content of uronic acid and alginates with low molecular weight have also been reported as good antioxidant potential.50 The antioxidant potential of S. muticum water extracts was higher in HWE especially in terms of total antioxidant capacity (p < 0.05) with 33.5% scavenging activity (corresponding to 37 μgascorbic acid equiv/mL) and hydroxyl radical scavenging activity (p < 0.05) with 50.3% scavenging activity (corresponding to 90 μgascorbic acid equiv/mL), respectively. Maximum DPPH free radical scavenging activity was observed for UAE with 20% scavenging activity (corresponding to 11 μgcatechol equiv/mL). In terms of antioxidant activity no increase was observed in the S. muticum extracts obtained from EAE. Higher values of free and hydroxyl scavenging activity were reported by Je et al.16 in EAE extracts with Alcalase and Viscozyme from brown seaweed Undaria pinnatifida, but no hydroxyl scavenging activity from EAE extracts with Flavourzyme was reported. Hydroxyl scavenging activity of S. muticum extracts produced with Flavourzyme was 42%, one of the lowest values together with those obtained for UAE extracts (Figure 2). In O. pinnatif ida, EAE extracts had, in general, higher scavenging potential than HWE or UAE. In terms of total antioxidant capacity, proteaseassisted extracts with 19% scavenging activity (corresponding to 15−16 μgascorbic acid equiv/mL) presented statistically significant higher values (p < 0.05) than the other extraction methods. According to Cian et al.,37 the low molecular weight peptides and polyphenols released during proteolysis were responsible for the DPPH radical scavenging activity of red seaweed Porphyra columbina. No radical scavenging activity toward ABTS was reported by Jiménez-Escrig et al.44 for aqueous extracts of O. pinnatif ida. For the hydroxyl radical, EAE extracts were capable of 42−45% scavenging activity (corresponding to 70−79 μgascorbic acid equiv/mL) (Figure 2). O. pinnatif ida EAE extracts from Cellulase had a statistically significant (p < 0.05) higher superoxide scavenger potential with 28% scavenging activity (corresponding to 54 μggallic acid equiv/mL) than the other extracts. The extraction procedures did not affect the total antioxidant capacity displayed by C. tomentosum extracts except for those obtained by protease EAE, where results were higher and statistically different (Figure 2). In terms of superoxide radical scavenging activity only slight differences were observed between the different C. tomentosum extracts. However, Cellulase and Viscozyme extracts of C. tomentosum, which had higher sugar content, had statistically significant (p < 0.05) higher DPPH free radical scavenger activity with 16−18% (corresponding to 9−10 μgcatechol acid equiv/mL). Valentão et al.51 also reported a weak concentration-dependent antioxidant capacity for C. tomentosum aqueous lyophilized extracts (10% of

