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Mar. Drugs 2013, 11, 1256-1270; doi:10.

3390/md11041256
OPEN ACCESS

Marine Drugs
ISSN 1660-3397 www.mdpi.com/journal/marinedrugs Article

Evaluation of the Antioxidant Activity of Cell Extracts from Microalgae
A. Catarina Guedes 1,2, Maria S. Giã o 1, Rui Seabra 3, A. C. Silva Ferreira 1, Paula Tamagnini 3,4, Pedro Moradas-Ferreira 3,5 and F. Xavier Malcata 2,6,*
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CBQF/Biotechnology College, Catholic University of Portugal, Rua Dr. Antó nio Bernardino de Almeida, Porto P-4200-072, Portugal; E-Mails: acatarinaguedes@gmail.com (A.C.G.); msilvagiao@hotmail.com (M.S.G.); acferreira@esb.ucp.pt (A.C.S.F.) CIMAR/CIIMAR—Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas nº 177, Porto P-4050-123, Portugal IBMC—Institute for Molecular and Cell Biology, University of Porto, Rua do Campo Alegre nº 823, Porto P-4150-180, Portugal; E-Mails: ruisea@gmail.com (R.S.); pmtamagn@ibmc.up.pt (P.T.); pmferrei@ibmc.up.pt (P.M.-F.) Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Edifí cio FC4, Porto P-4169-007, Portugal ICBAS—Institute of Biomedical Sciences Abel Salazar, University of Porto, Largo Abel Salazar nº 2, Porto P-4099-003, Portugal Department of Chemical Engineering, University of Porto, Rua Dr. Roberto Frias, Porto P-4200-465, Portugal

* Author to whom correspondence should be addressed; E-Mail: fmalcata@fe.up.pt; Tel.: +351-968-017-411. Received: 15 January 2013; in revised form: 6 March 2013 / Accepted: 7 March 2013 / Published: 17 April 2013

Abstract: A growing market for novel antioxidants obtained from non-expensive sources justifies educated screening of microalgae for their potential antioxidant features. Characterization of the antioxidant profile of 18 species of cyanobacteria (prokaryotic microalgae) and 23 species of (eukaryotic) microalgae is accordingly reported in this paper. The total antioxidant capacity, accounted for by both water- and lipid-soluble antioxidants, was evaluated by the (radical cation) ABTS method. For complementary characterization of cell extracts, a deoxyribose assay was carried out, as well as a bacteriophage P22/Salmonella-mediated approach. The microalga Scenedesmus obliquus strain M2-1 exhibited the highest (p > 0.05) total antioxidant capacity (149 ± 47 AAU) of intracellular extracts. Its scavenger activity correlated well with its protective effects against DNA

deoxyribose. Therefore. a favourable effect upon human life span via delays in ageing has been claimed. .Mar. via regular ingestion as part of formulated foods. Screening started with generic assays for antioxidant features. catalase.. they can use H2O and CO2 to synthesize complex organic compounds—and subsequently accumulate and/or secrete many primary and secondary metabolites of interest [4–6]. Furthermore. so as to comprehensively characterize the strains in terms of total antioxidant capacity. only those strains bearing a good potential were subjected to more detailed assays. Both intra. whereas the non-enzymatic counterpart includes such mediator compounds as ascorbic acid.and extra-cellular extracts were considered. but the latter would be more interesting from a processing point of view owing to easier recovery from the microbial broth. The research effort described in this paper attempts to contribute to expand the uses of microalgae. as required before any given microbial extract undergoes further in-depth characterization regarding high-added value functional compound(s). e. a preliminary test of mutagenicity was also carried out. as well as in the incidence and severity of cardiovascular and degenerative diseases [1–3]. Microalgae are the basis of the food chain in aquatic ecosystems. via stimulation of their antioxidant defence system [7] that consists of both enzymatic and non-enzymatic mechanisms: superoxide dismutase. DNA. less conventional sources therefore justifies comprehensive searches encompassing unusual sources—as is the case with microalgae. performance of an Ames test revealed no mutagenic effects of the said extract. Furthermore. Ames test 1257 1. Drugs 2013. ABTS•+. with the aid of solar energy. 11 oxidative damage induced by copper(II)-ascorbic acid. glutathione reductase and ascorbate peroxidase are key enzymes in the former. tocopherols. Finally. carotenoids and phycocyanin [8]. Keywords: 16S rDNA. phage P22/Salmonella. reduced glutathione.g. microalgae exhibit adaptative responses to oxidative stresses. and against decay in bacteriophage infection capacity induced by H2O2. The growing market for novel antioxidants obtained from non-expensive. the experimental work encompassed screening of several unusual microalgae for antioxidant behaviour using complementary analytical approaches. based on a decrease in such biomarkers as protein carbonyls produced via oxidative damage. by specifically taking advantage of their antioxidant features. namely in the food industry. the former are normally more abundant. Introduction The last decade has witnessed a growing interest in compounds from natural sources that bear antioxidant properties because these compounds may provide relevant contributions to maintain health. Indeed evidence gathered in a large number of worldwide studies supports the role of antioxidants in the prevention of and in the growth control of certain tumours. so as to reasonably comply with limited laboratory resources while maximizing experimental efficacy.

