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JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 18 (2005) 79–88
www.elsevier.com/locate/jfca

Original Article

Chemical composition and microbiological assays of marine

algae Enteromorpha spp. as a potential food source
M. Aguilera-Moralesa, M. Casas-Valdeza,*, S. Carrillo-Dom!ınguezb,
B. Gonza! lez-Acostaa,c, F. Pe! rez-Gilb
a
Laboratorio de Macroalgas, Centro Interdisciplinario de Ciencias Marinas, IPN, Becario COFAA y EDI Apdo.
Postal 592, 23000 La Paz, B.C.S., Mexico
b
Departamento de Nutricion! Animal, Instituto Nacional de Ciencias M!edicas y Nutricion
! Salvador Zubiran.
!
Vasco de Quiroga No. 15, 14000 D.F. Mexico
c
!
Laboratorio de Gen!etica Molecular, Centro de Investigaciones Biologicas del Noroeste A.C. Apdo,
Postal 128. 23000 La Paz, B.C.S., Mexico
Received 12 March 2002; received in revised form 19 December 2003; accepted 19 December 2003

Abstract

Enteromorpha spp. is a marine seaweed present almost year round. It often causes unsightly appearance
and foul odor from decomposition by micro-organisms. This generates expenses in cleaning beaches. This
work determines chemical and microbiological composition, toxicological evaluation of Enteromorpha spp.,
and recommends its use in human diets and promotion for commercial exploitation, and provides a
solution to an ecological problem. The seaweed was collected by hand on the beach during the winters of
1997 and 1998 along the Malecon (street and sidewalk adjacent to the beach) of La Paz, Baja California
Sur, Mexico. Chemical analysis indicated that Enteromorpha spp. has 9–14% protein; 2–3.6% ether extract;
32–36% ash, and n-3 and n-6 fatty acids 10.4 and 10.9 g/100 g of total fatty acid, respectively. The protein
of this seaweed has a high digestibility (98%). Salmonella was not found and mesophyllic aerobic and fecal
coliform did not exceed the norm. Alkaloids, cyanogenic glucids, saponins, and tannic acid were scarce to
null. Enteromorpha spp. is recommended for human consumption because it has several beneficial
components, such as minerals, protein, essential amino acids, essential fatty acid, and fiber.
r 2004 Elsevier Inc. All rights reserved.

Keywords: Enteromorpha spp.; Chemical composition; Microbiology analysis; Nutritional value; Marine algae

*Corresponding author. Tel.: +52-612-12-2-53-66; fax: +52-612-12-2-53-22.

E-mail address: [email protected] (M. Casas-Valdez).

0889-1575/$ - see front matter r 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2003.12.012
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80 M. Aguilera-Morales et al. / Journal of Food Composition and Analysis 18 (2005) 79–88

1. Introduction

Marine algae in human consumption has been documented since 600 BC. Seaweed is suitable
for human and animal feed, as well as for fertilizer, fungicides, herbicides, and phycocolloids
(algin, carrageenan, and agar) (Chapman and Chapman, 1980). The green algae genus
Enteromorpha has great potential for commercial exploitation because of its abundant and
varied chemical composition, and quality and concentration of basic nutriments for other living
organisms (Hoppe et al., 1979; Chapman and Chapman, 1980). In China, Japan, USA, France,
and Chile, this genus is harvested to prepare ‘‘aonori’’ (Nisizawa et al., 1987), which is included in
a great variety of dishes, including raw salads, soups, cookies, meals, and condiments (Ohono and
Critchley, 1993). Only a few studies deal with the chemical composition of this genus (Chapman
and Chapman, 1980; Gonza! lez, 1981; Lahaye and Jegou, 1992; Naidu et al., 1993). Data on the
cholesterol, fatty acid, anti-nutritional and anti-physiological factors, and microbial content are
scarce.
In Mexico, this marine algae is available almost throughout the year, and when present on
beaches, causes adverse public reactions because the decomposition leads to foul odors and
unpleasant appearance. Significant expense and work are required to keep the beaches clean. This
work determines the chemical composition, microbiological content, and anti-nutritional and
anti-physiological factors of Enteromorpha spp. Specimens were collected in 1997 and 1998 to
assay and to recommend its use, if warranted, for human consumption and commercial
exploitation and, at the same time, provide a financially profitable solution to a community
beautification problem.

2. Materials and methods

2.1. Sample collection

Sampling was carried out in the beaches along the Malecon in the city of La Paz, Baja
California Sur, Mexico (24
270 –24
060 N and 110
180 –110
400 W), in the winters of 1997 and 1998.
Samples of Enteromorpha spp. were collected manually from the intertidal zone, at depths
between 60 cm and 1.20 m.