reported that organic solvents such as ethanol are more efficient for polyphenol extraction than water.19 Variable efficiency of phenolic compound extraction was observed by the different extraction modes for each seaweed species. For S. muticum statistically higher values were obtained by HWE and by Viscozyme and Alcalase EAE (Table 2). For C. tomentosum, UAE and Flavourzyme EAE were responsible for higher efficiency, whereas for O. pinnatif ida no statistical difference were observed for the different extracts. No information on effects of water-based extraction mode on phenolic compounds was found in the literature. For example, higher content in total phenols values was reported by Plaza et al.36 in S. muticum extracts obtained by SWE water extracts at 100 and 200 °C ranging between 10.7 and 58.67 mggallic acid/ gextract, respectively, but no explanation is given for the increment in total phenols except that with a higher extraction temperature a higher mass transfer occurs, which increases extraction yield. Water and ethanolic extracts of 16 species collected at the Danish coast were screened for extraction yield and respective total phenolic content by Farvin and Jacobsen.19 For all species, water extracts showed higher yield, but in terms of phenolic content this was dependent on the species. In general, the ethanolic extracts had higher content of total phenolics except for four species including S. muticum.19 No explanation is yet available for the higher extractability of phenolic compounds by water in some species such as in S. muticum. In turn, for O. pinnatif ida, methanolic extraction rendered a higher extraction yield and higher polyphenol content than by aqueous extraction.44 According to Budhiyanti et al.45 phenolic content varied from 0.006 to 0.65 and from 6.72 to 21.99 gphloroglucinol equiv/100 gdried extract for cytoplasmic and membrane-bound extracts from Sargassum sp., respectively. These data show that the phenolic contents are extremely variable among species of Sargassum and cell structure. Antioxidant Activity. Figure 2 presents data for total antioxidant capacity and for DPPH free radical as well as hydroxyl and superoxide radical scavenging activities of the different extracts for each seaweed, evaluated both as micrograms of phenolic compound equivalent and as percent of scavenging activity. Because the diverse expression of results for the different antioxidant capacity evaluation methodologies found in the literature makes comparison between studies difficult,46 the authors think that the perception of the relative proportion between phenolic content (μg/mL) of extract and the corresponding value in terms of percentage of scavenging activity makes the interpretation more objective and more valuable (Figure 2). The four different antioxidant evaluations varied significantly among seaweed species and among extraction modes (p < 0.05). Maximum total antioxidant capacities as well as DPPH free radical and hydroxyl radical scavenging activities were observed in the extracts of S. muticum followed by C. tomentosum (Figure 2). In O. pinnatif ida extracts, lower total antioxidant capacity and DPPH free radical scavenging activity were observed in comparison to any of the two other seaweeds, but higher scavenging capacity for reactive oxygen species such as HO• and in particular of radical O2•− were achieved. Heo et al.18 also reported variable scavenging activities in brown seaweed extracts obtained by carbohydrases or proteases in terms of DPPH free, hydroxyl, and superoxide radicals. According to Freitas et al.46 no single assay seems to be able to accurately reflect the mechanism of action of all radicals and antioxidants, especially in complex systems. For example, some antioxidants may not be efficient 3183


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Figure 3. Mean and standard deviation of cell counts of B. animalis BB-12 (i) and L. acidophilus La-5 (ii) throughout 48 h of incubation at 37 °C in the presence or absence of sugars (a) or S. muticum extracts: hot water extraction (HWE) and ultrasound-assisted extraction (UAE) (b); enzymeassisted extraction (EAE) with Cellulase (Cell), Viscozyme L (Visc), Alcalase (Alc), and Flavourzyme (Flav) (c). Different letters indicate significant differences (p < 0.05) for the viable cells after 24 or 48 h of incubation at 37 °C in the presence or absence of sugars or S. muticum extracts, and ∗ indicates significant differences (p < 0.05) in comparison to values at 0 h.

Figure 4. Mean and standard deviation of cell counts of B. animalis BB-12 (i) and L. acidophilus La-5 (ii) throughout 48 h of incubation at 37 °C in the presence or absence of sugars (a) or O. pinnatif ida extracts: hot water extraction (HWE) and ultrasound-assisted extraction (UAE) (b); enzymeassisted extraction (EAE) with Cellulase (Cell), Viscozyme L (Visc), Alcalase (Alc), and Flavourzyme (Flav) (c). Different letters indicate significant differences (p < 0.05) for the viable cells after 24 or 48 h of incubation at 37 °C in the presence or absence of sugars or O. pinnatif ida extracts, and ∗ indicates significant differences (p < 0.05) in comparison to values at 0 h.

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Figure 5. Mean and standard deviation of cell counts of B. animalis BB-12 (i) and L. acidophilus La-5 (ii) throughout 48 h of incubation at 37 °C in the presence or absence of sugars (a) or C. tomentosum extracts: hot water extraction (HWE) and ultrasound-assisted extraction (UAE) (b); enzymeassisted extraction (EAE) with Cellulase (Cell), Viscozyme L (Visc), Alcalase (Alc), and Flavourzyme (Flav) (c). Different letters indicate significant differences (p < 0.05) for the viable cells after 24 or 48 h of incubation at 37 °C in the presence or absence of sugars or C. tomentosum extracts, and ∗ indicates significant differences (p < 0.05) in comparison to values at 0 h.

