Accepted Manuscript Chemical composition of red, brown and green macroalgae from Buarcos bay in Central West Coast of Portugal Dina Rodrigues, Ana C. Freitas, Leonel Pereira, Teresa A.P. Rocha-Santos, Marta W. Vasconcelos, Mariana Roriz, Luís M. Rodríguez-Alcalá, Ana M.P. Gomes, Armando C. Duarte PII: DOI: Reference:
S0308-8146(15)00429-X http://dx.doi.org/10.1016/j.foodchem.2015.03.057 FOCH 17304
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 March 2014 9 March 2015 18 March 2015
Please cite this article as: Rodrigues, D., Freitas, A.C., Pereira, L., Rocha-Santos, T.A.P., Vasconcelos, M.W., Roriz, M., Rodríguez-Alcalá, L.M., Gomes, A.M.P., Duarte, A.C., Chemical composition of red, brown and green macroalgae from Buarcos bay in Central West Coast of Portugal, Food Chemistry (2015), doi: http://dx.doi.org/ 10.1016/j.foodchem.2015.03.057
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Chemical composition of red, brown and green macroalgae from Buarcos bay
2
in Central West Coast of Portugal
3 4
Dina Rodrigues1*, Ana C. Freitas1,2, Leonel Pereira3, Teresa A. P. Rocha-Santos1,2, Marta W.
5
Vasconcelos4, Mariana Roriz4, Luís M. Rodríguez-Alcalá4, Ana M. P. Gomes4, Armando C.
6
Duarte1
7 8
1
9
University of Aveiro, 3810-193 Aveiro, Portugal
CESAM - Centre for Environmental and Marine Studies & Department of Chemistry,
10
2
11
Portugal;
12
3
13
Research, Department of Life Sciences, Faculty of Sciences and Technology, University of
14
Coimbra, P-3001-455 Coimbra, Portugal
15
4
16
de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino
17
Almeida, 4200-072 Porto, Portugal
ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa, Viseu,
MARE – Marine and Environmental Sciences Centre & IMAR – Institute of Marine
CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior
18 19
*Corresponding author: Mailing address: 1CESAM - Centre for Environmental and Marine
20
Studies & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
21
E-mail: [email protected])
22 23 24
This manuscript has been submitted for publication in Food Chemistry
25 26
It is not to be reproduced or cited without the written permission of the authors.
27
1
28 29
Abstract
30
Six representative edible seaweeds from the Central West Portuguese Coast, including the
31
less studied Osmundea pinnatifida, were harvested from Buarcos bay, Portugal and their
32
chemical characterization determined. Protein content, total sugar and fat contents ranged
33
between 14.4 and 23.8%, 32.4 and 49.3% and 0.6-3.6%. Highest total phenolic content was
34
observed in Codium tomentosum followed by S. muticum and O. pinnatifida. Fatty acid (FA)
35
composition covered the branched chain C13ai to C22:5 n3 with variable content in n6 and
36
n3 FA; low n6:n3 ratios were observed in O. pinnatifida, Grateloupia turuturu and C.
37
tomentosum. Some seaweed species may be seen as good sources of Ca, K, Mg and Fe,
38
corroborating their good nutritional value. According to FTIR-ATR spectra, G. turuturu was
39
associated with carrageenan seaweed producers whereas Gracilaria gracilis and O.
40
pinnatifida were mostly agar producers. In the brown algae, S. muticum and Saccorhiza
41
polyschides, alginates and fucoidans were the main polysaccharides found.
42 43 44
Keywords: Chemical composition, Elements, Fatty acids, FTIR-ATR characterization,
45
Seaweeds
46
2
47
1. Introduction
48
Algae, in particular, edible seaweeds, are a very interesting natural source of compounds
49
with biological activity that could be used as functional ingredients (Plaza, Cifuentes &
50
Ibánez, 2008). Considering their great taxonomic diversity, research on identification of
51
biologically active compounds is encouraging in such a vast untapped resource (Lordan,
52
Ross & Stanton, 2011). Some algae live in complex habitats and are often subject to extreme
53
conditions. Their metabolism can be influenced by parameters such as water temperature,
54
salinity, light and nutrients, being forced to quickly adapt to continuously new environmental
55
conditions and in order to survive they produce a wide variety of biologically active secondary
56
metabolites (Plaza et al., 2008). Seaweeds can in fact provide several compounds at
57
different quantities and for that, research on identification of bioactive compounds from algae
58
can be seen as an almost unlimited field (Lordan et al., 2011).