00 ±0.c 1. CCAP —Culture Collection of Algae and Protozoa (UK). Microorganisms 1258 The main objective of this research effort was to identify less conventional microalga cells harbouring compounds with antioxidant capacity. ACOI—Coimbra Collection of Algae (University of Coimbra.46 7.78 ±1.23 ±0.99 a.00 a.32 2.07 ±0.b.98 ±1.02 ±0.b.01 3. identified using morphological characteristics and partial sequencing of 16S rDNA.59 2.76 c. Total antioxidant capacity (average ± standard deviation) of cyanobacterial extracts.73 38. Results and Discussion 2.29 ±0. using both morphological characteristics and partial sequencing of 16S rRNA.46 ±1.c.) * Lyngbya majuscula Filamentous heterocystous Anabaena variabilis J52 (Anabaena planctonica) * Nodularia harveyana Nostoc carneum Nostoc muscorum Nostoc punctiforme Scytonema obscurum ATCC 29413 Maranhã o lagoon ACOI 729 ACOI 650 CCAP 1453/12 PCC 73102 ACOI 573 BG110 BG11 BG11 BG11 BG110 BG11 BG110 BG11 BG110 BG11 BG11 2. .c 5. in AAU ((mg/Lequivalent ascorbic acid)/μgchlorophyll a). Hence.b.33 5.97 ±0. without a common superscript.04 Extracellular nd nd nd 0. Synechocystis sp.34 14.05).09 2. nd (not detected).06 ±0. Gloeobacter violaceus Gloeothece sp.95 ±0.00 nd nd nd nd 0. Environmental isolates were identified via a polyphasic approach. Portugal).07 ±0. Table 1.60 2.46 3.1.43 2.01 ±0.80 ±1. Strain Unicellular Chlorococcus giganteus Cyanothece sp.48 3.90 ±6.68 ±0.00 nd nd nd nd nd 0. and PCC—Pasteur Culture Collection (France). Drugs 2013.42 ±1.01 nd nd Means within the ACOI 768 ATCC 51142 PCC 7421 ATCC 27152 PCC 6803 ACOI 48 ATCC 29408 PCC 73110 PCC 7410 Aquaculture biofilters CCAP 1446/4 * Wild strain. IPIMAR (Portugal).01 ±0.00 ±8. Leptolyngbya sp.78 ±0.10 ±6.12 ±1. the set of microorganisms tested included unusual strains/species maintained in culture collections.c nd nd nd 0. 11 2.89 35.19 9.d 3.31 17.d a–d Origin Culture Medium BG11 OHM BG11 BG11 BG110 BG11 BG11 ASW-BG11 BG11 BG11 BG11 BG11 Antioxidant Power Intracellular 2.32 ±0. ATCC—American Type Culture Collection (USA).Mar. as well as wild ones isolated from unique ecosystems in Portugal (see Tables 1 and 2).47 a. such isolates were tentatively named after their closest cultured relative.36 9.00 b 0. are significantly different (p < 0.01 b 0.92 1.57 6.22 ±0.01 ±0.03 ±0.78 ±0.03 a. Synechocystis salina Filamentous Arthrospira platensis Leptolyngbya sp.84 ±0. same column.01 ±0. M2-7 (Limnothrix sp. CBSC—Carolina Biological Supply Company (USA). expressed as ABTS radical scavenging activity.