DPPH free radical scavenging activity with 794 μg/mL extract). In terms of the hydroxyl radical, Alcalase and Flavourzyme extracts were able to inhibit >44% of scavenging activity corresponding to 76−83 gascorbic acid equiv/mL, which may indicate that such bioactivity is related not only to total phenolic content but also to proteinaceous compounds (Table 2). Nevertheless, given the statistically significant difference between Alcalase and Flavourzyme scavenging capacities and their respective phenolic and nitrogen contents shown in Table 2, it seems that not only the content but also the nature of the compounds is important to eventually justify the antioxidant properties. For example, Heo et al.18 reported that enzymatic extracts of brown algae Ecklonia cava with Alcalase did not possess DPPH free radical scavenging activity, although they contained as much phenolic compounds as other extracts with free radical scavenging activity. In turn, Farvin and Jacobsen19 reported some extracts with good antioxidant effects but with low total phenolic content, indicating that other compounds were coextracted in water extracts such as sulfate polysaccharides, proteins, or peptides. However, not much antioxidant potential was observed in crude sulfated polysaccharides from Sargassum plagiophyllum (2 mg/mL) for free radical (11.2%), hydroxyl (11.2%), or superoxide (0%) radical scavenging activities.29 In summary, the different extracts, with 2 mg lyophilized extract/mL, from S. muticum, O. pinnatif ida, and C. tomentosum did not exhibit high total antioxidant capacity or DPPH free radical scavenging activity but more prominent effects on hydroxyl radical scavenging activity (35−50%). Extracts from O. pinnatif ida with 0.2 mg lyophilized extract/mL, namely, HWE, UAE, and EAE with Cellulase and Alcalase, exhibited

moderate effects on superoxide radical scavenging activity (18− 26%). Prebiotic Potential of Seaweed Extracts. Recently, some research has been done in identifying potential prebiotic compounds in marine resources, namely, in seaweeds, given their rich profile in polysaccharides present in the cell walls;52 nonetheless, to our best of knowledge, no information is available regarding the prebiotic potential of the three seaweed species targeted in our study. Hence, the potential prebiotic effect of seaweed water-based extracts was evaluated by using pure cultures of representative beneficial bacteria including strains of lactobacilli and bifidobacteria (usual target genera for prebiotics) in parallel with well-established prebiotics, such as FOS, for comparison purposes. Both probiotic strains were also grown under the same conditions but in the absence of sugar (negative control) as well as in the presence of glucose or FOS at similar concentrations (positive controls) (Figures 3−5). Seaweed species, extraction mode, and incubation time were revealed to be significant factors for both probiotic strains viability (p < 0.05). Because significant differences were observed for each factor as well as significant interactions, one-way ANOVAs were carried out for each seaweed species to observe if the carbon source (glucose, FOS, or extract) was statistically significant for the viable cell numbers of L. acidophilus La-5 or B. animalis BB12, after 24 or 48 h of incubation, respectively. With regard to L. acidophilus La-5, significantly higher values (p < 0.05) of viable cell numbers were observed for the majority of culture media enriched with seaweed water-based extracts after 24 h of incubation in comparison to growth in media with glucose or FOS. In addition, some significant increases (p < 0.05) in viable cell numbers in comparison to those at 0 h were observed such as 3185