59
Seaweeds are known to be of low calorie content, rich in polysaccharides, minerals,
60
vitamins, proteins, steroids and dietary fibers (Lordan et al., 2011) making them increasingly
61
sought for commercial purposes. They are of potential interest to be incorporated in the diet
62
as added-value foods because they are claimed to contain low cholesterol content, fight
63
obesity, reduce blood pressure, tackle free radicals and promote healthy digestion (Plaza et
64
al., 2008); furthermore, some of them have demonstrated biological properties such as
65
antibacterial and anticoagulant activities (Alghazeer, Whida, Abduelrhman, Ammoudi &
66
Azwai, 2013; Chandía & Matsuhiro, 2008). The total concentration of polysaccharides in the
67
seaweeds can range from 4 to 76% of dry weight (Holdt and Kran, 2011) where agar, algins
68
carrageenans and fucoidans are some of the polysaccharides widely used by man, some
69
with various biological activities (Mak, Wang. Liu, Hamid, Li, Lu & White, 2014; Mak, Hamid,
70
Liu, Lu & White, 2013). Protein content is also variable and the highest contents are
71
generally found in green and red seaweeds (10-30% of dry weight) in comparison to brown
72
seaweeds (5-15% of dry weight) (Holdt and Kran, 2011; Zhou, Robertson, Hamid, Ma & Lu,
73
2014). Phospholipids and glycolipids are the major classes of lipids found in seaweeds
74
accounting for 1-5% of cell composition (Chojnacka, Saeid, Witkowska & Tuhy, 2012); 17 to 3
75
63 mg/g of lipid content has been reported for the brown seaweed Undaria pinnatifida
76
(Boulom, Robertson, Hamid, Ma & Lu, 2014). Phenolic compounds are another important
77
group of chemical compounds in seaweeds (Lordan et al. 2011) varying quantitatively and
78
qualitatively for each specimen of red, brown or green seaweeds. Seaweeds are also
79
recognized as sources rich in several elements such as Ca, Mg, Na, P and K or trace
80
elements such as Zn, I or Mn. This elemental richness is related to their capacity to retain
81
inorganic compounds accounting for up to 36% of dry matter in some species (Lordan et al.,
82
2011).
83
According to Chojnacka et al. (2012), there are about ten thousand identified algae
84
species, yet only about 5% thereof are used as human food or as animal feed. More than
85
one hundred seaweed species are used worldwide, especially in Asian countries, where they
86
are used as sea vegetables. In Southern Europe the use of edible seaweeds for food
87
purposes is still residual, not yet fitting with the regular consumption habits. However, this
88
trend is changing and currently researchers are searching for, and studying, less-known
89
seaweed species not only because of their potential biological properties due to specific
90
functional compounds but mostly for their nutrient profile, which is being considered as
91
important alternative protein and complex carbohydrate sources (Ibañez & Cifuentes, 2013).
92
The main objective of the present study was to determine the chemical composition of
93
six different edible species from each of the main seaweed groups (red, brown and green
94
algae) occurring at Buarcos Bay (Figueira da Foz, Portugal). The selection was based on
95
representative species from the Central West Portuguese Coast including the less studied
96
Osmundea pinnatifida. Information on the proximal composition, fatty acids profile and
97
elemental composition of the six seaweeds is presented as well as the identification of the
98
principal seaweed colloids by FTIR-ATR and FT-Raman.
99 100
2. Material and methods
101 102
2.1. Specimens of seaweeds 4
103
Specimens of red algae (Rhodophyta, Florideophyceae) Osmundea pinnatifida
104
(Ceramiales), Grateloupia turuturu (Halymeniales) and Gracilaria gracilis (Gracilariales),
105
brown algae (Heterokontophyta, Phaeophyceae) Sargassum muticum (Fucales) and
106
Saccorhiza polyschides (Tilopteridales) and of green algae (Chlorophyta, Ulvophyceae)
107
Codium tomentosum (Bryopsidales), were harvested in April 2012 from Buarcos bay (Figueira
108
da Foz, Portugal). The classification of these seaweeds was based on AlgaeBase (Guiry &
109
Guiry, 2013). All six selected species are edible being used as food for humans all around the
110
world (SIA, 2015; Munier, Dumay, Morançais, Jaouen & Fleurence, 2013; Lodeiro et al., 2012;
111
Roo, Mantri, Ganesan & Kumar, 2007). The seaweeds were first washed with running tap
112
water and then with deionised water to eliminate residues from the thalli surface and then
113
dried in an oven at 60 ºC. The dried seaweeds were milled to less than 1.0 mm particle size.