24 49.5 ±2.54 4.42 ±1.46 6.04 g/L SiO3.66 ±0.11 nd ASW 2 BG11 8.54 ±0.00 b nd nd 1.92 ±0. expressed as ABTS radical scavenging activity.91 a. without 0.2.04 ±0.50 1.27 ±1.05 0. 2. nd (not detected).52 8. vulgaris Haematococcus pluvialis Scenedesmus maximus M2-1 (Scenedesmus obliquus) M2-6 (S. obliquus F S. obliquus) M4-3 (S.35 ±0. are significantly different ( p < 0. Wild species. obliquus G Desmodesmus pleiomorphus A D. in AAU ((mg/Lequivalent ascorbic acid)/μgchlorophyll a).60 ±2. The first assay measured protection against oxidative damage by cell extracts and clearly indicated that isolate M2-1 (Scenedesmus obliquus) performed the best.05). obliquus) M2-5 (S.d f e nd nd nd nd 0. pleiomorphus H D. 11 Table 2.10 ±4. Hence.48 ±1.40 ±3. Antioxidant Capacity as ABTS Scavenging Selection of the most promising microorganism(s) was based on assays for their antioxidant activity via complementary approaches.86 1.79 ±2. this strategy was expected to effectively overcome the (obvious) limitations of each analytical method proposed when considered independently. without a common superscript.10 g 6.93 7. obliquus) 1 Scenedesmus obliquus B S.86 ±0.01 ±0.83 f Aquaculture biofilters OHM OHM OHM OHM OHM OHM OHM M2-18 (S.8 ±1.81 ±0. Total antioxidant capacity (average ± standard deviation) of microalgal extracts.10 ±6. obliquus) M4-5 (S.31 1 2 nd nd ASW medium Means within the same column. The results of the ABTS free radical scavenging . only this isolate was subjected to complementary antioxidant assays as discussed in the next subsections. pleiomorphus E D.25 2.91 10.2 ±1.00 ±46.39 21.47 63.76 ±0.13 ±1.84 nd OHM OHM OHM OHM OHM OHM OHM 10. identified using morphological characteristics and partial sequencing of chloroplastidial 16S rDNA.60 25. Class/Species Bacillariophyceae Phaeodactilum tricornutum Chlorophyceae Chlorella vulgaris C.66 3.34 ±0. Drugs 2013.83 4. pleiomorphus M Scenedesmus quadricauda Eustigmatophyceae Nannochloropsis sp.26 2.b c. Prymnesiophyceae Pavlova lutheri Rhodophyceae Porphyridium aerugineum a–g Estarreja wetlands OHM OHM OHM OHM CBSC SERI: NANNO-1 (GBSTICHO) IPIMAR: SMBA 60 ACOI 1332 OHM ASW 2 7.15 149.Mar. obliquus) 1 1 1 1 15. obliquus) 1 1 1259 Origin Culture Medium Antioxidant Power Intracellular Extracellular SERI-S/PHAEO-1 (TFX-1) CBSC ACOI 879 CCAP 34/7 ACOI 318 ASW 8.76 ± 1.32 nd nd nd nd nd nd nd nd nd nd nd M3-9 (S.80 ±10.

obliquus)..3.57 AAU (Antioxidant Activity Units) for Nodularia harveyana to 38. which is an indication of hydroxyl radical scavenger capacity (see Table 3).79 477. the antioxidant power correlated linearly (and positively) to ability to inhibit deoxyribose degradation. respectively.56 ±0. intracellular extracts of the most promising strains were able to protect deoxyribose and decreased significantly its extent of degradation.97 2. This method also addressed the question of whether a pro-oxidant effect was present (i.86 122.03 AAU for Gloeobacter violaceus.68 ±2. violaceus.85 ±46.81 9. The levels of antioxidant activity found in this initial screening revealed that the intracellular extracts of microalgae are typically stronger than those of cyanobacteria: this is notably the case for isolate M2-1 (S.04 16. Regarding intracellular extracts of microalgae. obliquus) b Antioxidant Power 20. the ascorbic acid-equivalent concentration ranged from 1. Hence. Since no extract exhibited pro-oxidant effects. besides assessment of the antioxidant capacity of M2-1 (S. The results are depicted in Figure 1.) J52 (Anabaena planctonica) Scytonema obscurum Antioxidant Power 113. obliquus) M2-5 (S. obliquus) M2-6 (S. 11 1260 assays are listed in Tables 1 and 2. For the extracellular extracts.46 ±30. The ascorbic acid-equivalent concentration in intracellular extracts ranged from 1. so only this extract underwent further assaying. Drugs 2013.45 ±5. Table 3. obliquus). violaceus.00 ±46. a Cyanobacterium Strains Gloeobacter violaceus M2-7 (Limnothrix sp.32 ±0. Total antioxidant capacity (average ± standard deviation) of the best (a) cyanobacterial and (b) microalgal intracellular extracts.34 ±0. The strongest protection was once again associated with strain M2-1. This test was applied only to the most promising strains as concluded from inspection of Tables 1 and 2.60 AAU for M2-1 (S. obliquus) extract.48 ±0.01 AAU for G. the level of which is almost four-fold the highest value obtained with G. obliquus) M3-9 (S. obliquus) M4-5 (S.65 Microalga Species Haematococcus pluvialis M2-1 (S.e.10 ±6.95 138. Antioxidant Capacity as DNA Protection It is known that active oxygen species react with DNA (besides deoxyribose). Following Perez et al.91 ±161.4.63 51. Antioxidant Capacity as Deoxyribose Protection The protection of DNA from oxidative damage was analytically determined via protection of microalgal and cyanobacterial intracellular extracts upon induced deoxyribose damage [10].54 ±0. in percent inhibition of degradation by induced oxidation. [11]. expressed as protection of deoxyribose. the values obtained were considerably lower [9]—ranging from (3. 2. ascorbic acid and copper were utilized to induce oxidation (and consequent DNA cleavage). .and extracellular extracts of cyanobacteria and microalgae.05 AAU for Desmodesmus pleiomorphus A to 149.75 ±2.60 121.07 ±0.84 ± 0.57 11.76) × 10−5 AAU for Nostoc muscorum to 0.Mar.92 ±9. for the intra. if it could cause oxidation by itself). and concomitantly produce strand breaks which can be detected using electrophoretic separation of the DNA fragments thus produced.

