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 Barbarino1,2 & Sergio O. Lourenço2,
1
Programa de Pós-Graduação 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, Niterói, 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 and inorganic nitrogen (nitrate, nitrite and ammonia)

(Lourenço et al., 1998; Conklin-Brittain et al., 1999;
Determination of protein content of algae can provide Fujihara et al., 2001) whose presence makes the factor
important information on the chemical characteristics 6.25 unsuitable since it overestimates the actual pro-
of algal biomass. The methods most commonly used tein content (Ezeagu et al., 2002). Specific nitrogen-
to quantify protein are: (i) the alkaline copper method to-protein conversion factors were recently proposed
(Lowry et al., 1951); (ii) the Coomassie Brilliant Blue for 12 marine microalgae (Lourenço et al., 2004) and
dye method (Bradford, 1976); or (iii) determination of 19 seaweeds (Lourenço et al., 2002), varying from 3.75
crude protein (N × 6.25). for Cryptonemia seminervis a red alga, to 5.72 for Pad-
The calculation of protein content by N × 6.25 re- ina gymnospora a brown alga.
quires some caution, not always considered by authors The determination of protein by the Lowry and
using this method. Plant materials, fungi and algae Bradford methods is carried out by spectrophotome-
commonly have high concentrations of non-protein ni- try. The Lowry method detects protein by a reaction
trogenaceous substances such as pigments (chlorophyll catalyzed by copper, a component of the Folin phe-
and phycoerythrin), nucleic acids, free amino acids nol reactions. The chemical reaction detects peptide
448

bonds and is also sensitive to some amino acids such trations of TCA between 0.18 and 0.34 M can be used
as tyrosine and tryptophan (Legler et al., 1985). In the to seperate protein from the other extract components,
Bradford method, the Coomassie Brilliant Blue dye because only protein is precipitated (Clayton et al.,
is bound to protein mainly by arginine residues and 1988). The physical separation among protein, small
to a lower degree by histidine, lysine, tyrosine, tryp- peptides and free amino acids is especially important,
tophan and phenylalanine residues. The binding be- since the analytical methods are sensitive to the last two
tween the dye and amino acids is attributed to van classes of substances. Thus, protein precipitation with
der Waals forces and hydrophobic interactions (Comp- TCA is strongly recommended to avoid the quantifica-
ton & Jones, 1985). As a consequence, the reactiv- tion of small peptides and free amino acids (Nguyen
ity of both methods in comparison to a specific pro- & Harvey, 1994) as well as interference by other
tein is strongly influenced by its amino acid compo- substances.
sition, since not all amino acids can oxidate equally As many variables are simultaneously involved with
the Folin phenol reactive or bind to the Coomassie dye protein analysis, the influence of specific factors may
(Stoscheck, 1990). The differences in the principles be neglected by authors, affecting the accuracy of pro-
of the methods contribute to making comparison of tein analysis. However, studies focussing on protein
results available in the literature even more difficult, analysis in algae are relatively uncommon and exper-
since the choice of the method to be used is an arbitrary imental data are needed to fill this gap. It is also very
decision. important to develop a simple and inexpensive pro-
Bovine serum albumin (BSA) is the most used pro- tocol, using low-cost equipment and consumables, in
tein standard for calibration curves in spectrophotome- order to make it accessible to everyone interested in
try, but many other proteins can be used. Several studies data on algal protein: researchers, algae producers, peo-
suggest that the Lowry and Bradford analyses produce ple interested on the nutritional value of algae and so
different measurements of protein when using BSA as on.
the protein standard for samples such as the gut fluid In this study, different procedures for extraction and
of fish (Crossman et al., 2000), marine invertebrates quantification of the protein content of marine algae
(Zamer et al., 1989), higher plants (Eze & Dumbroff, were evaluated. The specific aims of this study were: (i)
1982) and marine phytoplankton (Clayton et al., 1988). to create a protocol for the extraction and quantification
To obtain a more reliable measurement of protein, it of protein of marine algae; (ii) to compare the use of two
would be useful to identify the predominant proteins methods (Lowry and Bradford) for the determination
in the cells (Berges et al., 1993). However, this rec- of protein in marine algae; (iii) to evaluate the effects
ommendation has no practical value, considering the of the time of extraction, the use of grinding and the
difficulty of extracting, purifying and characterising precipitation of samples with TCA on the quantity of
the main proteins present in the cells and subsequently protein extracted; and (iv) to compare the amino acid
using them as protein standards. Nguyen & Harvey profile of the algal samples and the protein standards
(1994) suggested the use of ribulose-1,5-diphosphate (BSA and casein) used.
carboxylase (RuDPCase) for calibration curves to anal-
yse samples of photosynthetic organisms, since RuD-
PCase corresponds to about 15% of the total protein in Materials and methods
chloroplasts.
Several substances may interfere with both the Fifteen species of marine algae covering a wide taxo-
Lowry and Bradford method, such as phenol and phe- nomical range were analysed. The field-collected ma-
nolases (Mattoo et al., 1987), glucosamine and de- rine were identified following the checklist of Wynne
tergents (Peterson, 1979) and flavonoids (Compton & (1998). Marine microalgae were cultured in the labora-
Jones, 1985) among many others (see the comprehen- tory. The classification below is based on Lee (1999):
sive studies of Peterson, 1979; Stoscheck, 1990 on in- Chlorophyta
terfering substances). These substances could affect 1. Chlorophyceae: Dunaliella tertiolecta Butcher
analyses by either increasing the absorbance (overesti- (Volvocales).
mating values), or decreasing the measurements by in- 2. Ulvophyceae: Caulerpa fastigiata Montagne
hibiting the action of specific reagents. However, their (Bryopsidales), Codium decorticatum (Woodw.) M.
influence may be avoided by precipitation of the pro- Howe; (Bryopsidales) and Ulva fasciata Delile
tein sample with trichloroacetic acid (TCA). Concen- (Ulvales).
 449