114 115
2.2. Chemical characterization of seaweeds
116 117
2.2.1. Proximate composition
118
Content in moisture, organic matter and ash were determined according to AOAC methods,
119
(1990). Nitrogen content was determined by the Kjeldahl method adapted from where protein
120
content is estimated by multiplying the nitrogen content by 6.25 (Munier et al., 2013; Denis,
121
Morançais, Li, Deniaud, Gaudin & Wielgosz-Collin, 2010) whereas total fat content was
122
determined by Soxhlet extraction. Total sugar content was determined by calculation, i.e. by
123
subtracting protein content and fat content from total organic content. Total polyphenols were
124
extracted from 0.1 g of dry seaweed in 10 mL of ethyl acetate after 30 min of sonication (on a
125
water bath ultrasonicator, Ultrasonik 57H Ney). The extract was filtered with anhydrous
126
sodium sulfate (Sigma) and brought to dryness with a rotary evaporator (Laborato 4000,
127
Heidolph). The residue was re-dissolved in 5 ml of milliQ water and the content of total
128
polyphenols of 2 mL was determined by colorimetric method of Folin-Ciocalteu, using
129
cathecol (0 to 75 mg/L) as standard and expressed as g cathecol equivalent per g of dry
130
seaweed. 5
131 132
2.2.2 Analysis of fatty acids
133
Hexane and methanol were HPLC grade (VWR Scientific, West Chester, PA). Sodium
134
sulphate was analytic grade and purchased to Panreac (Barcelona, Spain). Methyl
135
tricosanoate (99%) and Supelco 37 FAME mix were obtained from Sigma (Sigma-Aldrich, St.
136
Louis, MO, USA). GLC-Nestlé36 was purchased to Nu-Chek Prep, inc. (Elysian, Minnesota,
137
USA) while butterfat CRM-164 (EU Commission; Brussels, Belgium) was from Fedelco Inc.
138
(Madrid, Spain).
139
For the analysis of the total fatty acid (FA) composition, 100 mg of sample were
140
accurately weighed and prepared according to Sánchez-Avila, Mata-Granados, Ruiz-
141
Jiménez & Luque de Castro (2009). For quantification purposes samples were added with
142
100 µL of methyl tricosanoate (1.28 mg/mL) prior to derivatization. FAME were analysed in a
143
gas chromatrograph HP6890A (Hewlett-Packard, Avondale, PA, USA), equipped with a
144
flame-ionization detector (GLC-FID) and a BPX70 capillary column (50m x 0.32 mm x 0.25
145
µm; SGE Europe Ltd, Courtaboeuf, France) according to the conditions described by
146
Vingering & Ledoux (2009). Supelco 37 and CRM-164 were used for identification of fatty
147
acids. GLC-Nestlé36 was assayed for calculation of response factors and detection and
148
quantification limits (LOD: 0.15 µg/mL; LOQ: 0.46 µg/mL).
149 150
2.2.3. Elemental composition
151 152
2.2.3.1. Microwave-assisted acid digestion procedure
153
The microwave-assisted digestion proposed by Speedwave MW-3+ (Berghof, Germany) for
154
dried plant samples with some modifications was used for determination of Mo, B, Zn, P, Cd,
155
Co, Ni, Mn, Fe, Mg, Ca, Cu, Na, Al and K in dried seaweed samples. A sample with up to 0.2
156
g dry seaweed was placed in the digestion vessel and added with 5 mL of concentrated nitric
157
acid. The vessels were capped and placed in a microwave pressure digestor Speedwave
6
158
MWS-3+ (Berghof) and subjected to microwave radiation at 20 bar according to the following
159
program: room temperature was raised first to 130 ºC at 22 ºC/min and 30% of irradiation
160
power, then to 160 ºC at 6 ºC/min and 40% of irradiation power, remaining 5 min at this
161
temperature, and to 170 ºC at 5 ºC/min and 50% of irradiation power, remaining 5 min at this
162
temperature. The cooling process consisted in decreasing temperature first to 100 ºC for 4
163
min and then to room temperature. After cooling, acid digests were made up to 20 mL with
164
Milli-Q water. Three replicates were performed for each seaweed sample as well as blanks.