2.2. Sample preparation

Samples were rinsed with fresh water to eliminate foreign materials such as sand,
shells, etc. Afterwards they were spread over a cement slab and sun-dried for three days.
The total sample collected each year was 25 kg. Whole plants were ground in a hammer mill (Jerza
model L) and then in a knife mill (Thomas Scientific model 5KH39QN5525) using a 1 mm mesh,
avoiding overheating that could lead to oxidation. They were stored in plastic bags at room
temperature and in the dark. A 1 kg subsample was taken for chemical and microbiological
analyses.
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2.3. Chemical analyses

The following analyses were carried out. Moisture (oven-drying at 60
C to constant weight),
ash (ignition at 550
C in an electric furnace) and ether extract (Soxhlet apparatus) were
determined using standard methods (AOAC, 1990). Nitrogen content was determined using a
micro-Kjeldahl method (AOAC, 1990). A conversion factor of 6.25 was used to calculate protein
content. Cell-wall fractions were analyzed according to Van Soest method described by Tejada
(1985). In this method to determine neutral detergent fiber (NDF), sodium lauryl sulfate is used to
break the cell-wall; this corresponds to the data of total fiber. To determine acid detergent fiber
(ADF), the algae was treated with cetyl trimethylamonium bromide to separate hemicellulose and
cellular content. Protein digestibility was determined by multienzymatic method described by Hsu
et al. (1977). Gross energy was determined using Calorimetric Parr Bomb.

2.4. Mineral analysis

Samples for mineral analysis were subjected to acid digestion and analyzed through atomic
absorption spectrophotometry following the procedures described by AOAC (1990).

2.5. Amino acids

Amino acids were analyzed by high-performance liquid chromatography, or HPLC (Waters

Instruments) using a fluorescence detector (Waters model 470) and derivatization with carbamate
in reversed-phase with an AccQ-Tag column (Waters). In each sample, the amino acid content
was measured twice, and values were expressed as mg/g of protein.

2.6. Anti-nutritional factors

Saponins and cyanogenic glycosides were determined using the qualitative tests described in the
National Institute of Nutrition’s Handbook of Analysis (INNSZ, 1984). Tannins were analyzed
through quantitative tests (INNSZ, 1984), and alkaloids were determined following the method
described by Dom!ınguez (1979).

2.7. Total lipids and cholesterol

These substances were determined following the method described by Folch et al. (1957).
Cholesterol was determined by direct saponification (Abell et al., 1951) using 5 alfa-cholestane as
an internal standard and through gas chromatography in a Varian 3400 CX chromatograph
equipped with a DB-5 capillary column (3 m  0.25 mm i.d.) and a flame-ionization detector.
Nitrogen was used as carrier gas, applying a flow rate of 30 ml/min. Temperatures were: column
280
C; injector 260
C and detector 280
C. Samples were analyzed in triplicate.
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2.8. Fatty acids

Methyl-ester fatty acids were obtained using boron trifluoride (Castro et al., 2001). Fatty acids
were quantified by gas chromatography using a DB23 column (30 m  0.25 mm i.d.) on a Varian
3400 CX gas–liquid chromatograph equipped with an autosampler and a flame-ionization
detector. Nitrogen was used as carrier gas at a flow rate of 30 mL/min. Temperatures were:
column 230
C, injector 150
C, detector 300
C. Retention times were compared with methyl-fatty
acids standards (Sigma). Samples were analyzed in triplicate.

2.9. Microbiological analysis

The presence of aerobic mesophyllic bacteria was determined by a viable count on agar plates
(Ferna! ndez, 1981). Fecal and total coliforms were estimated using the most-probable-number
technique (MPN) (Secretaria de Salud, 1995a). Salmonella was determined following the
procedures described in the Mexican Official Norm (Secretaria de Salud, 1995b). The analyses
were made in triplicate.

2.10. Statistical analysis

Student’s t-test was used for comparing samples from the two years (Steel and Torrie, 1985).

3. Results

The main constituents in Enteromorpha spp. were minerals, protein and ether extract. The gross
energy was 6.2471.62 kJ for 1997 and 10.073.51 kJ for 1998. A significant difference (Po0:05)
was found in nutriments levels of Enteromorpha spp. between the 2 years (Table 1). For amino
acids content, 9 of the 10 considered essential for humans are present in a higher quantity than in
an equivalent weight of soy (Table 2). Total lipids, cholesterol, and essential fatty acids content
are shown in Table 3. All such components, except ALA (alpha linolenic acid C18:3 n-3) were
higher in the 1997 samples. There were significant differences (Po0:05) in LN (linolenic acid
C18:2 n-6), ALA (alpha-linolenic acid C18:3 n-3), AA (arachidonic acid C20:4 n-6), and EPA
(eicosapentaenoic acid C20:5 n-3) content, between the samples of 1997 and 1998. Enteromorpha
spp. had high amount of C 18:2 n-6 and low concentrations of C 22:6 n-3, in both years.
Protein digestibility was 98% compared to the reference casein pattern. Anti-nutritional factors
(tannins, alkaloids, cyanogenic glucids, and saponin) that could affect digestibility were scarce to
none. Tannins were present at a concentration of 0.62–0.97 mg/g.
In the cellular wall of the analyzed algae, hemicellulose was dominant, with 16.64%70.64 and
13.04%70.52 in 1997 and 1998, respectively. There were significant differences (Po0:05) between
years. The values of total fiber determined were 38.4%70.63 and 33.1%70.77 for the years 1997
and 1998, respectively. The soluble fiber were 21.7%70.76 and 17.5%70.73. There was
significant difference in the previous values in both years (Po0:05), while for insoluble fiber
16.6%70.64 and 15.6%70.52 were obtained. Among the minerals (Table 4), sodium, potassium,
and phosphorus content were significantly different (Po0:05) between 1997 and 1998.
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Table 1
Chemical composition of the marine algae Enteromorpha spp.
(g/100 g) 1997 1998
Ash 36.3870.42a 32.6470.65b
Moisture 9.0070.74a 6.7070.68b
Crude protein 9.4570.52a 14.1070.85b
Ether extract 3.6070.73a 2.2070.41b
Gross energy (kJ/g) 6.2471.629a 10.073.51b
Mean and standard deviation is based on 3 samples from each year.
Letters represent significant differences (Po0:05) between the harvest years.