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from the enzymatic action will also be determinant to understand and correlate with the prebiotic activity. α-Glucosidase Inhibitory Activity. The α-glucosidase inhibitory activity, expressed as percent of inhibition, by S. muticum, O. pinnatif ida, and C. tomentosum water-based extracts is displayed in Figure 6. Extraction mode and seaweed species

in media containing HWE and EAE with Viscozyme, Cellulase, and Flavorzyme extracts of S. muticum, and EAE with Viscozyme and Alcalase extracts of O. pinnatif ida as well as EAE with Viscozyme and Cellulase extracts of C. tomentosum (Figures 3ii,a−c, 4ii,a−c, and 5ii,a−c). Despite the enhanced growth by 24 h of incubation, after 48 h of incubation lower viable cell numbers were observed, in particular for O. pinnatif ida extracts (Figure 4ii,b−c); end-product formation or substrate depletion could justify this decrease in viable cell numbers. In terms of seaweed extracts’ impact on B. animalis BB-12, it was found that, in general, they have a lower prebiotic potential (Figures 3i,a−c, 4i,a−c, and 5i,a−c). Nonetheless viable cell numbers of B. animalis BB-12 in all S. muticum and C. tomentosum extracts after 48 h of incubation were significantly higher than those obtained with prebiotic FOS and glucose (p < 0.05) (Figures 3i,a−c and 5i,a−c). Although of preliminary nature, these results show that the majority of the extracts obtained from the three seaweeds possess carbon sources that can be metabolized by L. acidophilus La-5 and B. animalis BB-12. A more promising prebiotic potential was observed for S. muticum and C. tomentosum extracts, especially for those obtained by enzymatic action. Because a lower potential was observed in extracts of O. pinnatif ida, this could be related to the largest amount of sulfated sugars observed in these extracts. Red seaweed O. pinnatif ida has been mostly characterized as an agar producer, and it is known that microorganisms are not able to hydrolyze and metabolize this polysaccharide, which is used as a solidifying agent of culture media. On the other hand, it should not be totally overlooked because it was shown recently that some low molecular weight polysaccharide fractions derived from seaweed agar and alginate by acid hydrolysis or degradation with hydrogen peroxide were fermented by gut microbiota, namely, bifidobacterial populations, exhibiting potential to be used as a novel source of prebiotics.53 Enzymatic extracts, especially those obtained with carbohydrases (Viscozyme and Cellulase), probably possess polysaccharides and oligosaccharides derived from hydrocolloids present in each type of seaweed that can act as sources of soluble fibers exerting a prebiotic effect. According to Wu et al.54 Bifidobacterium longum BCRC 11847 could utilize polysaccharide extracts of red seaweed Gracilaria sp. extracted with Cellulase R-10 and Macerozyme R-10 and display an increase in pH-lowering ability. Zaporozhets et al.52 recently reviewed the prebiotic potential of polysaccharides and extracts from seaweeds. In that review the authors included their research group studies’ results on the prebiotic potential of fucoidan and alginic acid derived from brown seaweed Fucus evanescens in vitro and in vivo with mice. These authors confirmed that fucoidan and alginic acid were used instead of lactose with a 3−5.8-fold increase in biomass of Bif idobacterium in comparison to the control. Given the promising reported results obtained in terms of growth promotion of L. acidophilus La-5 and B. animalis BB-12 and considering the prebiotic potential of polysaccharides and seaweed extracts reported in the literature concerning other seaweed species, further in vitro (nondigestibility and selective fermentation capacity) and in vivo studies must be performed with the enzymatic extracts assisted by Cellulase of S. muticum and C. tomentosum as well as in UAE extracts of C. tomentosum. The identification of polysaccharide (and associated molecular weight) and oligosaccharide fractions in the extracts derived

Figure 6. α-Glucosidase inhibitory activity, expressed as percent of inhibition, of S. muticum, O. pinnatif ida, and C. tomentosum extracts obtained by hot water extraction (HWE), ultrasound-assisted extraction (UAE), and enzyme-assisted extraction (EAE) with Cellulase (Cell), Viscozyme L (Visc), Alcalase (Alc), and Flavourzyme (Flav) at 50 °C. Different letters indicate significant differences (p < 0.05) between extracts for each seaweed.