165
The content of each element is expressed as the mean plus standard deviation
166 167
2.2.3.2. Determination of 15 elements
168
The elemental composition was determined using an inductively coupled plasma (ICP)
169
optical emission spectrometer model Optima™ 7000 DV ICP-OES (Dual View, PerkinElmer
170
Life and Analytical Sciences, Shelton, CT, USA) with radial plasma configuration.
171
Standard plasma conditions were used namely 1300 W for radio-frequency power, 1.5
172
mL/min pump rate, and 15.0, 0.2 and 0.8 L/min for plasma, auxiliary and nebulizer gas flow,
173
respectively. Detection wavelengths were 202.031, 208.889, 213.857, 214.914, 226.502,
174
228.616, 231.604, 257.610, 259.939, 279.955, 317.933, 324.752, 330.237, 394.401, 769.896
175
nm for Mo, B, Zn, P, Cd, Co, Ni, Mn, Fe, Mg, Ca, Cu, Na, Al and K, respectively.
176
A multi-element standard (Inorganic Ventures) containing up to 10 mg/L of Mo, 2,5 mg/L
177
of B, 15 mg/L of Zn, 750 mg/L of P, 0.2 mg/L of Co and Ni, 1 mg/L of Cd and Cu, 15 mg/L of
178
Mn and Fe, 1000 mg/L of Mg, 3000 mg/L of Ca, 50 mg/L of Na, 5 mg/L of Al and 2000 mg/L
179
of K was used for the preparation of standard solutions in 2% HNO 3. Successive dilutions of
180
the stock reference solution (100, 50 and 10 times) were prepared and used for calibration
181
models and the concentration of each element was determined by direct interpolation in the
182
standard curve within its linear dynamic range. The limits of detection (LODs) were
183
calculated using y=yB+3SB, where SB is the standard deviation (SD) of the blank signal
184
estimated as sy/x, the residual SD taken from the calibration line, and yB is the blank signal
185
estimated from the intercept, also taken from the calibration line. The LODs were found to be 7
186
0.010, 0.0009, 0.010, 0.40, 0.002, 0.002, 0.003, 0.014, 0.16, 0.18, 2.06, 3.32, 1.17, 0.037,
187
3.75 mg/L for Mo, B, Zn, P, Cd, Co, Ni, Mn, Fe, Mg, Ca, Cu, Na, Al and K, respectively. Acid
188
digests for some seaweed samples were diluted up 400 times to assess the content of some
189
elements in particular for Mg, Na, K and Fe within calibration curve range values.
190
The accuracy of the method (microwave acid digestion and ICP-OES analysis) was
191
assessed by analysis of certified reference material NIES-03 (Seaweed Chlorella; LGC
192
standards, UK). Five replicates of reference material were subject to microwave digestion
193
and analysed three times by ICP-OES. Recovery ranged between 89 and 108 %.
194 195
2.2.4. FTIR-ATR and FT-Raman Analysis
196
Samples of milled, dried algal material were analyzed by Fourier Transform Infrared
197
Spectroscopy with attenuated total reflectance (FTIR-ATR) and Fourier Transform Raman
198
spectroscopy (FT-Raman) according to the method described by Pereira (2006) and by
199
Pereira, Amado, Critchley, van de Velde & Ribeiro-Claro (2009).
200
The FTIR spectra of milled dried seaweed were recorded on an Bruker Tensor 27
201
spectrometer (Bruker Scientific Instruments, Massachusetts, USA), using a Golden Gate
202
single reflection diamond ATR system (Specac Lda, Cranston, USA), with no need for
203
sample preparation. All spectra resulted from the average of two counts, with 128 scans
204
each and a resolution of 2 cm−1.
205
The room temperature FT-Raman spectra were recorded on a RFS-100 Bruker FT-
206
spectrometer using a Nd:YAG laser with excitation wavelength of 1064 nm. Each spectrum
207
was the average of two repeated measurements, with 150 scans at a resolution of 4 cm−1.
208
For FT-Raman, the samples of seaweeds were first submitted to depigmentation by
209
immersion in cold calcium hypochlorite (4%) for 30-60 s at 4 ºC and then washed and dried
210
at 60 ºC.