Cryptophyta Conway nutrient solution (Walne, 1966). Each experi-

Cryptophyceae: Hillea sp. Schiller (Cryptomon- ment was carried out in four culture flasks, exposed to
adales). 300 µmol photons m−2 s−1 (measured with a Biospher-
Dinophyta ical Instruments quanta meter QLS100) from beneath,
Dinophyceae: Amphidinium carterae Hulburt provided by fluorescent lamps (Sylvania daylight
(Gymnodiniales). tubes), under a 12:12 h light:dark cycle. Mean temper-
Heterokontophyta atures were 23 ± 1 ◦ C in the light period and 20 ± 1 ◦ C
Bacillariophyceae: Skeletonema costatum (Gre- in the dark period. Salinity of the culture medium was
ville) Cleve (Biddulphiales). 32.0‰. Growth rates were calculated daily by direct
Phaeophyceae: Chnoospora minima (K. Hering) microscopic cell counting with Fuchs–Rosenthal or
Papenfuss (Scytosiphonales), Dictyota menstrualis Malassez chambers. Cultures were bubbled with fil-
(Hoyt) Schnetter, Hörnig et Weber-Peukert (Dic- tered air at a rate of 2 L min−1 . The culture medium
tyotales), Padina gymnospora (Kützing) Sonder was not buffered and pH was determined daily.
(Dictyotales) and Sargassum vulgare C. Agardh Each culture was sampled in the stationary growth
(Fucales). phase only. Cultures were concentrated by centrifuga-
Prymnesiophyta tion at 7000 g for 10 min at 15 ◦ C, at least once. Before
Prymnesiophyceae: Isochrysis galbana Parke the last centrifugation, cells were washed with artificial
(Pavlovales). seawater (Kester et al., 1967) prepared without nitro-
Rhodophyta gen, phosphorus and vitamins and adjusted to 15‰
Bangiophycidae: Porphyra acanthophora var. salinity to remove any residual nitrogen from the cul-
acanthophora E. C. Oliveira and Coll (Bangiales). ture medium. All supernatants obtained for each sam-
Florideophycidae: Aglaothamnion uruguayense ple were combined and the cell number was determined
(Taylor) Aponte, Ballantine et Norris (Ceramiales) in this pool to quantify possible cell losses. The pel-
and Pterocladiella capillacea (S. G. Gmel.) San- lets were frozen at −20 ◦ C, freeze-dried (as described
telices et Hommersand (Gelidiales). above), weighed and stored in desiccators under vac-
All species of marine macroalgae were collected uum and protected from light at room temperature until
in June 1998 at Rasa Beach (located in Armação de analysis was done.
Búzios, 22◦ 44 S and 41◦ 57 W) and Peró Beach (lo-
cated in Cabo Frio, 22◦ 51 S and 41◦ 58 W), Northern
Rio de Janeiro State, Brazil. Whole thalli of adult plants Amino acid analysis
were collected early in the morning and washed in the
field with seawater to remove epiphytes, sediment and Samples containing 5.0 mg of protein were acid hy-
organic matter. Algae were packed in plastic bags and drolysed with 1.0 mL of 6 N HCl in vacuum-sealed
kept on ice until returned to the laboratory. In the lab- hydrolysis vials at 110 ◦ C for 22 h. Norleucine was
oratory, samples were gently brushed under running added to the HCl as an internal standard. Although
seawater, rinsed with distilled water, dried with paper tryptophan was completely lost with acid hydrolysis
tissue, frozen at −20 ◦ C and freeze-dried. The dried and methionine and cystine + cysteine could be de-
material was powdered manually with the use of mor- stroyed to varying degrees by this procedure, the hy-
tar and pestle and kept in desiccators containing silica- drolysates were suitable for analysis of all other amino
gel and protected from light at room temperature until acids. The tubes were cooled after hydrolysis, opened,
chemical analysis. and placed in a descicator containing NaOH pellets
under vacuum until dry (5–6 days). The residue was
Culture of microalgae then dissolved in a suitable volume of a sample di-