the M2-1 extract protects calf-thymus DNA from induced oxidative damage. Drugs 2013.5 log units by 20 min—although an essentially nil effect was observed during the first 5 min. lanes A′). and A′—1 μg of DNA plus (A′1) 200. it was concluded that it did not act as either oxidant or antioxidant (data not shown). no significant pro-oxidant effect was detected (see Figure 1. Higher volumes (from 200 up to 400 μL) did not significantly affect the extent of protection (data not shown). and reached its maximum level when 200 μL of cell extract was incubated with DNA (see Figure 1. (A′2) 150. which reached 1.05 (see SPO in Figure 2). clearly unfolded a reduction in the number of phages available to infect Salmonella during the course of the experiment (see OP in Figure 2). The antioxidant effect using this specific assay was calculated as the difference between the observed extent of infection of the bacterium by the virus in the presence of both M2-1 extract and H2O2 (SPO). this reduction was used as base line to assess the antioxidant activity of the M2-1 extract. (A2) 150. This effect became gradually more visible as the sample volume increased (see Figure 1. a preliminary assay used only ethanol/water (1:1. (A′3) 100. Furthermore. A—1 μg of DNA plus Cu(II)-ascorbic acid. and its counterpart in the presence of H2O2 only (OP).5. By 20 min. an important protective effect was conveyed by M2-1 extracts as soon as after 10 min of contact. (A4) 75 or (A5) 50 μL of extract. yet results published elsewhere [12. C+—1 μg of DNA. the oxidant effect of H2O2 upon the phage was significantly reduced. C−—1 μg of DNA plus Cu(II)-ascorbic acid. p > 0.Mar. Upon adding 50 mg· mL−1 of the said extract. 11 Figure 1. the M2-1 extract itself did not produce any damage to the phage (see SP in Figure 2). 2. lane A1). v/v) to determine whether ethanol would influence phage growth. which was attained by adding 250 mM H2O2. a three log-cycle reduction had already been achieved relative to the initial phage numbers. and plus (A1) 200. The inactivation curve of the P22 bacteriophage. (A′4) 75 or (A′5) 50 μL of extract. (A3) 100. On the other hand.13] unfolded its suitability as analytical method. and the protective effect of various amounts of M2-1 extract: M—DNA molecular weight marker: λ DNA cut with HindIII. Agarose gel electrophoregram showing the damage induced to calf thimus DNA by Cu(II)-ascorbic acid. In order to ascertain the antioxidant effect of M2-1 extracts. 1261 According to this method. lanes A). Antioxidant Capacity as Bacteriophage Protection The use of bacteriophages as an in vivo system to assess antioxidant capacity is a relatively novel approach. . In all cases.