211 212
2.3. Statistical analysis
8
213
One-way analysis of variance (ANOVA) was carried out with SigmaStat™ (Systat Software,
214
Chicago, IL, USA), to assess differences between seaweed species in terms of proximate or
215
elemental composition at a significance level of P=0.05. The Holm-Sidak method was used
216
for pairwise comparisons at a significance level of P=0.05. FA data in turn, were analysed
217
using the IBM SPSS Statistics v22 for Mac. Normality and homogeneity were examined and
218
One way ANOVA with the Bonferroni test for post-hoc analyses was applied to evaluate
219
statistical differences between seaweeds (p
S0308-8146(15)00429-X http://dx.doi.org/10.1016/j.foodchem.2015.03.057 FOCH 17304
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
24 March 2014 9 March 2015 18 March 2015
Please cite this article as: Rodrigues, D., Freitas, A.C., Pereira, L., Rocha-Santos, T.A.P., Vasconcelos, M.W., Roriz, M., Rodríguez-Alcalá, L.M., Gomes, A.M.P., Duarte, A.C., Chemical composition of red, brown and green macroalgae from Buarcos bay in Central West Coast of Portugal, Food Chemistry (2015), doi: http://dx.doi.org/ 10.1016/j.foodchem.2015.03.057
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Chemical composition of red, brown and green macroalgae from Buarcos bay
2
in Central West Coast of Portugal
3 4
Dina Rodrigues1*, Ana C. Freitas1,2, Leonel Pereira3, Teresa A. P. Rocha-Santos1,2, Marta W.
5
Vasconcelos4, Mariana Roriz4, Luís M. Rodríguez-Alcalá4, Ana M. P. Gomes4, Armando C.
6
Duarte1
7 8
1
9
University of Aveiro, 3810-193 Aveiro, Portugal
CESAM - Centre for Environmental and Marine Studies & Department of Chemistry,
10
2
11
Portugal;
12
3
13
Research, Department of Life Sciences, Faculty of Sciences and Technology, University of
14
Coimbra, P-3001-455 Coimbra, Portugal
15
4
16
de Biotecnologia, Universidade Católica Portuguesa/Porto, Rua Dr. António Bernardino
17
Almeida, 4200-072 Porto, Portugal
ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa, Viseu,
MARE – Marine and Environmental Sciences Centre & IMAR – Institute of Marine
CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior
18 19
*Corresponding author: Mailing address: 1CESAM - Centre for Environmental and Marine
20
Studies & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal.
21
E-mail: [email protected])
22 23 24
This manuscript has been submitted for publication in Food Chemistry
25 26
It is not to be reproduced or cited without the written permission of the authors.
27
1
28 29
Abstract
30
Six representative edible seaweeds from the Central West Portuguese Coast, including the
31
less studied Osmundea pinnatifida, were harvested from Buarcos bay, Portugal and their
32
chemical characterization determined. Protein content, total sugar and fat contents ranged
33
between 14.4 and 23.8%, 32.4 and 49.3% and 0.6-3.6%. Highest total phenolic content was
34
observed in Codium tomentosum followed by S. muticum and O. pinnatifida. Fatty acid (FA)
35
composition covered the branched chain C13ai to C22:5 n3 with variable content in n6 and
36
n3 FA; low n6:n3 ratios were observed in O. pinnatifida, Grateloupia turuturu and C.
37
tomentosum. Some seaweed species may be seen as good sources of Ca, K, Mg and Fe,
38
corroborating their good nutritional value. According to FTIR-ATR spectra, G. turuturu was
39
associated with carrageenan seaweed producers whereas Gracilaria gracilis and O.
40
pinnatifida were mostly agar producers. In the brown algae, S. muticum and Saccorhiza
41
polyschides, alginates and fucoidans were the main polysaccharides found.
42 43 44
Keywords: Chemical composition, Elements, Fatty acids, FTIR-ATR characterization,
45
Seaweeds
46
2
47
1. Introduction
48
Algae, in particular, edible seaweeds, are a very interesting natural source of compounds
49
with biological activity that could be used as functional ingredients (Plaza, Cifuentes &
50
Ibánez, 2008). Considering their great taxonomic diversity, research on identification of
51
biologically active compounds is encouraging in such a vast untapped resource (Lordan,
52
Ross & Stanton, 2011). Some algae live in complex habitats and are often subject to extreme
53
conditions. Their metabolism can be influenced by parameters such as water temperature,
54
salinity, light and nutrients, being forced to quickly adapt to continuously new environmental
55
conditions and in order to survive they produce a wide variety of biologically active secondary
56
metabolites (Plaza et al., 2008). Seaweeds can in fact provide several compounds at
57
different quantities and for that, research on identification of bioactive compounds from algae
58
can be seen as an almost unlimited field (Lordan et al., 2011).