R
lution Na–S buffer (Beckman Instr.), pH 2.2, filtered
All microalgal strains used in this study are available through a Millipore membrane (0.22 µm pore size) and
at the Elizabeth Aidar Microalgae Culture Collection, analysed for amino acids by ion-exchange chromatog-
Department of Marine Biology, Federal Fluminense raphy in a Beckman, model 7300 instrument equipped
University, Brazil. Starter cultures of 50–100 mL in with an automatic integrator. Ammonia content is also
mid-exponential growth phase were inoculated into presented as it comes from the degradation of some
2.0 L of seawater, previously autoclaved at 121 ◦ C for amino acids (e.g. glutamine, asparagine) during acid
30 min in 3.0 L borosilicate flasks, and enriched with hydrolysis (Mossé, 1990).
450

Total nitrogen end of the incubation period. Seven millilitre of ultra-

pure water was added to the system to rinse the Potter
Total nitrogen (TN) content was determined by CHN homogeniser after grinding each sample to recover
analysis. 0.8–1.5 mg freeze-dried samples were com- all water-ground material. After this step, samples
busted in a CHN analyser (Perkin–Elmer, model 2400). were treated as described after the end of the incu-
Helium was used as carrier gas. Acetanilide (C = bation period for procedure I. The final volume of
71.09%; N = 10.36%; H = 6.71%) and/or benzoic the extract was 9.0 mL.
acid (C = 68.84%; H = 4.95%) were used to calibrate Procedure III. This treatment is similar to procedure
the instrument. I, differing by the use of 4 mL of water to incubate
dried samples, instead of 1 mL. The final volume of
Extraction of protein the extract was 5.0 mL.
Procedure IV. This treatment is similar to procedure II,
Eight procedures for protein extraction were tested in differing by the use of 4 mL of water for incubating
this study. All procedures start with 50 mg of freeze- dried samples, instead of 1 mL. Four millilitre of
dried algal sample, ground manually with pestle and ultra-pure water were added to the system to rinse
mortar. Two different volumes of water were tested the Potter homogeniser after grinding each sample
(1.0 and 4.0 mL), as well as two different incubation to recover all water-ground material. After this step,
periods of samples with water (6 and 12 h). In all the samples were centrifuged as described for procedure
cases samples were kept at 4 ◦ C during the incubation I. The final volume of the extract was 9.0 mL.
period. Procedure V. Similar to procedure I, differing only due
In four out of the eight extraction procedures, sam- the incubation period: 6 h instead of 12 h. The final
ples were also ground using a Potter homogeniser (Mar- volume of the extract was 2.0 mL.
coni, model MA099) after the incubation with water. Procedure VI. Dried samples are treated such as in pro-
Samples were water-ground with a glass pestle and a cedure II, except by the shorter incubation period: 6 h
teflon mortar at medium speed. During grinding, sam- instead of 12 h. The final volume of the extract was
ples were kept cool by the use of a circulating cooling 9.0 mL.
bath through the pestle. The grinding of samples was Procedure VII. Similar to procedure III, differing only
started 1 h before the end of incubation of the sam- due to the shorter incubation period: 6 h instead of
ples with water. Six replicates were prepared for each 12 h. The final volume of the extract was 5.0 mL.
treatment and each species. Procedures for protein ex- Procedure VIII. Samples were treated as described in
traction are based on Fleurence et al. (1995) with some procedure IV, differing only by the shorter incuba-
modifications (e.g. speed of centrifugation, grinding of tion period: 6 h instead of 12 h. The final volume of
the samples, time of incubation). The specific proce- the extract was 9.0 mL.
dures for protein extraction analysed in this study are
as follows:
A summary of the procedures to extract algal protein
Procedure I. Algal samples were immersed in 1 mL of is shown in Table 1.
ultra-pure water for 12 h. After the incubation pe-
riod, suspensions were centrifuged at 4 ◦ C, 15,000 g Table 1. Summary of the procedures for extracting protein used in
for 20 min. Supernatants were collected for protein this study
assay and the pellets re-extracted with 1.0 mL 0.1 N Volume of Period of
NaOH with 0.5% β-mercaptoethanol (v/v). The mix- Treatment water (ml) incubation (h) Grinding
ture of NaOH and pellets were kept at room tempera-
Procedure I 1.0 12 No
ture for 1 h with occasional manual shaking and then
Procedure II 1.0 12 Yes
centrifuged at 21 ◦ C, 15,000 g for 20 min. The sec-
Procedure III 4.0 12 No
ond supernatants were combined with the first ones
Procedure IV 4.0 12 Yes
and the pellets were discarded. The final volume of
Procedure V 1.0 6 No
the extract was 2.0 mL.
Procedure VI 1.0 6 Yes
Procedure II. Similar to procedure I, with one addi-
Procedure VII 4.0 6 No
tional step included: the grinding of samples with a
Procedure VIII 4.0 6 Yes
Potter tissue homogeniser for 5 min, 1 h before the
 451