standards obtained either from commercial sources (e.g. cryptoxanthin.56 ±0.6.5 14. alternatively. Drugs 2013. neoxanthin. Phage inactivation by externally induced chemical oxidation can accurately ascertain the effects of oxidative stress upon the biological integrity of DNA. Compound Neoxanthin Violaxanthin Lutein Zeaxanthin β-Apo-8′-carotenal β-Carotene Elution Time (min) 5.g.02 0. at 50 mg· mL−1. The antioxidant effect was assessed as the difference between the observed infection of the bacterium by the phage in the presence of sample and oxidant (SPO). The retention times of these standards are depicted in Table 4.. violaxanthin and neoxanthin concentrations were expressed as lutein-equivalents. or extracts of nettles and yellow pepper (e.69 ±0. in the presence of oxidant only (OP) and in the presence of sample only (SP). 1262 The M2-1 extract itself did not produce any damage to the virus.01 2.40 ±0. Antioxidant Concentration (mgequivalent lutein/gmicroalga) 0.9976).09 nd (internal standard) 0.6 15. nor did the ethanol used as solvent interfere with the assay.. several analyses were performed by HPLC. zeaxanthin.03 . violaxanthin and luteoxanthin) were accordingly employed as reference. expressed as mgequivalent lutein/gmicroalga. Our results showed that M2-1 extracts can effectively protect phage P22 against oxidative damage by H2O2. 11 Figure 2.Mar.1 34. whereas β-carotene..2 20. lutein).5 nd (not detected). it may reflect damage to the proteinaceous phage capsid that hampers delivery of the inner DNA material. Table 4. Lutein was quantified using a linear response factor. Evolution with time of the effect of M2-1 extract upon bacteriophage P22 viability (average ± standard deviation). 2. e. Antioxidant Compounds With the aim of identifying specific compounds that may account for the antioxidant capacity of M2-1 extract.9 6. calculated from reference solutions (characterized by a correlation factor of 0.14 ±0. echinenone.g. Identification and quantification (average ± standard deviation) of antioxidant compounds in M2-1 extract.

11 1263 A particularly high lutein content (i.03 mg· g−1) and neoxanthin (0.Mar.02 mg· g−1) cannot be ignored in this regard—including a putative synergistic effect with lutein [14]. using 1X TAE buffer [19]. The M2-1 extract did not produce evidence of any direct mutagenic effect. Variation with M2-1 extract concentration of the number of revertant colonies (average ±standard deviation) obtained with (a) TA 98 or (b) TA 100 strains. even at the highest concentrations tested. However. because the number of revertant bacteria was statistically identical (p > 0. In this test. even at the highest concentration tested. are plotted in Figure 3a. with and without the S9 (liver microssomal fraction) mixture [15]. besides other mutations that greatly increase their ability to detect chemical mutagens: e. whereas strain TA 100 detects mutagens that cause base-pair substitutions [16].69 ± 0. histidine-requiring strains were used—each one having a different type of mutation on the histidine operon.18]. Note that the Ames reversion assay was carried out on the sample solutions with TA98 and TA100. The results of this test on M2-1 extract.g. 2. strain TA 98 detects various frameshift mutagens. since the number of revertants was statistically identical to that of spontaneous ones. DNA Extraction and Agarose Gel Electrophoresis Genomic cyanobacterial DNA and total microalgal DNA were isolated using the phenol/chloroform method. in either pure or crude forms.09 mg· g−1) was recorded in the M2-1 extract.05) to that of the control.7. DNA bands were visualized with ethidium bromide.1. 2.56 ± 0. Mutagenicity The Ames test has been classically used to assess the mutagenic effects of various compounds.40 ± 0.b. followed by ethanol precipitation [17. the said extract did not lead to evidence of any direct mutagenic effect. Figure 3. which suggests that the high antioxidant capacity observed is likely associated therewith. 3. .. using the aforementioned TA98 and TA100 strains. ( ) without S9 and ( ) with S9. Experimental Section 3. Agarose gel electrophoresis was performed following standard protocols.e. Drugs 2013. under UV light.. respectively. presence of β-carotene (0.

according to the manufacturer’s instructions. WI. Computer-assisted sequence analysis and comparisons were performed using Vector NTI Advance 10 (Invitrogen Corporation. Plasmid DNA was isolated using the GenEluteTM Plasmid Miniprep Kit (Sigma-Aldrich. under the accession numbers depicted in Table 5. DQ396875) S. AJ293108) Limnothrix sp. Novel sequences associated with this study are available from GenBank. DQ396875) S. Portugal). obliquus strain UTEX 393 (99%. UK). 50 ° C for 1 min and 72 ° C for 1 min.Mar. and sequencing was performed at STAB Vida (Lisbon. obliquus strain UTEX 393 (99%.2. Madison. accession number) a Anabaena planctonica strain 71 (99%. using the following profile: 40 cycles of 94 ° C for 1 min. USA). DQ396875) S. USA). The purified PCR products were cloned into the pGEM®-T Easy vector (Promega Corporation. DQ396875) Levels of similarity determined by BLAST. followed by an extension at 72 ° C for 7 min. CA. USA). In addition. obliquus strain UTEX 393 (92%. EF088338) Scenedesmus obliquus strain UTEX 393 (98%. . DQ396875) S. Carlsbad. Colonies were screened for presence of the insert via colony-PCR. coli DH5α competent cells—following again the manufacturer’s instructions. obliquus strain UTEX 393 (92%. Table 5. and closest cultured relatives. obliquus strain UTEX 393 (92%. DNA Amplification. Drugs 2013. 11 3. Visualization of the amplified DNA products was performed using 1. and subsequently grown overnight in liquid LB medium at 37 ° C with shaking. CYA359F and CYA781R [18]. DQ396875) S. List of GenBank accession numbers of novel 16S rDNA sequences obtained. St. Purification. Louis. DQ396875) S. obliquus strain UTEX 393 (92%. the universal bacterial primer 1541R (5′ AAGGAGGTGATCCAGCC 3′) was employed to obtain longer sequences of the cyanobacterial 16S rRNA genes. Isolate J52 M2-7 M2-1 M2-6 M3-9 M4-3 M4-5 M2-5 M2-18 GenBank Accession Number EU073188 EF634458 EU073189 EU073191 EU073193 EU073194 EU073195 EU073190 EU073192 a Closest Cultured Relative (% similarity. Cloning and Sequencing 1264 The oligonucleotide primers used to amplify both the cyanobacterial 16S rDNA and the microalgal chloroplast 16S rDNA were CYA106F. and further used to transform E. MO. strain CENA110 (99%.5% agarose gel electrophoresis. The Polymerase Chain Reaction (PCR) assays were performed as described elsewhere [17]. DNA fragments were isolated from agarose gels using the GFX PCR-DNA and Gel Band Purification kit (GE Healthcare.

