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Journal of Applied Phycology (2005) 17: 447–460 DOI: 10.1007/s10811-005-1641-4 C Springer 2005 An evaluation of methods for extraction and quantification of protein from marine macro- and microalgae Elisabete Barbarino 1,2 & Sergio O. Louren¸ co 2, 1 Programa de P´ os-Graduac ¸˜ ao em Biotecnologia Vegetal, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil; 2 Departamento de Biologia Marinha, Universidade Federal Fluminense, Caixa Postal 100644, CEP 24001-970, Niter ´ oi, RJ, Brazil ( Author for correspondence: e-mail: [email protected], fax: +5521-2629-2292) Received 19 December 2004; accepted 27 June 2005 Key words: amino acids, marine macroalgae, marine microalgae, nitrogen, protein determination, seaweeds Abstract Comparison of data of protein content in algae is very difficult, primarily due to differences in the analytical meth- ods employed. The different extraction procedures (exposure to water, grinding, etc.), protein precipitation using different amounts of 25% trichloroacetic acid and quantification of protein by two different methods and using two protein standards were evaluated. All procedures were tested using freeze-dried samples of three macroalgae: Porphyra acanthophora var. acanthophora, Sargassum vulgare and Ulva fasciata. Based on these results, a protocol for protein extraction was developed, involving the immersion of samples in 4.0 mL ultra-pure water for 12 h, fol- lowed by complete grinding of the samples with a Potter homogeniser. The precipitation of protein should be done with 2.5:1 25% TCA:homogenate (v/v). The protocol for extraction and precipitation of protein developed in this study was tested with other macroalgae (Aglaothamnion uruguayense, Caulerpa fastigiata, Chnoospora minima, Codium decorticatum, Dictyota menstrualis, Padina gymnospora and Pterocladiella capillacea) and microalgae (Amphidinium carterae, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana and Skeletonema costatum). Compar- ison with the actual protein content determined from the sum of amino acid residues, suggests that Lowry’s method should be used instead of Bradford’s using bovine serum albumin (BSA) as protein standard instead of casein. This may be related to the reactivity of the protein standards and the greater similarity in the amino acid composition of BSA and algae. The current results should contribute to more accurate protein determinations in marine algae. Introduction Determination of protein content of algae can provide important information on the chemical characteristics of algal biomass. The methods most commonly used to quantify protein are: (i) the alkaline copper method (Lowry et al., 1951); (ii) the Coomassie Brilliant Blue dye method (Bradford, 1976); or (iii) determination of crude protein (N × 6.25). The calculation of protein content by N × 6.25 re- quires some caution, not always considered by authors using this method. Plant materials, fungi and algae commonly have high concentrations of non-protein ni- trogenaceous substances such as pigments (chlorophyll and phycoerythrin), nucleic acids, free amino acids and inorganic nitrogen (nitrate, nitrite and ammonia) (Louren¸ co et al., 1998; Conklin-Brittain et al., 1999; Fujihara et al., 2001) whose presence makes the factor 6.25 unsuitable since it overestimates the actual pro- tein content (Ezeagu et al., 2002). Specific nitrogen- to-protein conversion factors were recently proposed for 12 marine microalgae (Louren¸ co et al., 2004) and 19 seaweeds (Louren¸ co et al., 2002), varying from 3.75 for Cryptonemia seminervis a red alga, to 5.72 for Pad- ina gymnospora a brown alga. The determination of protein by the Lowry and Bradford methods is carried out by spectrophotome- try. The Lowry method detects protein by a reaction catalyzed by copper, a component of the Folin phe- nol reactions. The chemical reaction detects peptide

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Page 1: Protein Extraction From Algae Method

Journal of Applied Phycology (2005) 17: 447–460DOI: 10.1007/s10811-005-1641-4 C© Springer 2005

An evaluation of methods for extraction and quantification of protein frommarine macro- and microalgae

Elisabete Barbarino1,2 & Sergio O. Lourenco2,∗

1Programa de Pos-Graduacao em Biotecnologia Vegetal, Universidade Federal do Rio de Janeiro, Rio de Janeiro,RJ, Brazil; 2Departamento de Biologia Marinha, Universidade Federal Fluminense, Caixa Postal 100644, CEP24001-970, Niteroi, RJ, Brazil

(∗Author for correspondence: e-mail: [email protected], fax: +5521-2629-2292)

Received 19 December 2004; accepted 27 June 2005

Key words: amino acids, marine macroalgae, marine microalgae, nitrogen, protein determination, seaweeds

Abstract

Comparison of data of protein content in algae is very difficult, primarily due to differences in the analytical meth-ods employed. The different extraction procedures (exposure to water, grinding, etc.), protein precipitation usingdifferent amounts of 25% trichloroacetic acid and quantification of protein by two different methods and usingtwo protein standards were evaluated. All procedures were tested using freeze-dried samples of three macroalgae:Porphyra acanthophora var. acanthophora, Sargassum vulgare and Ulva fasciata. Based on these results, a protocolfor protein extraction was developed, involving the immersion of samples in 4.0 mL ultra-pure water for 12 h, fol-lowed by complete grinding of the samples with a Potter homogeniser. The precipitation of protein should be donewith 2.5:1 25% TCA:homogenate (v/v). The protocol for extraction and precipitation of protein developed in thisstudy was tested with other macroalgae (Aglaothamnion uruguayense, Caulerpa fastigiata, Chnoospora minima,Codium decorticatum, Dictyota menstrualis, Padina gymnospora and Pterocladiella capillacea) and microalgae(Amphidinium carterae, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana and Skeletonema costatum). Compar-ison with the actual protein content determined from the sum of amino acid residues, suggests that Lowry’s methodshould be used instead of Bradford’s using bovine serum albumin (BSA) as protein standard instead of casein. Thismay be related to the reactivity of the protein standards and the greater similarity in the amino acid composition ofBSA and algae. The current results should contribute to more accurate protein determinations in marine algae.

Introduction

Determination of protein content of algae can provideimportant information on the chemical characteristicsof algal biomass. The methods most commonly usedto quantify protein are: (i) the alkaline copper method(Lowry et al., 1951); (ii) the Coomassie Brilliant Bluedye method (Bradford, 1976); or (iii) determination ofcrude protein (N × 6.25).

The calculation of protein content by N × 6.25 re-quires some caution, not always considered by authorsusing this method. Plant materials, fungi and algaecommonly have high concentrations of non-protein ni-trogenaceous substances such as pigments (chlorophylland phycoerythrin), nucleic acids, free amino acids

and inorganic nitrogen (nitrate, nitrite and ammonia)(Lourenco et al., 1998; Conklin-Brittain et al., 1999;Fujihara et al., 2001) whose presence makes the factor6.25 unsuitable since it overestimates the actual pro-tein content (Ezeagu et al., 2002). Specific nitrogen-to-protein conversion factors were recently proposedfor 12 marine microalgae (Lourenco et al., 2004) and19 seaweeds (Lourenco et al., 2002), varying from 3.75for Cryptonemia seminervis a red alga, to 5.72 for Pad-ina gymnospora a brown alga.