The use of 1.0 mL 0.1 N NaOH with 0.5% β- 50 mL 95% ethanol (Merck Co.) with a further addi-
mercaptoethanol (v/v) for re-extracting the pellets was tion of 100 mL 85% H3 PO4 (Merck Co.). The solution
adopted in all the eight procedures tested, independent was diluted with ultra-pure water to 1.0 L. Five millil-
of the time of incubation and volume of ultra-pure wa- itre of the reactive was used for each 0.1 mL sample.
ter used to incubate the samples. Absorbance was measured at 595 nm, 5 min after the
start of the chemical reaction at room temperature.
Precipitation of protein Calibration curves were prepared using bovine
serum albumin (BSA) (Sigma Co.) and casein (Sigma
Protein precipitation was followed Berges et al. (1993). Co.) at maximum concentrations of 100 µg mL−1
Two proportions of cold 25% trichloroacetic acid (Lowry method) and 100 µg 0.1 mL−1 (Bradford
(TCA) (4 ◦ C) added to the extracts were tested: 2.5:1 method). Casein was diluted in ultra-pure water plus
and 3.0:1 (TCA:homogenate, v/v). Tubes contain- some drops of 0.1 N NaOH. All measurements were
ing TCA and homogenate were kept in an ice bath done using a Shimadzu, model UV Mini 1240 spec-
for 30 min and then centrifuged for 20 min at 4 ◦ C trophotometer.
(15,000 g). Supernatants were discarded, pellets were In addition to colorimetric assays of protein, crude
washed with cold 10% TCA (4 ◦ C) and centrifuged protein for each species was also calculated using spe-
again. Pellets formed after the second centrifugation cific nitrogen-to-protein conversion factors proposed
were suspended in 5% TCA at room temperature, in a by Lourenço et al. (2002, 2004) as follows: A. carterae
proportion of 5:1 (5% TCA:precipitate, v/v) and cen- (5.13), A. uruguayense (3.94), C. decorticatum (5.34),
trifuged at 21 ◦ C (15,000 g) for 20 min. Supernatants C. fastigiata (4.52), C. minima (5.70), D. menstrualis
were discarded and pellets were kept in the tubes un- (4.55), D. tertiolecta (4.39), Hillea sp. (4.93), I. gal-
til quantification of protein was done a few minutes bana (5.07), P. acanthophora var. acantophora (4.47),
later. When the protein analysis was not performed im- P. capillacea (4.78), P. gymnospora (5.72), S. costatum
mediately, pellets were stored at −20 ◦ C until further (4.53), S. vulgare (5.53), and U. fasciata (5.59).
analysis, following Dortch et al. (1984).
Precipitated protein was suspended in 0.5 mL 1.0 N
NaOH and 2.0 mL 0.1 N NaOH for the a Bradford and Statistical analysis
Lowry assays, respectively. Aliquots were also col-
The results were analysed by one-way analysis of vari-
lected from the crude extracts obtained before the pre-
ance (ANOVA) with significance level α = 0.05 (Zar,
cipitation with TCA to perform protein analysis by the
1996) followed, where applicable, with Tukey’s mul-
Lowry method without previous precipitation.
tiple comparison test. In some cases, Student’s t-test
was used instead of ANOVA when comparing only two
Protein analysis
treatments for each variable.
In the Lowry method, the Folin–Ciocalteu reactive
(Folin & Ciocalteu, 1927) (Sigma Co.) was diluted in
two volumes of ultra-pure water (1:2) and 0.5 mL of Results
the diluted reactive was added to 1.0 mL of sample,
previously mixed with 5.0 mL of the reactive “C” [50 Tests of protein extraction
volumes of reactive “A” (2.0% Na2 CO3 + 0.1 N NaOH)
+ 1 volume of reactive “B” (1/2 volume of 0.5% CuSO4 The use of the Potter homogeniser produced remark-
5H2 O + 1/2 volume of 1.0% C4 H4 NaO6 4H2 O)]. After able differences in the extraction of protein for the three
the addition of each reactive, samples were stirred for species tested. In all cases, significantly ( p < 0.001)
2 s in a test tube stirrer. Absorbance was measured at higher concentrations of protein was obtained in the
750 nm, 35 min after the start of the chemical reaction treatment with the use of the Potter homogeniser (Fig-
at room temperature. ures 1A–C). For Sargassum vulgare (Figure 1B) dif-
In the Bradford assay, the Coomassie Brilliant Blue ferences in values obtained for samples extracted with
dye G-250 (CBBG) binds to the protein. The bind- and without the Potter homogeniser were about 50%.
ing of the dye with the protein is very quick and the Higher values of protein were also obtained for samples
protein-dye complex remains soluble for 1 h. One hun- of Porphyra acanthophora var. acanthophora and Ulva
dred milligram of CBBG (Sigma Co.) was dissolved in fasciata when extracted with the Potter homogeniser.
452