and the supernatant (i.and intra-cellular extracts of microalgae and cyanobacteria were evaluated via the ABTS radical cation (ABTS •+) assay. ASW [22] and ASW-BG11 [23] media were used instead. the supernatant (i. but also allows more meaningful discussion vis a vis with available data in the literature (owing to it being a regular trophic indicator). this is so because there is a strong correlation between dry weight and chlorophyll a content. the cells were crushed in an Ultra Turrax T 18 homogenizer (Ika. Growth Conditions and Microorganisms 1265 Environmental isolates and culture collection species/strains tested in this study are listed in Table 1 for cyanobacteria. In short. for microalgae and cyanobacteria. and in Table 2 for microalgae.000 rpm for 30 s. plain . Batch cultures were grown at 25 ° C. parallel determination of the content of chlorophyll a per dry biomass of a few species taken at random from each genus would not indeed alter the decision on selection of strains throughout screening. nor would it reverse any major conclusion. Drugs 2013. Genomic cyanobacterial DNA and total microalgal DNA were extracted according to Tamagnini et al. NC.6. was taken advantage of for quantification as reference basis. The known dependence of chlorophyll a levels on the physiological status of the cells did not apparently interfere with the figures generated. respectively. Novel sequences associated with this study were made available from GenBank. extracellular extract) was collected. purification. as originally detailed by Re et al. Wilmington..7 × Abs663nm—previously proposed by Meeks and Castenholz [25]. for 5 min at 15 ° C. at 14. The pellet was then resuspended and homogenized in 5 mL of a mixture of ethanol and water (1:1 v/v). [17] and Burja et al. Portugal). [18].. Extracellular and Intracellular Extraction Extracellular and intracellular extracts were collected according to Guedes et al. Although a per biomass weight basis would in principle have been preferable because secondary rather than primary production was at stake. computer-assisted sequence analysis and comparisons were performed using Vector NTI Advance 10 (Invitrogen Corporation. USA).3. and then subjected to centrifugation at 4000 rpm.5. ABTS Scavenging Assay The radical-scavenging capacities of both extra. for 7 min at 15 ° C. 11 3. 3. Chlorophyll a Content The total chlorophyll a content was determined after extraction of the cyanobacterial cells using 90%(v/v) methanol. DNA amplification. For marine isolates. finally.4.Mar. as in our case the cultures were all in their early stationary phase when harvested. under continuous illumination with −2 −1 fluorescent daylight (35 μmolphoton m s ). USA). absorbance was measured at 663 nm. [26] and recently refined by Giã o et al. [27]—using. CA. 5 mL of a 30 day-old culture was centrifuged at 4000 rpm. 3. The basis chosen is not only easier to quantitate.e. in the case of extracellular extracts. [24]. in 200 mL of medium. cloning and sequencing was performed at STAB Vida (Lisbon. using preferentially OHM [20] and BG11/BG110 [21] media. 3. and the equation μgchlorophyll a mL−1 = 12. Carlsbad. the intracellular extract) was collected.e.