The determination of protein by the Lowry andBradford methods is carried out by spectrophotome-try. The Lowry method detects protein by a reactioncatalyzed by copper, a component of the Folin phe-nol reactions. The chemical reaction detects peptide

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bonds and is also sensitive to some amino acids suchas tyrosine and tryptophan (Legler et al., 1985). In theBradford method, the Coomassie Brilliant Blue dyeis bound to protein mainly by arginine residues andto a lower degree by histidine, lysine, tyrosine, tryp-tophan and phenylalanine residues. The binding be-tween the dye and amino acids is attributed to vander Waals forces and hydrophobic interactions (Comp-ton & Jones, 1985). As a consequence, the reactiv-ity of both methods in comparison to a specific pro-tein is strongly influenced by its amino acid compo-sition, since not all amino acids can oxidate equallythe Folin phenol reactive or bind to the Coomassie dye(Stoscheck, 1990). The differences in the principlesof the methods contribute to making comparison ofresults available in the literature even more difficult,since the choice of the method to be used is an arbitrarydecision.

Bovine serum albumin (BSA) is the most used pro-tein standard for calibration curves in spectrophotome-try, but many other proteins can be used. Several studiessuggest that the Lowry and Bradford analyses producedifferent measurements of protein when using BSA asthe protein standard for samples such as the gut fluidof fish (Crossman et al., 2000), marine invertebrates(Zamer et al., 1989), higher plants (Eze & Dumbroff,1982) and marine phytoplankton (Clayton et al., 1988).To obtain a more reliable measurement of protein, itwould be useful to identify the predominant proteinsin the cells (Berges et al., 1993). However, this rec-ommendation has no practical value, considering thedifficulty of extracting, purifying and characterisingthe main proteins present in the cells and subsequentlyusing them as protein standards. Nguyen & Harvey(1994) suggested the use of ribulose-1,5-diphosphatecarboxylase (RuDPCase) for calibration curves to anal-yse samples of photosynthetic organisms, since RuD-PCase corresponds to about 15% of the total protein inchloroplasts.

Several substances may interfere with both theLowry and Bradford method, such as phenol and phe-nolases (Mattoo et al., 1987), glucosamine and de-tergents (Peterson, 1979) and flavonoids (Compton &Jones, 1985) among many others (see the comprehen-sive studies of Peterson, 1979; Stoscheck, 1990 on in-terfering substances). These substances could affectanalyses by either increasing the absorbance (overesti-mating values), or decreasing the measurements by in-hibiting the action of specific reagents. However, theirinfluence may be avoided by precipitation of the pro-tein sample with trichloroacetic acid (TCA). Concen-

trations of TCA between 0.18 and 0.34 M can be usedto seperate protein from the other extract components,because only protein is precipitated (Clayton et al.,1988). The physical separation among protein, smallpeptides and free amino acids is especially important,since the analytical methods are sensitive to the last twoclasses of substances. Thus, protein precipitation withTCA is strongly recommended to avoid the quantifica-tion of small peptides and free amino acids (Nguyen& Harvey, 1994) as well as interference by othersubstances.

As many variables are simultaneously involved withprotein analysis, the influence of specific factors maybe neglected by authors, affecting the accuracy of pro-tein analysis. However, studies focussing on proteinanalysis in algae are relatively uncommon and exper-imental data are needed to fill this gap. It is also veryimportant to develop a simple and inexpensive pro-tocol, using low-cost equipment and consumables, inorder to make it accessible to everyone interested indata on algal protein: researchers, algae producers, peo-ple interested on the nutritional value of algae and soon.

In this study, different procedures for extraction andquantification of the protein content of marine algaewere evaluated. The specific aims of this study were: (i)to create a protocol for the extraction and quantificationof protein of marine algae; (ii) to compare the use of twomethods (Lowry and Bradford) for the determinationof protein in marine algae; (iii) to evaluate the effectsof the time of extraction, the use of grinding and theprecipitation of samples with TCA on the quantity ofprotein extracted; and (iv) to compare the amino acidprofile of the algal samples and the protein standards(BSA and casein) used.

Materials and methods

Fifteen species of marine algae covering a wide taxo-nomical range were analysed. The field-collected ma-rine were identified following the checklist of Wynne(1998). Marine microalgae were cultured in the labora-tory. The classification below is based on Lee (1999):Chlorophyta1. Chlorophyceae: Dunaliella tertiolecta Butcher

(Volvocales).2. Ulvophyceae: Caulerpa fastigiata Montagne

(Bryopsidales), Codium decorticatum (Woodw.) M.Howe; (Bryopsidales) and Ulva fasciata Delile(Ulvales).

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CryptophytaCryptophyceae: Hillea sp. Schiller (Cryptomon-adales).

DinophytaDinophyceae: Amphidinium carterae Hulburt(Gymnodiniales).

HeterokontophytaBacillariophyceae: Skeletonema costatum (Gre-ville) Cleve (Biddulphiales).Phaeophyceae: Chnoospora minima (K. Hering)Papenfuss (Scytosiphonales), Dictyota menstrualis(Hoyt) Schnetter, Hornig et Weber-Peukert (Dic-tyotales), Padina gymnospora (Kutzing) Sonder(Dictyotales) and Sargassum vulgare C. Agardh(Fucales).

PrymnesiophytaPrymnesiophyceae: Isochrysis galbana Parke(Pavlovales).

RhodophytaBangiophycidae: Porphyra acanthophora var.acanthophora E. C. Oliveira and Coll (Bangiales).Florideophycidae: Aglaothamnion uruguayense(Taylor) Aponte, Ballantine et Norris (Ceramiales)and Pterocladiella capillacea (S. G. Gmel.) San-telices et Hommersand (Gelidiales).All species of marine macroalgae were collected

in June 1998 at Rasa Beach (located in Armacao deBuzios, 22◦44′S and 41◦57′W) and Pero Beach (lo-cated in Cabo Frio, 22◦51′S and 41◦58′W), NorthernRio de Janeiro State, Brazil. Whole thalli of adult plantswere collected early in the morning and washed in thefield with seawater to remove epiphytes, sediment andorganic matter. Algae were packed in plastic bags andkept on ice until returned to the laboratory. In the lab-oratory, samples were gently brushed under runningseawater, rinsed with distilled water, dried with papertissue, frozen at −20 ◦C and freeze-dried. The driedmaterial was powdered manually with the use of mor-tar and pestle and kept in desiccators containing silica-gel and protected from light at room temperature untilchemical analysis.

Culture of microalgae

All microalgal strains used in this study are availableat the Elizabeth Aidar Microalgae Culture Collection,Department of Marine Biology, Federal FluminenseUniversity, Brazil. Starter cultures of 50–100 mL inmid-exponential growth phase were inoculated into2.0 L of seawater, previously autoclaved at 121 ◦C for30 min in 3.0 L borosilicate flasks, and enriched with

Conway nutrient solution (Walne, 1966). Each experi-ment was carried out in four culture flasks, exposed to300 µmol photons m−2 s−1 (measured with a Biospher-ical Instruments quanta meter QLS100) from beneath,provided by fluorescent lamps (Sylvania daylighttubes), under a 12:12 h light:dark cycle. Mean temper-atures were 23 ± 1 ◦C in the light period and 20 ± 1 ◦Cin the dark period. Salinity of the culture medium was32.0‰. Growth rates were calculated daily by directmicroscopic cell counting with Fuchs–Rosenthal orMalassez chambers. Cultures were bubbled with fil-tered air at a rate of 2 L min−1. The culture mediumwas not buffered and pH was determined daily.