The precipitation of protein

No differences in the precipitation of protein were ob-

served for the two TCA:homogenate ratios using the
Lowry (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 three

species 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. acanthophora
var. acanthophora ( p < 0.02). However for U. fasci-
ata, using the Lowry method ( p = 0.56) and S. vulgare
with the Bradford method ( p = 0.06), no differences
were found when comparing results with BSA or casein
as 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 endogenous
algal proteases in the samples which could partially de-
stroy protein during incubation (Pérez-Llorénz et al.,
2003). Tests were carried out for all the three algae at
same 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 in
which the algal material was incubated, such as in pro-
cedure IV. Controls included incubation of BSA with
no algae, following the same steps described for algal
Figure 1. Quantification of protein in three species of marine
samples and direct dilution and measurement of BSA,
macroalgae (A) Porphyra acanthophora var. acanthophora, (B) Sar- without incubation. The results (data not presented)
gassum vulgare and (C) Ulva fasciata by the Lowry (Lwy) and Brad- showed no loss of protein after the incubation period
ford (Bdf) methods, using bovine serum albumin (BSA) and casein in all experiments carried out ( p ≥ 0.083), indicating
(CAS) as protein standards in the calibration curves. Three vari- no activity of algal proteases at 4 ◦ C, the incubation
ables evaluated: (i) the water volume used to incubate samples (1
and 4 mL); (ii) the length of the incubation period for the extraction
temperature used.
of protein (6 and 12 h); and (iii) the use of a Potter homogenizer
for grinding samples. All assays included the precipitation of pro-
tein with 25% TCA, in a proportion of 2.5:1 (TCA:homogenate). Protocol for extraction and precipitation of protein
Mean ± S.D. (n = 6).
Results suggest the use of a general protocol involving
Higher concentrations of protein were obtained extraction of protein from algal samples using 4.0 mL
when 4.0 mL of water was used for U. fasciata ( p < of ultra-pure water, for 12 h and grinding of samples
0.001) (Figure 1C), and a longer period of incubation with a Potter homogeniser. Samples should be precipi-
(12 h) for S. vulgare ( p = 0.002) (Figure 1B). There tated with TCA:homogenate (2.5:1 v/v). A diagram of
were no differences for P. acanthophora var. acan- the protocol is shown in Figure 2. This protocol was
thophora (Figure 1A) incubated in wither 1.0 or 4.0 mL used for protein determination of the other algal species
for 6 h ( p = 0.52). using both the Bradford and Lowry methods, using
 453

Figure 2. Diagrammatic representation of the protocol for extraction and precipitation of algal protein developed in this study.
454

BSA and casein as protein standards in the calibration fastigiata, casein as protein standard). For all species,
curves. values obtained with BSA as the protein standard were
higher than those obtained with casein (Table 3). Crude
extracts analysed using the Lowry method always gave
Amino acid profile of the algae and protein standards
higher values than those obtained with precipitated
samples ( p < 0.01), except for A. uruguayense and
The comparison of the amino acid profile of the algal
Hillea sp. (Table 3).
species (Table 2) shows great differences for aspartic
The 15 algae species showed great differences re-
acid, glutamic acid and arginine. On the other hand,
garding total nitrogen and crude protein (Table 3). Mi-
some amino acids, such as glycine and leucine, had
croalgae had smaller variations in the total N, vary-
similar values for the 15 species studied.
ing from 3.35% (I. galbana) to 4.69% (A. carterae).
The comparison of the algal amino acid profile with
Variations in the total N were greater in the macroal-
the protein standards show that the concentration of
gae, ranging from 1.94% (C. minima) to 5.68% (A.
glutamic acid in casein, and lysine in BSA, are higher
uruguayense). The calculations of crude protein gave
than those reported for all the algae. Both protein stan-
wide variations among species, varying from 10.52%
dards were lower in methionine compared to the algae.
in C. decorticatum to 25.04% in Hillea sp. (Table 3).
Casein and BSA also show remarkable differences with
The sum of amino acid residues varied from 9.99% (C.
each other regarding some amino acids, such as alanine
minima) to 20.11% (Hillea sp.). Microalgae tended to
(higher concentration in BSA) and proline (higher con-
have higher percentages of amino acid residues than
centration in casein).
macroalgae.