Deoxyribose Protection Assay The deoxyribose protection was quantified via a method described in detail elsewhere [28. 10 μL of 1 mmol L−1 33% (w/v) H2O2.22 μm). at 37 ° C for 1 h.. The cation ABTS•+ was diluted with ultra-pure water. 3.e. For that purpose. where 1 AAU is defined as 1 mg L−1 of equivalent ascorbic acid per μg of chlorophyll a. Details of this method were described previously [12. thus decreasing the extent of fragmentation of deoxyribose. 150 and 200 μL). each reaction mixture was prepared so as to contain 50 μg of calf thymus DNA. the final volume was in all cases adjusted to 1 mL with 100 mM sodium phosphate buffer (pH 7.and lipid-soluble antioxidants was possible. in the presence of 1 mL of 1% (v/v) TBA in 0. [30] and Perez et al. In brief. and triplicate samples were taken. and incubated for 1 h at 37 ° C—in the presence of 10 μL of 10 mmol L−1 Fe3+.Mar. 100 ° C for 15 min). Their protective effects against DNA oxidative damage induced by Cu(II)-ascorbic acid were assessed following Muñ iz et al.9.1% (w/v) agarose gel. .. followed by cell disruption via sonication for 15 min. 11 1266 growth medium as control. 3.4) containing 15 mmol L−1 NaCl. the sample was then filtered through a sterile filter (0. give rise to a chromophore (malonaldehyde).5 mL of 28% (v/v) tricloroacetic acid) and high temperature (i. and final stirring for 10 min.05 mol L−1 NaOH. to generate hydroxyl radicals. under acidic conditions (i. When antioxidants are present. resuspended in 12 mL of ethanol/water (1:1. Bacteriophage Protection Assay Cell-free microalga extracts were prepared using 250 mg of lyophilized culture. v/v) and disrupted by ultrasonication for 15 min. and 10 μL of 10 mmol L−1 EDTA. Drugs 2013. The reaction mixtures were incubated in a shaking water bath. as well as various volumes of cell-free extract (i. hence full quantification of both water..4). 3.7. Electrophoresis was performed according to the protocol described by Sambrook and Russell [19].e.29]. 10 mM of ascorbic acid and 100 μM of Cu(II). Cambridge. [11]. and in a mixture of ethanol and water (1:1 v/v).13]. The results were expressed in Antioxidant Activity Units (AAU). in a 24 mmol L−1 sodium phosphate buffer (pH 7. and DNA was visualized using ethidium bromide. for intracellular extracts. resuspended in 5 mL of a mixture of ethanol/water (1:1. All measurements were made against adequate blanks. 100. DNA Protection Assay Cell-free microalga extracts were prepared using 400 mg of lyophilized culture. under UV light. v/v). this adduct was quantified by absorbance at 532 nm. 75. for extracellular extracts.8. The aforementioned radicals break deoxyribose into fragments which.e. with analyses run in quadruplicate for each one. 50. they compete for hydroxyl radicals. UK). a 200 μL-sample of the extract of interest was added to 10 μL of 100 mmol L−1 deoxyribose. and 20 μL of each was loaded onto a 1. 1. using 1×TAE buffer. using a Heλios α spectrophotometer (Unicam.

previous experience indicated that the compounds most likely to account for the antioxidant activity (i. v/v) or acetone/hexane (1:1. zeaxanthin. were characterized elsewhere [15]. More specifically. and only in the presence of oxidant (OP). at room temperature (in duplicate).e. and was then quenched via addition of 50 μL of 500 U mL−1 catalase (Sigma). with and without the S9 (liver microssomal fraction) mixture [15]. carotenoids) are quantitatively extracted irrespective of whether ethanol/water (1:1. and disrupted via ultrasonication for 15 min. 100 and 150 μL/plate) using two types of reference strains. in a water bath kept at 37 °C. Reversion of specificity and activity of the S9 mix was confirmed with . v/v). Full description of the HPLC procedure is available elsewhere [24. Mutagenicity Assessment An Ames reversion assay was carried out. Each mixture was left to react for 20 min. when a challenging oxidant (250 mM H2O2) is initially applied. owing to their known antioxidant features. harvested in the exponential phase—i. the stronger the infection capacity of the virus. and serial decimal dilutions were performed up to 10−6. lutein.. the bacteriophage infection capacity decreases. 3. and incubated at 37 ° C for 24 h. the stocks of phage P22 and Salmonella Typhimurium (ATCC 19585 P1) were prepared according to ATCC indications. Dilutions of the virus up to 10–12 were performed (in duplicate) in tryptone soy broth (TSB. a 1% (v/v) inoculum of an overnight culture in TSB at 37 ° C. however. 100 μL was spread onto pre-dried TSA plates (in duplicate). Cell-free microalga extracts were accordingly prepared as described for the bacteriophage protection assay. on the M2-1 extract (25. from Biokar Diagnostic). violaxanthin. the viable −1 phage number was again expressed as PFU mL . Drugs 2013. 100 μL of each dilution was then mixed with 300 μL of Salmonella culture. The difference between the viable phage numbers in the presence of sample and oxidant (SOP).10. and incubated at 37 ° C for 10 min.31].5 h. Following incubation. For the infection stage. then.11. The tester bacteria.. and negative values indicated obviously otherwise. then samples exhibited an antioxidant effect. and β -carotene were assayed for. PFU mL−1). the higher this figure. on the other hand. Although the type of compounds extracted depends in general on the nature of the extracting solvent(s).Mar. Aliquots of 100 μL were collected every 5 min for a period of 20 min. 11 1267 The rationale for this methodology is a virucidal attack on Salmonella Typhimurium cells. the plaques were counted and the total viable phage numbers were accordingly calculated (and expressed in plaque-forming units per unit volume. Infection was allowed to proceed for 10 min. were considered as a datum point. water was not allowed in the aforementioned HPLC protocol. the virus will recover its normal infection capacity. neoxanthin. from LAB M). 100 μL of each phage dilution was added at a time to 300 μL of the suspension of Salmonella in the exponential phase (as described above). Salmonella Typhimurium strains TA 98 and TA 100. into fresh TSB was incubated at 37 ° C for a further 2.e. 3. 50. and incubated at 37 ° C for 24 h. v/v) are employed. in duplicate. 100 μL was spr ead onto pre-dried plates of tryptone soy agar (TSA. Antioxidant Identification Microalgal cell-free extracts were prepared using 40 mg of lyophilized culture. when these were positive. resuspended in 5 mL of acetone/hexane (1:1. if protection occurs in the presence of antioxidant compound(s). Then.