Each culture was sampled in the stationary growthphase only. Cultures were concentrated by centrifuga-tion at 7000 g for 10 min at 15 ◦C, at least once. Beforethe last centrifugation, cells were washed with artificialseawater (Kester et al., 1967) prepared without nitro-gen, phosphorus and vitamins and adjusted to 15‰salinity to remove any residual nitrogen from the cul-ture medium. All supernatants obtained for each sam-ple were combined and the cell number was determinedin this pool to quantify possible cell losses. The pel-lets were frozen at −20 ◦C, freeze-dried (as describedabove), weighed and stored in desiccators under vac-uum and protected from light at room temperature untilanalysis was done.

Amino acid analysis

Samples containing 5.0 mg of protein were acid hy-drolysed with 1.0 mL of 6 N HCl in vacuum-sealedhydrolysis vials at 110 ◦C for 22 h. Norleucine wasadded to the HCl as an internal standard. Althoughtryptophan was completely lost with acid hydrolysisand methionine and cystine + cysteine could be de-stroyed to varying degrees by this procedure, the hy-drolysates were suitable for analysis of all other aminoacids. The tubes were cooled after hydrolysis, opened,and placed in a descicator containing NaOH pelletsunder vacuum until dry (5–6 days). The residue wasthen dissolved in a suitable volume of a sample di-lution Na–S

R©buffer (Beckman Instr.), pH 2.2, filtered

through a Millipore membrane (0.22 µm pore size) andanalysed for amino acids by ion-exchange chromatog-raphy in a Beckman, model 7300 instrument equippedwith an automatic integrator. Ammonia content is alsopresented as it comes from the degradation of someamino acids (e.g. glutamine, asparagine) during acidhydrolysis (Mosse, 1990).

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Total nitrogen

Total nitrogen (TN) content was determined by CHNanalysis. 0.8–1.5 mg freeze-dried samples were com-busted in a CHN analyser (Perkin–Elmer, model 2400).Helium was used as carrier gas. Acetanilide (C =71.09%; N = 10.36%; H = 6.71%) and/or benzoicacid (C = 68.84%; H = 4.95%) were used to calibratethe instrument.

Extraction of protein

Eight procedures for protein extraction were tested inthis study. All procedures start with 50 mg of freeze-dried algal sample, ground manually with pestle andmortar. Two different volumes of water were tested(1.0 and 4.0 mL), as well as two different incubationperiods of samples with water (6 and 12 h). In all thecases samples were kept at 4 ◦C during the incubationperiod.

In four out of the eight extraction procedures, sam-ples were also ground using a Potter homogeniser (Mar-coni, model MA099) after the incubation with water.Samples were water-ground with a glass pestle and ateflon mortar at medium speed. During grinding, sam-ples were kept cool by the use of a circulating coolingbath through the pestle. The grinding of samples wasstarted 1 h before the end of incubation of the sam-ples with water. Six replicates were prepared for eachtreatment and each species. Procedures for protein ex-traction are based on Fleurence et al. (1995) with somemodifications (e.g. speed of centrifugation, grinding ofthe samples, time of incubation). The specific proce-dures for protein extraction analysed in this study areas follows:

Procedure I. Algal samples were immersed in 1 mL ofultra-pure water for 12 h. After the incubation pe-riod, suspensions were centrifuged at 4 ◦C, 15,000 gfor 20 min. Supernatants were collected for proteinassay and the pellets re-extracted with 1.0 mL 0.1 NNaOH with 0.5%β-mercaptoethanol (v/v). The mix-ture of NaOH and pellets were kept at room tempera-ture for 1 h with occasional manual shaking and thencentrifuged at 21 ◦C, 15,000 g for 20 min. The sec-ond supernatants were combined with the first onesand the pellets were discarded. The final volume ofthe extract was 2.0 mL.

Procedure II. Similar to procedure I, with one addi-tional step included: the grinding of samples with aPotter tissue homogeniser for 5 min, 1 h before the

end of the incubation period. Seven millilitre of ultra-pure water was added to the system to rinse the Potterhomogeniser after grinding each sample to recoverall water-ground material. After this step, sampleswere treated as described after the end of the incu-bation period for procedure I. The final volume ofthe extract was 9.0 mL.

Procedure III. This treatment is similar to procedureI, differing by the use of 4 mL of water to incubatedried samples, instead of 1 mL. The final volume ofthe extract was 5.0 mL.

Procedure IV. This treatment is similar to procedure II,differing by the use of 4 mL of water for incubatingdried samples, instead of 1 mL. Four millilitre ofultra-pure water were added to the system to rinsethe Potter homogeniser after grinding each sampleto recover all water-ground material. After this step,samples were centrifuged as described for procedureI. The final volume of the extract was 9.0 mL.

Procedure V. Similar to procedure I, differing only duethe incubation period: 6 h instead of 12 h. The finalvolume of the extract was 2.0 mL.

Procedure VI. Dried samples are treated such as in pro-cedure II, except by the shorter incubation period: 6 hinstead of 12 h. The final volume of the extract was9.0 mL.

Procedure VII. Similar to procedure III, differing onlydue to the shorter incubation period: 6 h instead of12 h. The final volume of the extract was 5.0 mL.

Procedure VIII. Samples were treated as described inprocedure IV, differing only by the shorter incuba-tion period: 6 h instead of 12 h. The final volume ofthe extract was 9.0 mL.

A summary of the procedures to extract algal proteinis shown in Table 1.

Table 1. Summary of the procedures for extracting protein used inthis study

Volume of Period ofTreatment water (ml) incubation (h) Grinding

Procedure I 1.0 12 No

Procedure II 1.0 12 Yes

Procedure III 4.0 12 No

Procedure IV 4.0 12 Yes

Procedure V 1.0 6 No

Procedure VI 1.0 6 Yes

Procedure VII 4.0 6 No

Procedure VIII 4.0 6 Yes

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The use of 1.0 mL 0.1 N NaOH with 0.5% β-mercaptoethanol (v/v) for re-extracting the pellets wasadopted in all the eight procedures tested, independentof the time of incubation and volume of ultra-pure wa-ter used to incubate the samples.

Precipitation of protein

Protein precipitation was followed Berges et al. (1993).Two proportions of cold 25% trichloroacetic acid(TCA) (4 ◦C) added to the extracts were tested: 2.5:1and 3.0:1 (TCA:homogenate, v/v). Tubes contain-ing TCA and homogenate were kept in an ice bathfor 30 min and then centrifuged for 20 min at 4 ◦C(15,000 g). Supernatants were discarded, pellets werewashed with cold 10% TCA (4 ◦C) and centrifugedagain. Pellets formed after the second centrifugationwere suspended in 5% TCA at room temperature, in aproportion of 5:1 (5% TCA:precipitate, v/v) and cen-trifuged at 21 ◦C (15,000 g) for 20 min. Supernatantswere discarded and pellets were kept in the tubes un-til quantification of protein was done a few minuteslater. When the protein analysis was not performed im-mediately, pellets were stored at −20 ◦C until furtheranalysis, following Dortch et al. (1984).

Precipitated protein was suspended in 0.5 mL 1.0 NNaOH and 2.0 mL 0.1 N NaOH for the a Bradford andLowry assays, respectively. Aliquots were also col-lected from the crude extracts obtained before the pre-cipitation with TCA to perform protein analysis by theLowry method without previous precipitation.