were both statistically significant factors (p < 0.05). Extracts obtained by enzymatic action possess higher inhibition activity especially for O. pinnatif ida, with 38−40% inhibition in the extracts obtained by Cellullase and Viscozyme, and for C. tomentosum with 45−49% inhibition in the extracts obtained by Alcalase and Flavourzyme. To our knowledge there are no reports on potential antidiabetic activity of water-based enzymatic seaweed extracts, in particular for the studied species, and therefore no direct comparisons with other works with only other seaweed species and other extraction modes are possible. For example, Senthil et al.22 reported that ethyl acetate extracts of red seaweeds of Chondrococcus harnmanni and Padina gymnospora (81 and 94%, respectively) possesses high levels of α-amylase inhibitory activity. Lower values of α-glucosidase inhibitory activity were observed for S. muticum extracts with a maximum of inhibition in the extracts obtained with Alcalase (39%). This is an interesting observation because the S. muticum extract showed the highest protein content among all other extracts (Table 2). Other research works have studied the role of seaweed polysaccharides, from other Sargassum species, or of phenolic compounds on the inhibition activity of α-amylase and α-glucosidase but less on the role of protein or peptides for the same purpose. For example, Hardoko et al.55 reported inhibitory activity on αglucosidase by laminaran and fucoidan fractions from two brown seaweeds (Sargassum duplicatum and Turbinaria decurens) but not by the alginate fraction. According to Motshakeri et al.,56 35−36% blood glucose reduction was observed in type 2 diabetic rats after 22 days of feeding with 300 mg/kg body weight ethanolic or water extracts of Sargassum polycystum. In the present study, EAE extracts from O. pinnatif ida and C. tomentosum showed promising inhibitory 3186


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Journal of Agricultural and Food Chemistry potential against α-glucosidase and, although further in vivo studies are required to test for effective hypoglycaemic activity, these may prove to be important strategies in the prevention (functional food) or management (oral therapeutic agent) of blood glucose level in type 2 diabetic and borderline patients. In conclusion, this study demonstrates once more that edible seaweeds such as S. muticum (brown alga), O. pinnatif ida (red alga), and C. tomentosum (green alga) are natural sources of compounds with important biological interest. In the present study, water-based crude extracts were obtained from efficient methods that are also food grade compatible and sustainable. Enzyme-assisted extraction was revealed to be a promising tool to obtain extracts with attractive biological properties. According to the general results achieved, it becomes difficult to pinpoint the most efficient extraction method with the highest biological potential because it depends on several factors: (i) seaweed species; (ii) extraction mode considering the duality extraction yield versus chemical/biological properties; and (iii) the type of analysis made. From this perspective, S. muticum EAE presented interesting antioxidant and prebiotic potentials, O. pinnatif ida enzyme-assisted free radical scavenging activity given its high sulfated sugar content, and C. tomentosum EAE a promising α-glucosidase inhibitory activity and consequently attractive antidiabetic potential. In general, it can be pointed out that the enzyme-assisted extraction seems to be one of the best options in terms of food compatible extraction method with higher yield rates, and through the selection of a specific enzyme some biological properties may be favored over other properties. It is our opinion that, for future studies with these seaweeds in terms of functional food development, the selection of the most efficient extraction mode will need to be coupled to the best results in terms of biological properties according to the intended purpose associated with the identification of polysaccharides, peptides, and phenolic compounds.

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AUTHOR INFORMATION

Corresponding Author

*(D.R.) Phone: (+351) 232 910 117. Fax: (+351) 232 910 193. E-mail: [email protected]. Funding

This work was supported by the Portuguese Science Foundation (FCT − Fundaçaõ para a Ciência e Tecnologia) through individual research grant references SFRH/BPD/ 73781/2010 and SFRH/BD/77647/2011 under QREN − POPH funds, cofinanced by the European Social Fund and Portuguese National Funds from MCTES and projects PEst-C/ MAR/LA0017/2013 and PEst-OE/EQB/LA0016/2013 supported by European Funds through COMPETE and by National Funds through the FCT. Notes

The authors declare no competing financial interest.

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on March 18, 2015, without an update to reference 35. The corrected version was reposted on March 23, 2015.

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