59
Seaweeds are known to be of low calorie content, rich in polysaccharides, minerals,
60
vitamins, proteins, steroids and dietary fibers (Lordan et al., 2011) making them increasingly
61
sought for commercial purposes. They are of potential interest to be incorporated in the diet
62
as added-value foods because they are claimed to contain low cholesterol content, fight
63
obesity, reduce blood pressure, tackle free radicals and promote healthy digestion (Plaza et
64
al., 2008); furthermore, some of them have demonstrated biological properties such as
65
antibacterial and anticoagulant activities (Alghazeer, Whida, Abduelrhman, Ammoudi &
66
Azwai, 2013; Chandía & Matsuhiro, 2008). The total concentration of polysaccharides in the
67
seaweeds can range from 4 to 76% of dry weight (Holdt and Kran, 2011) where agar, algins
68
carrageenans and fucoidans are some of the polysaccharides widely used by man, some
69
with various biological activities (Mak, Wang. Liu, Hamid, Li, Lu & White, 2014; Mak, Hamid,
70
Liu, Lu & White, 2013). Protein content is also variable and the highest contents are
71
generally found in green and red seaweeds (10-30% of dry weight) in comparison to brown
72
seaweeds (5-15% of dry weight) (Holdt and Kran, 2011; Zhou, Robertson, Hamid, Ma & Lu,
73
2014). Phospholipids and glycolipids are the major classes of lipids found in seaweeds
74
accounting for 1-5% of cell composition (Chojnacka, Saeid, Witkowska & Tuhy, 2012); 17 to 3
75
63 mg/g of lipid content has been reported for the brown seaweed Undaria pinnatifida
76
(Boulom, Robertson, Hamid, Ma & Lu, 2014). Phenolic compounds are another important
77
group of chemical compounds in seaweeds (Lordan et al. 2011) varying quantitatively and
78
qualitatively for each specimen of red, brown or green seaweeds. Seaweeds are also
79
recognized as sources rich in several elements such as Ca, Mg, Na, P and K or trace
80
elements such as Zn, I or Mn. This elemental richness is related to their capacity to retain
81
inorganic compounds accounting for up to 36% of dry matter in some species (Lordan et al.,
82
2011).
83
According to Chojnacka et al. (2012), there are about ten thousand identified algae
84
species, yet only about 5% thereof are used as human food or as animal feed. More than
85
one hundred seaweed species are used worldwide, especially in Asian countries, where they
86
are used as sea vegetables. In Southern Europe the use of edible seaweeds for food
87
purposes is still residual, not yet fitting with the regular consumption habits. However, this
88
trend is changing and currently researchers are searching for, and studying, less-known
89
seaweed species not only because of their potential biological properties due to specific
90
functional compounds but mostly for their nutrient profile, which is being considered as
91
important alternative protein and complex carbohydrate sources (Ibañez & Cifuentes, 2013).
92
The main objective of the present study was to determine the chemical composition of
93
six different edible species from each of the main seaweed groups (red, brown and green
94
algae) occurring at Buarcos Bay (Figueira da Foz, Portugal). The selection was based on
95
representative species from the Central West Portuguese Coast including the less studied
96
Osmundea pinnatifida. Information on the proximal composition, fatty acids profile and
97
elemental composition of the six seaweeds is presented as well as the identification of the
98
principal seaweed colloids by FTIR-ATR and FT-Raman.
99 100
2. Material and methods
101 102
2.1. Specimens of seaweeds 4
103
Specimens of red algae (Rhodophyta, Florideophyceae) Osmundea pinnatifida
104
(Ceramiales), Grateloupia turuturu (Halymeniales) and Gracilaria gracilis (Gracilariales),
105
brown algae (Heterokontophyta, Phaeophyceae) Sargassum muticum (Fucales) and
106
Saccorhiza polyschides (Tilopteridales) and of green algae (Chlorophyta, Ulvophyceae)
107
Codium tomentosum (Bryopsidales), were harvested in April 2012 from Buarcos bay (Figueira
108
da Foz, Portugal). The classification of these seaweeds was based on AlgaeBase (Guiry &
109
Guiry, 2013). All six selected species are edible being used as food for humans all around the
110
world (SIA, 2015; Munier, Dumay, Morançais, Jaouen & Fleurence, 2013; Lodeiro et al., 2012;
111
Roo, Mantri, Ganesan & Kumar, 2007). The seaweeds were first washed with running tap
112
water and then with deionised water to eliminate residues from the thalli surface and then
113
dried in an oven at 60 ºC. The dried seaweeds were milled to less than 1.0 mm particle size.