Quantification of protein
Discussion
The highest percent of protein was measured in the red
algae A. uruguayense (15.6 ± 0.3% of the dry matter), Extraction and precipitation of algal protein
followed by the cryptomonad Hillea sp. (15.3 ± 0.6%)
(Table 3). The other microalgae had similar protein Protein content of the three main algae species tested in
contents, varying from 11.4 ± 1.0%. (D. tertiolecta) this study varied greatly depending on the different ex-
to 10.1 ± 0.8% (I. galbana). The edible red algae P. traction procedures tested. The efficiency of extraction
acanthophora var. acanthophora had 8.9 ± 0.7% of seems to be influenced directly by two main factors:
protein and P. capillacea had the lowest protein content the chemical composition of the species and its mor-
among the red algae (4.2 ± 0.3%). In the brown algae, phological and structural characteristics. The chemi-
small variations in protein content were found (8.7, cal composition of the three species is very distinct
7.8 and 6.9% for P. gymnospora, C. minima and S. (Lourenço et al., 2002) and, in theory, this may lead
vulgare, respectively), except for D. menstrualis, which to differences in the protein content. S. vulgare is a
had a lower protein content (4.0 ± 0.2%). The green branched algae and possesses a hard and leathery thal-
macroalgae had protein contents varying from 5.5% lus, while P. acanthophora var. acanthophora and U.
(C. fastigiata) to 7.3% (U. fasciata) (Table 3). fasciata have flattened soft thalli.
Confirming the same trend obtained with the ini- In the present study, the effects of lyophylisation on
tial results for the three macroalgae (P. acanthophora the thalli should be also considered since freeze-dried
var. acanthophora, S. vulgare and U. fasciata), all other samples tend to be more difficult to extract, especially
species gave significantly higher values of protein when leathery species such as C. minima and S. vulgare. This
quantified using the Lowry method compared to the means that better preservation by freeze-drying makes
Bradford method. In some cases (e.g. C. fastigiata), dif- them more difficult for further protein extraction. This
ferences between mean values were higher than 50% problem can be solved by grinding samples with Potter
(Table 3). For the microalgae, the variation between homogeniser.
the two methods was less, varying from 1.5 (I. gal- For P. acanthophora var. acanthophora lyophilisa-
bana, BSA as protein standard) to 1.9 (A. carterae, tion, the main factor influencing the yield in the pro-
BSA and casein as protein standard). For the macroal- tein extraction was the use of grinding. This is prob-
gae, the Lowry:Bradford ratio varied from 1.5 (D. men- ably related to the thallus form of this species. Ulva
strualis, BSA and casein as protein standard) to 3.2 (C. fasciata has the same kind of thallus, but the use of
 455

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
a Resultsare expressed as percentage of amino acid per 100 g of algal protein (or pure protein for the two standards) and represent the real
recovery of amino acids after analysis. Concentrations of ammonia correspond to nitrogen recovery from some amino acids destroyed during
acid hydrolysis. Values indicate the mean of three replicates ± S.D. (n(3). N.D.: not detected.
456

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 Crude
Species 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
a Proteinwas determined by different methods, with BSA and casein as protein standards. Analysis by Lowry’s method was also made with
notprecipitated 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- Amino acid profile, non-protein nitrogen and protein
traction of protein. Differences in the behaviour of content of algae
the two flattened algae may be related to the chemi-
cal composition and thallus morphology, since U. fas- In this study we assume that the actual concentra-
ciata has two layers of cells compared to P. acan- tions of protein in the samples are calculated from
thophora var. acanthophora with only one layer of the sum of amino acid residues (Tables 1 and 2), a
cells. widely accepted procedure since the 1970s (Heidel-
For the extraction of protein from the branched and baugh et al., 1975). After acid hydrolysis, all proteins
hard thallus of the brown algae S. vulgare, 12 h in- are destroyed, even those associated with other macro-
cubation in water seems to be important. Even us- molecules and biological membranes. The values for
ing a greater volume of water (4 mL), an incubation the total amino acid residues were calculated by sum-
period of 6 h was not enough to soften the thalli. ming up the amino acid masses retrieved after acid hy-
Fragments of S. vulgare thallus were ground more eas- drolysis (total amino acid), less the water mass (18 g in
ily with the Potter homogeniser after 12 h of exposure to 1 M of each amino acid) incorporated into each amino
water. acid after disruption of the peptide bonds. Total amino
As the precipitation of protein with TCA: ho- acid analysis involves some errors, such as the total
mogenate (3.0:1 and 2.5:1 v/v) gave no significantly (tryptophan) or partial (methionine and cysteine) de-
different results and therefore a TCA:homogenate struction of some amino acids, as well as the impossi-
ratio of 2.5:1 (v/v) is recommended in order to save bility of identifying the contribution of free amino acids
reagent. in the samples. However, it indicates the maximum
 457