Protein analysis

In the Lowry method, the Folin–Ciocalteu reactive(Folin & Ciocalteu, 1927) (Sigma Co.) was diluted intwo volumes of ultra-pure water (1:2) and 0.5 mL ofthe diluted reactive was added to 1.0 mL of sample,previously mixed with 5.0 mL of the reactive “C” [50volumes of reactive “A” (2.0% Na2CO3 + 0.1 N NaOH)+ 1 volume of reactive “B” (1/2 volume of 0.5% CuSO4

5H2O + 1/2 volume of 1.0% C4H4NaO6 4H2O)]. Afterthe addition of each reactive, samples were stirred for2 s in a test tube stirrer. Absorbance was measured at750 nm, 35 min after the start of the chemical reactionat room temperature.

In the Bradford assay, the Coomassie Brilliant Bluedye G-250 (CBBG) binds to the protein. The bind-ing of the dye with the protein is very quick and theprotein-dye complex remains soluble for 1 h. One hun-dred milligram of CBBG (Sigma Co.) was dissolved in

50 mL 95% ethanol (Merck Co.) with a further addi-tion of 100 mL 85% H3PO4 (Merck Co.). The solutionwas diluted with ultra-pure water to 1.0 L. Five millil-itre of the reactive was used for each 0.1 mL sample.Absorbance was measured at 595 nm, 5 min after thestart of the chemical reaction at room temperature.

Calibration curves were prepared using bovineserum albumin (BSA) (Sigma Co.) and casein (SigmaCo.) at maximum concentrations of 100 µg mL−1

(Lowry method) and 100 µg 0.1 mL−1 (Bradfordmethod). Casein was diluted in ultra-pure water plussome drops of 0.1 N NaOH. All measurements weredone using a Shimadzu, model UV Mini 1240 spec-trophotometer.

In addition to colorimetric assays of protein, crudeprotein for each species was also calculated using spe-cific nitrogen-to-protein conversion factors proposedby Lourenco et al. (2002, 2004) as follows: A. carterae(5.13), A. uruguayense (3.94), C. decorticatum (5.34),C. fastigiata (4.52), C. minima (5.70), D. menstrualis(4.55), D. tertiolecta (4.39), Hillea sp. (4.93), I. gal-bana (5.07), P. acanthophora var. acantophora (4.47),P. capillacea (4.78), P. gymnospora (5.72), S. costatum(4.53), S. vulgare (5.53), and U. fasciata (5.59).

Statistical analysis

The results were analysed by one-way analysis of vari-ance (ANOVA) with significance level α = 0.05 (Zar,1996) followed, where applicable, with Tukey’s mul-tiple comparison test. In some cases, Student’s t-testwas used instead of ANOVA when comparing only twotreatments for each variable.

Results

Tests of protein extraction

The use of the Potter homogeniser produced remark-able differences in the extraction of protein for the threespecies tested. In all cases, significantly (p < 0.001)higher concentrations of protein was obtained in thetreatment with the use of the Potter homogeniser (Fig-ures 1A–C). For Sargassum vulgare (Figure 1B) dif-ferences in values obtained for samples extracted withand without the Potter homogeniser were about 50%.Higher values of protein were also obtained for samplesof Porphyra acanthophora var. acanthophora and Ulvafasciata when extracted with the Potter homogeniser.

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Figure 1. Quantification of protein in three species of marinemacroalgae (A) Porphyra acanthophora var. acanthophora, (B) Sar-gassum vulgare and (C) Ulva fasciata by the Lowry (Lwy) and Brad-ford (Bdf) methods, using bovine serum albumin (BSA) and casein(CAS) as protein standards in the calibration curves. Three vari-ables evaluated: (i) the water volume used to incubate samples (1and 4 mL); (ii) the length of the incubation period for the extractionof protein (6 and 12 h); and (iii) the use of a Potter homogenizerfor grinding samples. All assays included the precipitation of pro-tein with 25% TCA, in a proportion of 2.5:1 (TCA:homogenate).Mean ± S.D. (n = 6).

Higher concentrations of protein were obtainedwhen 4.0 mL of water was used for U. fasciata (p <

0.001) (Figure 1C), and a longer period of incubation(12 h) for S. vulgare (p = 0.002) (Figure 1B). Therewere no differences for P. acanthophora var. acan-thophora (Figure 1A) incubated in wither 1.0 or 4.0 mLfor 6 h (p = 0.52).

The precipitation of protein

No differences in the precipitation of protein were ob-served for the two TCA:homogenate ratios using theLowry (0.12 ≤ p ≤ 0.70) and Bradford (0.14 ≤ p ≤0.97) methods.

The quantification of protein in the tests

The results show large differences for all the threespecies between the two protein quantification meth-ods; values obtained with the Lowry method were al-ways higher in all comparisons evaluated (p < 0.01)(Figures 1A–C).

The use of BSA in calibration curves seems to gen-erate higher values in the samples of P. acanthophoravar. acanthophora (p < 0.02). However for U. fasci-ata, using the Lowry method (p = 0.56) and S. vulgarewith the Bradford method (p = 0.06), no differenceswere found when comparing results with BSA or caseinas calibration standards.

In addition to the general analyses described above,some tests on samples spiked with protein were per-formed to assess any possible effects of endogenousalgal proteases in the samples which could partially de-stroy protein during incubation (Perez-Llorenz et al.,2003). Tests were carried out for all the three algae atsame dilutions used to extract and quantify algal pro-tein. Controlled amounts of BSA were used as an inter-nal standard: 2.5 mg of BSA were added to each flask inwhich the algal material was incubated, such as in pro-cedure IV. Controls included incubation of BSA withno algae, following the same steps described for algalsamples and direct dilution and measurement of BSA,without incubation. The results (data not presented)showed no loss of protein after the incubation periodin all experiments carried out (p ≥ 0.083), indicatingno activity of algal proteases at 4 ◦C, the incubationtemperature used.

Protocol for extraction and precipitation of protein

Results suggest the use of a general protocol involvingextraction of protein from algal samples using 4.0 mLof ultra-pure water, for 12 h and grinding of sampleswith a Potter homogeniser. Samples should be precipi-tated with TCA:homogenate (2.5:1 v/v). A diagram ofthe protocol is shown in Figure 2. This protocol wasused for protein determination of the other algal speciesusing both the Bradford and Lowry methods, using

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Figure 2. Diagrammatic representation of the protocol for extraction and precipitation of algal protein developed in this study.

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BSA and casein as protein standards in the calibrationcurves.

Amino acid profile of the algae and protein standards

The comparison of the amino acid profile of the algalspecies (Table 2) shows great differences for asparticacid, glutamic acid and arginine. On the other hand,some amino acids, such as glycine and leucine, hadsimilar values for the 15 species studied.

The comparison of the algal amino acid profile withthe protein standards show that the concentration ofglutamic acid in casein, and lysine in BSA, are higherthan those reported for all the algae. Both protein stan-dards were lower in methionine compared to the algae.Casein and BSA also show remarkable differences witheach other regarding some amino acids, such as alanine(higher concentration in BSA) and proline (higher con-centration in casein).