114 115
2.2. Chemical characterization of seaweeds
116 117
2.2.1. Proximate composition
118
Content in moisture, organic matter and ash were determined according to AOAC methods,
119
(1990). Nitrogen content was determined by the Kjeldahl method adapted from where protein
120
content is estimated by multiplying the nitrogen content by 6.25 (Munier et al., 2013; Denis,
121
Morançais, Li, Deniaud, Gaudin & Wielgosz-Collin, 2010) whereas total fat content was
122
determined by Soxhlet extraction. Total sugar content was determined by calculation, i.e. by
123
subtracting protein content and fat content from total organic content. Total polyphenols were
124
extracted from 0.1 g of dry seaweed in 10 mL of ethyl acetate after 30 min of sonication (on a
125
water bath ultrasonicator, Ultrasonik 57H Ney). The extract was filtered with anhydrous
126
sodium sulfate (Sigma) and brought to dryness with a rotary evaporator (Laborato 4000,
127
Heidolph). The residue was re-dissolved in 5 ml of milliQ water and the content of total
128
polyphenols of 2 mL was determined by colorimetric method of Folin-Ciocalteu, using
129
cathecol (0 to 75 mg/L) as standard and expressed as g cathecol equivalent per g of dry
130
seaweed. 5
131 132
2.2.2 Analysis of fatty acids
133
Hexane and methanol were HPLC grade (VWR Scientific, West Chester, PA). Sodium
134
sulphate was analytic grade and purchased to Panreac (Barcelona, Spain). Methyl
135
tricosanoate (99%) and Supelco 37 FAME mix were obtained from Sigma (Sigma-Aldrich, St.
136
Louis, MO, USA). GLC-Nestlé36 was purchased to Nu-Chek Prep, inc. (Elysian, Minnesota,
137
USA) while butterfat CRM-164 (EU Commission; Brussels, Belgium) was from Fedelco Inc.
138
(Madrid, Spain).
139
For the analysis of the total fatty acid (FA) composition, 100 mg of sample were
140
accurately weighed and prepared according to Sánchez-Avila, Mata-Granados, Ruiz-
141
Jiménez & Luque de Castro (2009). For quantification purposes samples were added with
142
100 µL of methyl tricosanoate (1.28 mg/mL) prior to derivatization. FAME were analysed in a
143
gas chromatrograph HP6890A (Hewlett-Packard, Avondale, PA, USA), equipped with a
144
flame-ionization detector (GLC-FID) and a BPX70 capillary column (50m x 0.32 mm x 0.25
145
µm; SGE Europe Ltd, Courtaboeuf, France) according to the conditions described by
146
Vingering & Ledoux (2009). Supelco 37 and CRM-164 were used for identification of fatty
147
acids. GLC-Nestlé36 was assayed for calculation of response factors and detection and
148
quantification limits (LOD: 0.15 µg/mL; LOQ: 0.46 µg/mL).
149 150
2.2.3. Elemental composition
151 152
2.2.3.1. Microwave-assisted acid digestion procedure
153
The microwave-assisted digestion proposed by Speedwave MW-3+ (Berghof, Germany) for
154
dried plant samples with some modifications was used for determination of Mo, B, Zn, P, Cd,
155
Co, Ni, Mn, Fe, Mg, Ca, Cu, Na, Al and K in dried seaweed samples. A sample with up to 0.2
156
g dry seaweed was placed in the digestion vessel and added with 5 mL of concentrated nitric
157
acid. The vessels were capped and placed in a microwave pressure digestor Speedwave
6
158
MWS-3+ (Berghof) and subjected to microwave radiation at 20 bar according to the following
159
program: room temperature was raised first to 130 ºC at 22 ºC/min and 30% of irradiation
160
power, then to 160 ºC at 6 ºC/min and 40% of irradiation power, remaining 5 min at this
161
temperature, and to 170 ºC at 5 ºC/min and 50% of irradiation power, remaining 5 min at this
162
temperature. The cooling process consisted in decreasing temperature first to 100 ºC for 4
163
min and then to room temperature. After cooling, acid digests were made up to 20 mL with
164
Milli-Q water. Three replicates were performed for each seaweed sample as well as blanks.
165
The content of each element is expressed as the mean plus standard deviation
166 167
2.2.3.2. Determination of 15 elements
168
The elemental composition was determined using an inductively coupled plasma (ICP)
169
optical emission spectrometer model Optima™ 7000 DV ICP-OES (Dual View, PerkinElmer
170
Life and Analytical Sciences, Shelton, CT, USA) with radial plasma configuration.