3. Amaro. and plain solvent as negative one.C. Drugs 2013. Haematococcus astaxanthin: Applications for human health and nutrition.. Abe. References 1.12. strain M2-1 appears to be a particularly promising source of antioxidant additives for food formulation. M. 656–661. 45.Y. This antioxidant activity—assessed by such alternative methods as scavenging of free radicals. Rev.M. 1865–1872. Although low-cost cultivation media were utilized and no major constraints existed regarding growth. Chen..G. Pan...H.. SFRH/BPD/72777/ 2010)—supervised also by author F. 100.C. M.. 210–216. Process Biochem. as assessed with specific Salmonella strains. (grant ref. J.C. are hereby gratefully acknowledged. Hiran. 2.X..Mar. Tandeau-de-Marsac. 27. Malcata. so non-parametric tests were applied to ascertain whether the type of microalga is a statistically significant parameter upon antioxidant activity. Accumulation and antioxidant activity of secondary carotenoids in the aerial microalga Coelastrella striolata var. 1993. Chang. 3. and biological protection of bacteriophage upon Salmonella infection—seems to be associated chiefly with lutein. Olaizola.L. H. M.0 software [32]. H. Microalgae as sources of high added-value compounds—A brief review of recent work. a careful economic analysis is still required prior to drawing any definite conclusions on industrial feasibility. A. among 23 microalgae and 18 cyanobacteria screened for that purpose. Yang. Identification of anti-lung cancer extract from Chlorella vulgaris C-C by antioxidant property using supercritical carbon dioxide extraction.. Hattor. POCTI/CTA/44600/2002). 2010. 4. Acknowledgments Funding by projects BIOALGA—(grant ref.. chemical protection of deoxyribose and DNA.0.X.M. . Biotechnol.X.M. Wang. In addition.. and by a postdoctoral fellowship for author A. J.. PTDC/EBB-EBI/102728/ 2008). 21.S. obliquus) exhibits a particularly high antioxidant activity. multistriata. the antioxidant-rich extract of the M2-1 strain does not entertain any mutagenic effect. C. N. The tests were performed using SPSS. Prog. H.M. K. Chiu. as well as by projects MICROPESCA (grant ref. FEMS Microbiol.W. 11 1268 5 μg of benzo-[a]-pyrene per plate. Trends Biotechnol. 4.. Chang. C... Guerin. Statistical Analyses The experimental data did not typically follow a normal distribution. J. Huntley. and MICROPHYTE (grant ref. or active principles for therapeutic use. F. Food Chem. 5. 104. under the auspices of ESF (III Quadro Comunitá rio de Apoio/Portuguese State) and coordinated by author F. 2011. Quercetin (1 μg/plate) was used as positive control. 2005. Therefore. Guedes. Conclusions The microalga M2-1 (S. 2003. MARE/0072/04). 119–190. H. Houmard. Adaptation of cyanobacteria to environmental stimuli: New steps towards molecular mechanisms. M. M. version 16. in its various forms.E. 597–613.

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