Quantification of protein

The highest percent of protein was measured in the redalgae A. uruguayense (15.6 ± 0.3% of the dry matter),followed by the cryptomonad Hillea sp. (15.3 ± 0.6%)(Table 3). The other microalgae had similar proteincontents, varying from 11.4 ± 1.0%. (D. tertiolecta)to 10.1 ± 0.8% (I. galbana). The edible red algae P.acanthophora var. acanthophora had 8.9 ± 0.7% ofprotein and P. capillacea had the lowest protein contentamong the red algae (4.2 ± 0.3%). In the brown algae,small variations in protein content were found (8.7,7.8 and 6.9% for P. gymnospora, C. minima and S.vulgare, respectively), except for D. menstrualis, whichhad a lower protein content (4.0 ± 0.2%). The greenmacroalgae had protein contents varying from 5.5%(C. fastigiata) to 7.3% (U. fasciata) (Table 3).

Confirming the same trend obtained with the ini-tial results for the three macroalgae (P. acanthophoravar. acanthophora, S. vulgare and U. fasciata), all otherspecies gave significantly higher values of protein whenquantified using the Lowry method compared to theBradford method. In some cases (e.g. C. fastigiata), dif-ferences between mean values were higher than 50%(Table 3). For the microalgae, the variation betweenthe two methods was less, varying from 1.5 (I. gal-bana, BSA as protein standard) to 1.9 (A. carterae,BSA and casein as protein standard). For the macroal-gae, the Lowry:Bradford ratio varied from 1.5 (D. men-strualis, BSA and casein as protein standard) to 3.2 (C.

fastigiata, casein as protein standard). For all species,values obtained with BSA as the protein standard werehigher than those obtained with casein (Table 3). Crudeextracts analysed using the Lowry method always gavehigher values than those obtained with precipitatedsamples (p < 0.01), except for A. uruguayense andHillea sp. (Table 3).

The 15 algae species showed great differences re-garding total nitrogen and crude protein (Table 3). Mi-croalgae had smaller variations in the total N, vary-ing from 3.35% (I. galbana) to 4.69% (A. carterae).Variations in the total N were greater in the macroal-gae, ranging from 1.94% (C. minima) to 5.68% (A.uruguayense). The calculations of crude protein gavewide variations among species, varying from 10.52%in C. decorticatum to 25.04% in Hillea sp. (Table 3).The sum of amino acid residues varied from 9.99% (C.minima) to 20.11% (Hillea sp.). Microalgae tended tohave higher percentages of amino acid residues thanmacroalgae.

Discussion

Extraction and precipitation of algal protein

Protein content of the three main algae species tested inthis study varied greatly depending on the different ex-traction procedures tested. The efficiency of extractionseems to be influenced directly by two main factors:the chemical composition of the species and its mor-phological and structural characteristics. The chemi-cal composition of the three species is very distinct(Lourenco et al., 2002) and, in theory, this may leadto differences in the protein content. S. vulgare is abranched algae and possesses a hard and leathery thal-lus, while P. acanthophora var. acanthophora and U.fasciata have flattened soft thalli.

In the present study, the effects of lyophylisation onthe thalli should be also considered since freeze-driedsamples tend to be more difficult to extract, especiallyleathery species such as C. minima and S. vulgare. Thismeans that better preservation by freeze-drying makesthem more difficult for further protein extraction. Thisproblem can be solved by grinding samples with Potterhomogeniser.

For P. acanthophora var. acanthophora lyophilisa-tion, the main factor influencing the yield in the pro-tein extraction was the use of grinding. This is prob-ably related to the thallus form of this species. Ulvafasciata has the same kind of thallus, but the use of

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Table 2. Amino acid profile of 15 species of marine algae and two standards of pure protein: bovine serum albumin (BSA) and caseina

Protein standards Chlorophyta Cryptophyta Dinophyta

Amino acid BSA Casein C. fastigiata C. decorticatum D. tertiolecta U. fasciata Hillea sp. A. carterae

Cisteic acid 4.5 ± 0.4 0.9 ± 0.5 2.8 ± 0.3 1.1 ± 0.3 0.7 ± 0.2 0.5 ± 0.1 0.8 ± 0.2 0.2 ± 0.0

Aspartic acid 9.6 ± 0.2 6.4 ± 0.3 8.8 ± 0.8 10.7 ± 0.1 12.3 ± 0.5 13.4 ± 1.1 13.1 ± 0.2 9.1 ± 0.2

Treonine 4.8 ± 0.2 3.7 ± 0.2 4.7 ± 0.5 6.0 ± 0.1 4.6 ± 0.2 5.2 ± 0.2 4.6± 0.0 5.1 ± 0.1

Serine 3.8 ± 0.4 4.8 ± 0.3 6.0 ± 0.6 5.0 ± 0.2 3.6 ± 0.1 5.9 ± 0.7 3.8 ± 0.0 5.5 ± 0.3

Glutamic acid 16.7 ± 0.3 21.1 ± 0.8 10.4 ± 1.1 12.0 ± 0.9 12.8 ± 0.4 12.9 ±0.4 12.1 ± 0.0 13.6 ± 0.3

Proline 4.7 ± 0.3 11.3 ± 0.6 5.1 ± 0.4 4.8 ± 0.7 4.9 ± 0.2 4.7 ± 0.1 3.5± 0.0 4.2 ± 0.3

Glycine 1.7 ± 0.3 1.6 ± 0.2 6.9 ± 0.8 7.2 ± 0.5 5.8 ± 0.1 6.7 ± 0.2 7.2± 0.5 5.1 ± 0.2

Alanine 5.4 ± 0.2 2.6 ± 0.3 6.0 ± 0.6 8.8 ± 0.6 7.1 ± 0.2 8.7 ± 1.1 6.9± 0.0 7.3 ± 0.2

Valine 5.7 ± 0.1 6.0 ± 0.2 6.0 ± 0.5 6.2 ± 0.0 5.7 ± 0.8 5.9 ± 0.6 5.9 ± 0.0 6.2 ± 0.1

Methionine N.D. 0.5 ± 0.0 1.0 ± 0.2 0.7 ± 0.5 2.8 ± 0.3 0.9 ± 0.1 2.8 ±0.0 1.9 ± 0.2

Isoleucine 2.4 ± 0.1 4.7 ± 0.2 3.9 ± 0.4 3.8 ± 0.6 4.3 ± 0.1 4.0 ± 0.7 4.9± 0.1 4.0 ± 0.1

Leucine 10.9 ± 0.1 8.8 ± 0.3 8.5 ± 0.9 8.4 ± 1.1 8.3 ± 0.1 7.9 ± 0.7 7.9± 0.3 8.4 ± 0.1

Tyrosine 3.8 ± 0.1 4.1 ± 0.1 3.8 ± 0.4 2.1 ± 0.3 3.2 ± 0.1 3.3 ± 0.7 5.5± 0.2 3.8 ± 0.3

Phenylalanine 5.8 ± 0.1 4.8 ± 0.2 6.4 ± 0.6 5.0 ± 0.7 5.6 ± 0.2 5.3 ± 0.1 5.6 ± 0.4 5.4 ± 0.1

Histidine 3.5 ± 0.6 4.0 ± 0.8 2.2 ± 0.7 3.3 ± 0.2 2.1 ± 1.2 2.5 ± 0.5 1.9± 0.1 3.0 ± 0.4