possible concentration of protein in the sample con- tein, which possesses 36.0% of the total amino acid
sidering that all amino acid are in protein, providing (Galland-Irmouli et al., 1999).
a good reference point for the protein concentrations High concentrations of non-protein N may result
measured by the Bradford and Lowry assays. In our in overestimation of protein (Zamer et al., 1989). Ac-
results, values for amino acid residues were similar to cording to Lourenço et al. (2004), concentrations of
the estimated crude protein, suggesting the suitability non-protein N vary widely during growth in cultures
of the nitrogen-to-protein conversion factors calculated of microalgae, commonly fluctuating from 15 to 30%
by Lourenço et al. (2002, 2004). Exceptions to this are of the total N. In the present study, the occurrence
C. fastigiata and A. carterae (Table 3), which showed of high concentrations of the total N was not mir-
values for crude protein ca. 30% higher than those for rored by high total amino acid concentrations in some
the sum of amino acid residues. This difference prob- species such as A. carterae, A. uruguayense, C. fasti-
ably results from the presence of high concentrations giata, and Hillea sp., which is explained by the pres-
of non-protein nitrogen, presumably transient stocks ence of large amounts on non-protein N. The deter-
of inorganic nitrogen (Lavı́n & Lourenço, unpublished mination of crude protein should be based on the use
data). of specific nitrogen-to-protein conversion factors as
The sum of amino acid residues indicates that mi- proposed by Lourenço et al. (2002, 2004). In addi-
croalgae, independent of the taxonomic group, tend tion, results obtained for the total amino acid residues
to accumulate higher concentrations of protein than and precipitated and non-precipitated extracts with the
macroalgae. This fact may be related to the high con- Lowry method suggest the influence of variable con-
centration of nitrogen in the culture medium (as well as centrations of free amino acids and small peptides in
other dissolved nutrients), growth conditions in the lab- the samples. This finding indicates that precipitation
oratory and the higher surface area:volume ratios found of the samples is a fundamental step during protein
in microalgae (Hein et al., 1995). Dried samples of mi- analysis.
croalgae are better exposed to solvents and to grind- Data of protein content in macroalgae from the trop-
ing during the extraction procedures, while macroalgal ical and subtropical coastal environments frequently
samples have to be powdered before the start of the show lower concentrations (Kaehler & Kennish, 1996;
extraction. This factor may produce a more efficient Wong & Cheung, 2000). In some Brazilian environ-
protein extraction. ments Ramos et al. (2000) found that the percentage
For many macroalgae, the combined concentration of protein (N × 6.25) in 14 seaweeds varied from 2.30
of glutamic acid and aspartic acid represents 40% of to 25.6% of dry weight. Despite the overestimation of
total amino acids, agreeing with data obtained for the protein content caused by the use of the factor 6.25
edible red algae Palmaria palmata, in which glu and (Lourenço et al., 2002), values obtained by Ramos
asp represent 39.6% of total amino acids (Galland- et al. (2000) indicated predominantly low concentra-
Irmouli et al., 1999). For microalgae, the sum of asp tions of protein. This trend may be related to the nat-
and glu represent mean values of about 20% of the total ural characteristics of Brazilian marine environments;
amino acids for Skeletonema costatum, Dunaliella ter- predominantly oligotrophic, with low availability of N
tiolecta and Thalassiosira pseudonana (Brown, 1991). (Oliveira et al., 1997; Ovalle et al., 1999). As a conse-
For Ulva rigida and U. rotundata, percentages of these quence, low concentrations of protein would be accu-
two amino acid may represent from 26 to 32% of mulated by natural populations of macroalgae. In this
the total amino acids (Fleurence et al., 1995). In the context, our data of protein concentration in macroal-
present study, values for asp + glu varied from 19.2% gae are in accordance with the information available
(C. fastigiata) to 28.1% (A. uruguayense) (Table 2). in the literature (e.g. Wong & Cheung, 2001; McDer-
For microalgae, the fraction represented by these two mid & Stuercke, 2003). On the other hand, the relative
amino acids varied from 23.1% (A. carterae) to 26.9% low content of protein in microalgae results from the
(S. costatum) of the total amino acids (Table 2). The physiological state of the species. All microalgae were
set composed of the essential amino acids in samples sampled in stationary growth phase when percentages
varied from 36.3% (S. vulgare) to 44.0% (C. fasti- of protein in cells decreased due to depletion of dis-
giata), with a mean value of 40.2% of the total amino solved nutrients in the culture medium (Lourenço et al.,
acids. Concerning nutritional properties, these species 1998).
show concentrations of essential amino acids compa- Low protein levels were found in Pterocladiella
rable to those commonly described to soybean pro- capillacea using both the Lowry and Bradford
458

methods. The values obtained are equivalent to 1/3 with protein occurs mainly with the two amino acids,
of those determined from the sum of the amino arginine and phenylalanine, and this fact seems to
acid residues, being the lowest protein concentrations contribute to lower protein measurements. Our results
among all red macroalgae tested. This seems to indicate with Bradford’s method agree with Kaehler and
inefficient extraction using the procedures developed in Kennish (1996). These authors found predominantly
this study for this macroalgae. We hypothesise that the low values for some seaweeds (from 1.3 to 12.6%)
extraction of protein in this species might be influenced from Hong Kong using the Bradford method. In
by the presence of phycocolloids, especially agarans. contrast, the Folin–Ciocalteu reagent used in the
P. capillacea is a good source of agar (McHugh, 1991) Lowry assay interacts with all peptide bonds and also
and is probably the most abundant macromolecule in with some amino acids. As a result, the quantification
this alga. During the water extraction step, it was possi- of protein tends to be greater.
ble to extract variable quantities of agar visible, as the The differences in amino acid composition among
occurrence of gels in several steps of protein extrac- protein standards and algae have important implica-
tion, mainly when using the Potter homogeniser and tions regarding protein reactivity and quantification in
after the centrifugation at 4 ◦ C of the ground samples. algal samples. Despite the good linearity obtained with
It is possible that the gels can trap part of the protein both protein standards (BSA and casein), our data sug-
extracted, giving low values in the spectrophotometric gest that casein has a slightly smaller reactivity than
determination of protein. This kind of analytical prob- BSA resulting in a smaller quantification of protein.
lem may be present in other agarophytes as well as The two protein standards have extremely different
carragheenan-producing species. The presence of large amino acid composition and the reactivity of them in
amounts of anionic polysaccharide in the cell walls re- each method tends to be different due the functional
duces protein solubility during extraction (Fleurence, groups that they present (Morrison & Boyd, 2003).
1999). Further studies are needed to develop better Functional groups change the charge and the geom-
procedures for protein extraction in phycocolloid-rich etry of neighbouring atoms, affecting the reactivity of
species. the whole molecule (Morrison & Boyd, 2003).