171
Standard plasma conditions were used namely 1300 W for radio-frequency power, 1.5
172
mL/min pump rate, and 15.0, 0.2 and 0.8 L/min for plasma, auxiliary and nebulizer gas flow,
173
respectively. Detection wavelengths were 202.031, 208.889, 213.857, 214.914, 226.502,
174
228.616, 231.604, 257.610, 259.939, 279.955, 317.933, 324.752, 330.237, 394.401, 769.896
175
nm for Mo, B, Zn, P, Cd, Co, Ni, Mn, Fe, Mg, Ca, Cu, Na, Al and K, respectively.
176
A multi-element standard (Inorganic Ventures) containing up to 10 mg/L of Mo, 2,5 mg/L
177
of B, 15 mg/L of Zn, 750 mg/L of P, 0.2 mg/L of Co and Ni, 1 mg/L of Cd and Cu, 15 mg/L of
178
Mn and Fe, 1000 mg/L of Mg, 3000 mg/L of Ca, 50 mg/L of Na, 5 mg/L of Al and 2000 mg/L
179
of K was used for the preparation of standard solutions in 2% HNO 3. Successive dilutions of
180
the stock reference solution (100, 50 and 10 times) were prepared and used for calibration
181
models and the concentration of each element was determined by direct interpolation in the
182
standard curve within its linear dynamic range. The limits of detection (LODs) were
183
calculated using y=yB+3SB, where SB is the standard deviation (SD) of the blank signal
184
estimated as sy/x, the residual SD taken from the calibration line, and yB is the blank signal
185
estimated from the intercept, also taken from the calibration line. The LODs were found to be 7
186
0.010, 0.0009, 0.010, 0.40, 0.002, 0.002, 0.003, 0.014, 0.16, 0.18, 2.06, 3.32, 1.17, 0.037,
187
3.75 mg/L for Mo, B, Zn, P, Cd, Co, Ni, Mn, Fe, Mg, Ca, Cu, Na, Al and K, respectively. Acid
188
digests for some seaweed samples were diluted up 400 times to assess the content of some
189
elements in particular for Mg, Na, K and Fe within calibration curve range values.
190
The accuracy of the method (microwave acid digestion and ICP-OES analysis) was
191
assessed by analysis of certified reference material NIES-03 (Seaweed Chlorella; LGC
192
standards, UK). Five replicates of reference material were subject to microwave digestion
193
and analysed three times by ICP-OES. Recovery ranged between 89 and 108 %.
194 195
2.2.4. FTIR-ATR and FT-Raman Analysis
196
Samples of milled, dried algal material were analyzed by Fourier Transform Infrared
197
Spectroscopy with attenuated total reflectance (FTIR-ATR) and Fourier Transform Raman
198
spectroscopy (FT-Raman) according to the method described by Pereira (2006) and by
199
Pereira, Amado, Critchley, van de Velde & Ribeiro-Claro (2009).
200
The FTIR spectra of milled dried seaweed were recorded on an Bruker Tensor 27
201
spectrometer (Bruker Scientific Instruments, Massachusetts, USA), using a Golden Gate
202
single reflection diamond ATR system (Specac Lda, Cranston, USA), with no need for
203
sample preparation. All spectra resulted from the average of two counts, with 128 scans
204
each and a resolution of 2 cm−1.
205
The room temperature FT-Raman spectra were recorded on a RFS-100 Bruker FT-
206
spectrometer using a Nd:YAG laser with excitation wavelength of 1064 nm. Each spectrum
207
was the average of two repeated measurements, with 150 scans at a resolution of 4 cm−1.
208
For FT-Raman, the samples of seaweeds were first submitted to depigmentation by
209
immersion in cold calcium hypochlorite (4%) for 30-60 s at 4 ºC and then washed and dried
210
at 60 ºC.
211 212
2.3. Statistical analysis
8
213
One-way analysis of variance (ANOVA) was carried out with SigmaStat™ (Systat Software,
214
Chicago, IL, USA), to assess differences between seaweed species in terms of proximate or
215
elemental composition at a significance level of P=0.05. The Holm-Sidak method was used
216
for pairwise comparisons at a significance level of P=0.05. FA data in turn, were analysed
217
using the IBM SPSS Statistics v22 for Mac. Normality and homogeneity were examined and
218
One way ANOVA with the Bonferroni test for post-hoc analyses was applied to evaluate
219
statistical differences between seaweeds (p