Lisine 11.4 ± 0.1 7.4 ± 0.2 6.9 ± 0.6 6.3 ± 0.1 5.5 ± 0.3 5.2 ± 0.4 5.3± 0.0 7.1 ± 0.3

Arginine 5.1 ± 0.5 3.7 ± 0.7 6.4 ± 0.9 5.0 ± 0.4 5.6 ± 0.6 5.7 ± 0.9 4.0± 0.3 6.5 ± 0.2

Ammonia 1.0 ± 0.2 1.5 ± 0.2 1.0 ± 0.2 1.6 ± 0.1 2.5 ± 0.1 1.8 ± 0.1 1.8± 0.0 0.6 ± 0.0

Total 99.8 ± 3.0 96.5 ± 6.7 94.8 ± 5.5 96.4 ± 4.7 94.9 ± 5.5 98.7 ± 2.3 95.2 ± 2.5 98.8 ± 3.6

Heterokontophyta Prymnesiophyta Rhodophyta

Amino acid C. minima D. menstrualis P. gymnospora S. costatum S. vulgare I. galbana A. uruguayense P. acanthophora P. capillacea

Cisteic acid 0.5 ± 0.1 0.5 ± 0.0 0.9 ± 0.3 0.3 ± 0.0 0.6 ± 0.2 0.6 ± 0.0 0.9 ± 0.3 1.2 ± 0.3 0.7 ± 0.1

Aspartic acid 12.0 ± 0.8 14.5 ± 0.7 12.8 ± 2.5 13.4 ± 0.1 10.6 ± 1.3 12.6 ±0.7 13.2 ± 1.8 12.5 ± 2.1 11.6 ± 2.8

Treonine 5.1 ± 0.3 5.0 ± 0.0 5.1 ± 0.6 5.2 ± 0.1 4.4 ± 0.7 5.1 ± 0.4 5.4± 0.6 5.8 ± 0.3 5.2 ± 0.9

Serine 6.0 ± 0.5 6.8 ± 0.4 5.0 ± 0.6 4.7 ± 0.1 4.7 ± 0.7 4.1 ± 0.4 5.2 ± 0.2 5.3 ± 0.4 5.7 ± 1.4

Glutamic acid 14.8 ± 1.4 12.6 ± 0.4 13.1 ± 1.4 13.5 ± 0.0 17.4 ± 0.4 12.1 ±0.2 14.9 ± 1.5 12.9 ± 4.3 14.7 ± 0.2

Proline 4.3 ± 0.4 4.8 ± 0.1 4.3 ± 0.7 3.7 ± 0.0 4.2 ± 0.7 4.1 ± 0.5 4.9± 0.4 4.6 ± 0.1 4.9 ± 0.6

Glycine 6.0 ± 0.4 6.0 ± 0.0 6.0 ± 0.9 6.2 ± 0.1 5.3 ± 0.9 5.8 ± 0.2 6.5± 0.1 7.1 ± 1.4 6.0 ± 0.9

Alanine 7.9 ± 0.8 6.6 ± 0.2 6.9 ± 0.5 6.7 ± 0.1 6.8 ± 1.1 7.4 ± 0.3 7.5± 0.7 8.8 ± 1.2 7.2 ± 1.4

Valine 5.7 ± 0.4 5.2 ± 0.1 5.3 ± 0.6 5.9 ± 0.0 5.4 ± 0.9 6.4 ± 0.2 6.0 ± 0.7 6.4 ± 0.2 5.5 ± 1.8

Methionine 2.0 ± 0.3 1.3 ± 0.2 1.0 ± 0.4 2.6 ± 0.1 1.7 ± 0.3 2.6 ± 0.1 0.7± 0.3 1.1 ± 0.1 1.1 ± 0.1

Isoleucine 3.9 ± 0.4 4.3 ± 0.0 4.3 ± 0.3 5.7 ± 0.0 4.3 ± 0.8 5.1 ± 0.2 4.7± 0.2 4.1 ± 0.8 3.7 ± 0.6

Leucine 7.9 ± 0.6 8.6 ± 0.1 8.5 ± 1.1 8.3 ± 0.1 8.2 ± 1.4 9.3 ± 0.3 8.2± 1.2 8.1 ± 0.9 6.8 ± 1.3

Tyrosine 1.8 ± 0.3 2.6 ± 0.1 2.1 ± 0.4 3.2 ± 0.1 1.8 ± 0.2 3.4 ± 0.2 2.4± 0.3 2.4 ± 0.4 3.7 ± 0.4

Phenylalanine 4.9 ± 0.3 5.5 ± 0.1 5.2 ± 0.7 6.1 ± 0.1 4.9 ± 0.8 5.9 ± 0.1 5.2 ± 0.6 4.7 ± 1.0 5.3 ± 0.9

Histidine 2.0 ± 0.2 2.2 ± 0.1 2.1 ± 0.5 1.6 ± 0.1 1.6 ± 0.3 2.0 ± 0.2 2.4± 0.5 3.0 ± 0.6 3.5 ± 0.5

Lisine 5.0 ± 0.4 4.6 ± 0.2 5.4 ± 0.8 4.6 ± 1.2 5.0 ± 0.9 5.4 ± 0.4 6.2 ± 0.9 6.3 ± 1.4 7.9 ± 0.8

Arginine 4.2 ± 0.3 5.1 ± 0.2 5.0 ± 0.6 4.1 ± 0.0 3.9 ± 0.6 5.8 ± 0.9 4.7± 0.2 4.8 ± 0.1 5.6 ± 0.6

Ammonia 1.4 ± 0.2 1.2 ± 0.1 1.5 ± 0.2 2.4 ± 0.0 1.3 ± 0.1 1.9 ± 0.1 1.8± 0.3 1.9 ± 0.1 1.7 ± 0.56

Total 95.2 ± 4.7 98.0 ± 2.6 94.5 ± 6.4 95.8 ± 2.3 94.0 ± 4.6 98.3 ± 3.2 96.6 ± 4.6 99.2 ± 6.1 99.0 ± 5.3

aResults are expressed as percentage of amino acid per 100 g of algal protein (or pure protein for the two standards) and represent the realrecovery of amino acids after analysis. Concentrations of ammonia correspond to nitrogen recovery from some amino acids destroyed duringacid hydrolysis. Values indicate the mean of three replicates ± S.D. (n(3). N.D.: not detected.