Lowry × Bradford methods and the influence of the Conclusions

amino acid profile of samples and standards
The use of 4.0 mL of water to incubate algal samples for
Some authors suggest that Bradford’s method would 12 h, combined with grinding of the samples with a Pot-
generate lower protein values for a large number of or- ter homogeniser is strongly recommended. This proce-
ganisms compared to Lowry’s method. Calculating the dure results in better extraction of protein from algal
Lowry:Bradford ratio from data by Eze & Dumbroff samples of different species independent of their mor-
(1982) for leaves of bean plants gives a ratio of 1.4. For phological and biochemical characteristics. As a conse-
the diatom T. pseudonana, Clayton et al. (1988) found quence, this procedure can be applied widely to many
ratios varying from 1.8 to 2.0, while Berges et al. (1993) algal species. The precipitation of protein should be
determined a ratio of 1.2 for the same microalgae. The done with 25% trichloroacetic acid in the ratio of 2.5:1
present results confirm the general trends found by (TCA:homogenate). Results generated with Lowry’s
those authors, but we found higher Lowry:Bradford method are more similar to the data obtained from the
ratios for most of the species. Our results varied from sum of the amino acid residues which is considered
1.5 (I. galbana, BSA as the protein standard) to 3.2 (C. the most reliable way of determining the actual pro-
fastigiata, casein as the protein standard). tein content. Protein values obtained with BSA as a
The trend of obtaining lower concentrations of protein standard were closer to those calculated from
protein using Bradford’s method may be related to the the sum of amino acid residues, suggesting that the
binding of the dye Coomassie Brilliant Blue-G250 to use of BSA is more suitable for the Lowry method.
both basic and aromatic amino acid residues (Compton The procedures proposed here can contribute to better
& Jones, 1985). Most of the algae show relatively results, since protein is extracted efficiently and po-
low concentrations of the two amino acids (tyrosine tential interference from compounds such as pigments,
and tryptophan) as well as the two basic amino acids lipids, phenolics, small peptides and free amino acids is
(lysine and histidine). Thus, the binding of the dye eliminated.
 459

Acknowledgments Fleurence J, Le Couer C, Mabeau S, Maurice M, Landrein A (1995)

Comparison of different extractive procedures for proteins from
We are indebted to FAPERJ (Foundation for Re- the edible seaweeds Ulva rigida and Ulva rotundata. J. Appl.
Phycol. 7: 577–582.
search Support of Rio de Janeiro State, grant E- Folin O, Ciocalteu V (1927) On tyrosine and tryptophane determi-
26.170.041/98) for the financial support to this study. nations in proteins. J. Biol. Chem. 73: 627–650.
Special acknowledgements are due to Dr. Yocie Fujihara S, Kasuga A, Aoyagi Y (2001) Nitrogen-to-protein conver-
Yoneshigue-Valentin (Universidade Federal do Rio de sion factors for common vegetables in Japan. J. Food Sci. 66:
Janeiro) and Dr. Carlos Logullo de Oliveira (Uni- 412–415.
Galland-Irmouli A-V, Fleurence J, Lamghari R, Luçon M, Rouxel
versidade Estadual do Norte Fluminense) for offer- C, Barbaroux O, Bronowicki J-P, Villaume C, Guéant J-L (1999)
ing us laboratory facilities to perform this study. We Nutritional value of proteins from edible seaweed Palmaria pal-
thank Dr Ursula M. Lanfer Marquez (Universidade de mata (Dulse). J. Nutr. Biochem. 10: 353–359.
São Paulo) for her support in the amino acid analy- Heidelbaugh ND, Huber CS, Bednarczyk JF, Smith MC, Rambaut
sis. E.B. acknowledges CAPES and S.O.L. acknowl- PC, Wheeler HO (1975) Comparison of three methods for
calculating protein content of foods. J. Agric. Food Chem. 23:
edges FAPERJ and CNPq for providing them research 611–613.
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nitrogen uptake in micro- and macroalgae. Mar. Ecol. Prog. Ser.
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