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Table 3. Total nitrogen, total amino acid residues and protein content of marine algae, as percentage of the dry mattera

Lowry precipitation Bradford precipitation Lowry row extract,with TCA 2.5:1 with TCA 2.5:1 no precipitation

Total Total amino CrudeSpecies nitrogen BSA Casein BSA Casein BSA Casein acid residues protein

Chlorophyta

C. fastigiata 4.32 ± 0.36 5.47 ± 0.34 5.29 ± 0.33 1.74 ± 0.08 1.63 ± 0.07 7.52 ± 0.58 7.27 ± 0.56 13.50 ± 2.31 19.53

C. decorticatum 2.13 ± 0.10 7.12 ± 0.53 6.88 ± 0.51 4.49 ± 0.37 4.24 ±0.35 7.55 ± 0.08 7.30 ± 0.08 10.93 ± 1.06 11.37

D. tertiolecta 4.18 ± 0.09 11.4 ± 0.99 11.0 ± 0.95 6.86 ± 0.26 6.48 ±0.25 11.0 ± 0.13 10.6 ± 0.12 17.14 ± 0.70 18.35

U. fasciata 2.29 ± 0.02 7.30 ± 0.84 7.05 ± 0.81 2.60 ± 0.19 2.43 ± 0.23 7.55 ± 0.10 7.37 ± 0.09 11.03 ± 0.98 12.80

Cryptophyta

Hillea sp. 5.08 ± 0.26 15.3 ± 0.60 14.8 ± 0.58 8.54 ± 0.35 8.07 ± 0.33 13.1 ± 0.51 12.7 ± 0.49 20.11 ± 2.22 25.04

Dinophyta

A. carterae 4.69 ± 0.05 10.2 ± 0.09 9.85 ± 0.09 5.49 ± 0.26 5.18 ± 0.25 10.8 ± 0.12 10.5 ± 0.12 15.84 ± 1.72 24.06

Heterokontophyta

C. minima 1.94 ± 0.10 7.83 ± 0.33 7.57 ± 0.32 3.56 ± 0.23 3.36 ± 0.22 10.0 ± 0.20 9.67 ± 0.20 9.99 ± 0.80 11.06

D. menstrualis 3.26 ± 0.15 4.04 ± 0.17 3.90 ± 0.16 2.72 ± 0.21 2.56 ±0.20 7.01 ± 0.13 6.77 ± 0.13 10.35 ± 0.31 14.83

P. gymnospora 2.41 ± 0.14 8.69 ± 0.72 8.40 ± 0.70 4.79 ± 0.45 4.53 ± 0.43 11.9 ± 0.50 11.5 ± 0.48 12.55 ± 1.55 13.78

S. costatum 3.41 ± 0.22 11.1 ± 0.68 10.7 ± 0.66 6.43 ± 0.39 6.07 ± 0.37 11.5 ± 0.75 11.1 ± 0.72 14.30 ± 1.76 15.40

S. vulgare 2.08 ± 0.14 6.91 ± 0.15 6.68 ± 0.14 3.19 ± 0.19 3.00 ± 0.18 8.77 ± 0.12 8.47 ± 0.12 11.0 ± 1.54 11.50

Prymnesiophyta

I. galbana 3.35 ± 0.20 10.1 ± 0.85 9.75 ± 0.82 6.58 ± 0.49 6.22 ± 0.46 11.1 ± 0.26 10.7 ± 0.25 15.83 ± 0.09 16.98

Rhodophyta

A. uruguayense 5.68 ± 0.03 15.7 ± 0.33 15.1 ± 0.32 10.2 ± 0.32 9.48 ±0.30 12.2 ± 0.22 11.7 ± 0.21 17.22 ± 1.88 22.38

P. acanthophora 3.68 ± 0.04 4.19 ± 0.29 4.05 ± 0.28 2.60 ± 0.13 2.45 ±0.12 6.26 ± 0.18 6.05 ± 0.17 11.83 ± 1.58 16.45

P. capillacea 3.24 ± 0.10 8.94 ± 0.53 7.98 ± 0.49 4.62 ± 0.18 4.23 ± 0.19 11.7 ± 0.38 10.9 ± 0.35 12.11 ± 3.00 15.49

aProtein was determined by different methods, with BSA and casein as protein standards. Analysis by Lowry’s method was also made withnotprecipitated samples. Data represent the mean of six replicates ± S.D. (n(6), except for total nitrogen and amino acid residues (n(3).

a greater volume of water seems to improve the ex-traction of protein. Differences in the behaviour ofthe two flattened algae may be related to the chemi-cal composition and thallus morphology, since U. fas-ciata has two layers of cells compared to P. acan-thophora var. acanthophora with only one layer ofcells.

For the extraction of protein from the branched andhard thallus of the brown algae S. vulgare, 12 h in-cubation in water seems to be important. Even us-ing a greater volume of water (4 mL), an incubationperiod of 6 h was not enough to soften the thalli.Fragments of S. vulgare thallus were ground more eas-ily with the Potter homogeniser after 12 h of exposure towater.

As the precipitation of protein with TCA: ho-mogenate (3.0:1 and 2.5:1 v/v) gave no significantlydifferent results and therefore a TCA:homogenateratio of 2.5:1 (v/v) is recommended in order to savereagent.

Amino acid profile, non-protein nitrogen and proteincontent of algae

In this study we assume that the actual concentra-tions of protein in the samples are calculated fromthe sum of amino acid residues (Tables 1 and 2), awidely accepted procedure since the 1970s (Heidel-baugh et al., 1975). After acid hydrolysis, all proteinsare destroyed, even those associated with other macro-molecules and biological membranes. The values forthe total amino acid residues were calculated by sum-ming up the amino acid masses retrieved after acid hy-drolysis (total amino acid), less the water mass (18 g in1 M of each amino acid) incorporated into each aminoacid after disruption of the peptide bonds. Total aminoacid analysis involves some errors, such as the total(tryptophan) or partial (methionine and cysteine) de-struction of some amino acids, as well as the impossi-bility of identifying the contribution of free amino acidsin the samples. However, it indicates the maximum

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possible concentration of protein in the sample con-sidering that all amino acid are in protein, providinga good reference point for the protein concentrationsmeasured by the Bradford and Lowry assays. In ourresults, values for amino acid residues were similar tothe estimated crude protein, suggesting the suitabilityof the nitrogen-to-protein conversion factors calculatedby Lourenco et al. (2002, 2004). Exceptions to this areC. fastigiata and A. carterae (Table 3), which showedvalues for crude protein ca. 30% higher than those forthe sum of amino acid residues. This difference prob-ably results from the presence of high concentrationsof non-protein nitrogen, presumably transient stocksof inorganic nitrogen (Lavın & Lourenco, unpublisheddata).

The sum of amino acid residues indicates that mi-croalgae, independent of the taxonomic group, tendto accumulate higher concentrations of protein thanmacroalgae. This fact may be related to the high con-centration of nitrogen in the culture medium (as well asother dissolved nutrients), growth conditions in the lab-oratory and the higher surface area:volume ratios foundin microalgae (Hein et al., 1995). Dried samples of mi-croalgae are better exposed to solvents and to grind-ing during the extraction procedures, while macroalgalsamples have to be powdered before the start of theextraction. This factor may produce a more efficientprotein extraction.

For many macroalgae, the combined concentrationof glutamic acid and aspartic acid represents 40% oftotal amino acids, agreeing with data obtained for theedible red algae Palmaria palmata, in which glu andasp represent 39.6% of total amino acids (Galland-Irmouli et al., 1999). For microalgae, the sum of aspand glu represent mean values of about 20% of the totalamino acids for Skeletonema costatum, Dunaliella ter-tiolecta and Thalassiosira pseudonana (Brown, 1991).For Ulva rigida and U. rotundata, percentages of thesetwo amino acid may represent from 26 to 32% ofthe total amino acids (Fleurence et al., 1995). In thepresent study, values for asp + glu varied from 19.2%(C. fastigiata) to 28.1% (A. uruguayense) (Table 2).For microalgae, the fraction represented by these twoamino acids varied from 23.1% (A. carterae) to 26.9%(S. costatum) of the total amino acids (Table 2). Theset composed of the essential amino acids in samplesvaried from 36.3% (S. vulgare) to 44.0% (C. fasti-giata), with a mean value of 40.2% of the total aminoacids. Concerning nutritional properties, these speciesshow concentrations of essential amino acids compa-rable to those commonly described to soybean pro-

tein, which possesses 36.0% of the total amino acid(Galland-Irmouli et al., 1999).

High concentrations of non-protein N may resultin overestimation of protein (Zamer et al., 1989). Ac-cording to Lourenco et al. (2004), concentrations ofnon-protein N vary widely during growth in culturesof microalgae, commonly fluctuating from 15 to 30%of the total N. In the present study, the occurrenceof high concentrations of the total N was not mir-rored by high total amino acid concentrations in somespecies such as A. carterae, A. uruguayense, C. fasti-giata, and Hillea sp., which is explained by the pres-ence of large amounts on non-protein N. The deter-mination of crude protein should be based on the useof specific nitrogen-to-protein conversion factors asproposed by Lourenco et al. (2002, 2004). In addi-tion, results obtained for the total amino acid residuesand precipitated and non-precipitated extracts with theLowry method suggest the influence of variable con-centrations of free amino acids and small peptides inthe samples. This finding indicates that precipitationof the samples is a fundamental step during proteinanalysis.

Data of protein content in macroalgae from the trop-ical and subtropical coastal environments frequentlyshow lower concentrations (Kaehler & Kennish, 1996;Wong & Cheung, 2000). In some Brazilian environ-ments Ramos et al. (2000) found that the percentageof protein (N × 6.25) in 14 seaweeds varied from 2.30to 25.6% of dry weight. Despite the overestimation ofprotein content caused by the use of the factor 6.25(Lourenco et al., 2002), values obtained by Ramoset al. (2000) indicated predominantly low concentra-tions of protein. This trend may be related to the nat-ural characteristics of Brazilian marine environments;predominantly oligotrophic, with low availability of N(Oliveira et al., 1997; Ovalle et al., 1999). As a conse-quence, low concentrations of protein would be accu-mulated by natural populations of macroalgae. In thiscontext, our data of protein concentration in macroal-gae are in accordance with the information availablein the literature (e.g. Wong & Cheung, 2001; McDer-mid & Stuercke, 2003). On the other hand, the relativelow content of protein in microalgae results from thephysiological state of the species. All microalgae weresampled in stationary growth phase when percentagesof protein in cells decreased due to depletion of dis-solved nutrients in the culture medium (Lourenco et al.,1998).

Low protein levels were found in Pterocladiellacapillacea using both the Lowry and Bradford

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methods. The values obtained are equivalent to 1/3of those determined from the sum of the aminoacid residues, being the lowest protein concentrationsamong all red macroalgae tested. This seems to indicateinefficient extraction using the procedures developed inthis study for this macroalgae. We hypothesise that theextraction of protein in this species might be influencedby the presence of phycocolloids, especially agarans.P. capillacea is a good source of agar (McHugh, 1991)and is probably the most abundant macromolecule inthis alga. During the water extraction step, it was possi-ble to extract variable quantities of agar visible, as theoccurrence of gels in several steps of protein extrac-tion, mainly when using the Potter homogeniser andafter the centrifugation at 4 ◦C of the ground samples.It is possible that the gels can trap part of the proteinextracted, giving low values in the spectrophotometricdetermination of protein. This kind of analytical prob-lem may be present in other agarophytes as well ascarragheenan-producing species. The presence of largeamounts of anionic polysaccharide in the cell walls re-duces protein solubility during extraction (Fleurence,1999). Further studies are needed to develop betterprocedures for protein extraction in phycocolloid-richspecies.

Lowry × Bradford methods and the influence of theamino acid profile of samples and standards

Some authors suggest that Bradford’s method wouldgenerate lower protein values for a large number of or-ganisms compared to Lowry’s method. Calculating theLowry:Bradford ratio from data by Eze & Dumbroff(1982) for leaves of bean plants gives a ratio of 1.4. Forthe diatom T. pseudonana, Clayton et al. (1988) foundratios varying from 1.8 to 2.0, while Berges et al. (1993)determined a ratio of 1.2 for the same microalgae. Thepresent results confirm the general trends found bythose authors, but we found higher Lowry:Bradfordratios for most of the species. Our results varied from1.5 (I. galbana, BSA as the protein standard) to 3.2 (C.fastigiata, casein as the protein standard).

The trend of obtaining lower concentrations ofprotein using Bradford’s method may be related to thebinding of the dye Coomassie Brilliant Blue-G250 toboth basic and aromatic amino acid residues (Compton& Jones, 1985). Most of the algae show relativelylow concentrations of the two amino acids (tyrosineand tryptophan) as well as the two basic amino acids(lysine and histidine). Thus, the binding of the dye

with protein occurs mainly with the two amino acids,arginine and phenylalanine, and this fact seems tocontribute to lower protein measurements. Our resultswith Bradford’s method agree with Kaehler andKennish (1996). These authors found predominantlylow values for some seaweeds (from 1.3 to 12.6%)from Hong Kong using the Bradford method. Incontrast, the Folin–Ciocalteu reagent used in theLowry assay interacts with all peptide bonds and alsowith some amino acids. As a result, the quantificationof protein tends to be greater.

The differences in amino acid composition amongprotein standards and algae have important implica-tions regarding protein reactivity and quantification inalgal samples. Despite the good linearity obtained withboth protein standards (BSA and casein), our data sug-gest that casein has a slightly smaller reactivity thanBSA resulting in a smaller quantification of protein.The two protein standards have extremely differentamino acid composition and the reactivity of them ineach method tends to be different due the functionalgroups that they present (Morrison & Boyd, 2003).Functional groups change the charge and the geom-etry of neighbouring atoms, affecting the reactivity ofthe whole molecule (Morrison & Boyd, 2003).

Conclusions

The use of 4.0 mL of water to incubate algal samples for12 h, combined with grinding of the samples with a Pot-ter homogeniser is strongly recommended. This proce-dure results in better extraction of protein from algalsamples of different species independent of their mor-phological and biochemical characteristics. As a conse-quence, this procedure can be applied widely to manyalgal species. The precipitation of protein should bedone with 25% trichloroacetic acid in the ratio of 2.5:1(TCA:homogenate). Results generated with Lowry’smethod are more similar to the data obtained from thesum of the amino acid residues which is consideredthe most reliable way of determining the actual pro-tein content. Protein values obtained with BSA as aprotein standard were closer to those calculated fromthe sum of amino acid residues, suggesting that theuse of BSA is more suitable for the Lowry method.The procedures proposed here can contribute to betterresults, since protein is extracted efficiently and po-tential interference from compounds such as pigments,lipids, phenolics, small peptides and free amino acids iseliminated.

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Acknowledgments

We are indebted to FAPERJ (Foundation for Re-search Support of Rio de Janeiro State, grant E-26.170.041/98) for the financial support to this study.Special acknowledgements are due to Dr. YocieYoneshigue-Valentin (Universidade Federal do Rio deJaneiro) and Dr. Carlos Logullo de Oliveira (Uni-versidade Estadual do Norte Fluminense) for offer-ing us laboratory facilities to perform this study. Wethank Dr Ursula M. Lanfer Marquez (Universidade deSao Paulo) for her support in the amino acid analy-sis. E.B. acknowledges CAPES and S.O.L. acknowl-edges FAPERJ and CNPq for providing them researchfellowships.

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