Table 2
Amino acids profile of Enteromorpha spp.
Amino acids (g/100 g protein) Enteromorpha spp. 1997 Enteromorpha spp. 1998 Soybean
Asp 4.40 4.91 NR
Ser 7.76 2.73 NR
Glu 9.60 7.55 NR
Gli 4.61 2.37 NR
His 1.33 3.14 NR
Arg 3.81 3.09 NR
Thr 4.38 3.69 4.60
Ala 4.77 4.84 NR
Pro 4.34 5.10 NR
Cys 4.29 4.20 NR
Tyr 2.44 2.94 NR
Val 6.99 4.00 4.90
Met 2.45 2.83 1.00
Lys 3.55 2.85 7.20
Iso 3.52 2.91 4.90
Leu 4.59 4.27 7.50
Phe 3.70 3.72 4.80
Try — — 1.10
NR=Not reported in the food tables from Morales de Leon et al. (2000).
The amino acid in bold are essential for humans.

Microbial analyses did not find any Salmonella spp., mesophyllic aerobic bacteria were 90,000
and 93,000 UFC/g in 1997 and 1998, respectively, and were below the norm of the Superior
Council of Public Hygiene of France (Mabeau, 1991), for the export of edible marine algae. Fecal
and total coliform did not exceed the norm listed by the Secretaria de Salud (1995a).

4. Discussion

The difference between the nutrient contents of the samples from the harvest years may result
from the extraordinary capability of seaweed to accumulate elements present in water (Carrillo
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Table 3
Lipids, cholesterol, and fatty acids of marine algae Enteromorpha spp.
Enteromorpha spp. 1997 Enteromorpha spp. 1998
Lipids (g/100 g dry algae) 2.24 2.27
Cholesterol (g/100 g dry algae) 0.11 0.08

Fatty acids (g/100 g of FA)

LA 9.11 6.93
ALA 3.54 6.39
AA 1.80 1.32
EPA 5.73 2.88
DHA 1.11 0.92
Total n-3 10.38 10.19
Total n-6 10.90 8.25

Table 4
Mineral content of Enteromorpha spp. with respect to other species
Mineral (g/100 g DM) Enteromorpha spp. 1997 Enteromorpha spp. 1998 E. proliferaa E. prolifera var. tubulosab
Na 7.7070.74 9.2070.62 0.001 3.53
K 1.1070.56 1.8070.77 0.022 3.30
Ca 2.1070.79 2.4970.69 0.023 1.50
Mg 0.4370.92 0.7170.55 0.007 2.20
P 2.7070.41 3.5070.71 0.006 NR
Zn (ppm) 0.0570.87 0.0270.85 NR NR
NR=not reported.
a
Tewari (1972) (source: India).
b
Ho (1979) (source: Hong Kong).

et al., 1992). However, mineral concentrations vary seasonally and annually. Barber and Cha! vez
(1983) pointed out that as a consequence of the increase in water temperature from ‘‘El Niño’’,
nutrients transported to the surface water decreases, which affects the metabolism of marine algae
photosynthesis, reproduction, and growth. At the end of 1997, ‘‘El Niño’’ was present, and its
effects were reflected in a decline in the abundance of Enteromorpha in 1998. On the other hand,
Dawes (1991) pointed out that seasonal changes in lipid, carbohydrate, and protein quantities are
possible indicators of the physiologic state of the algae.
The ash obtained in marine algae Enteromorpha spp. was higher than Enteromorpha prol!ıfera
var. tubulosa (20.9%) obtained by Tewari (1972) and Enteromorpha compressa (26%) obtained by
Lahaye and Jegou (1992), but similar to brown algae Macrocystis pyrifera (34.2%) and Sargassum
sinicola (37.2%) recommended for human consumption by Manzano and Rosales (1989).
Protein in the samples was similar to that of Enteromorpha prolifera var. tubulosa (12% to 15%)
reported by Tewari (1972) and to that of grains like oats (13.3%) and other cereals (Morales de
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! et al., 2000). Furthermore, 9 of 10 essential amino acids were also found, and in
Leon
amounts greater than in soy. The concentration of isoleucine, leucine, lysine and threonine
are higher to the FAO-OMS pattern (Young, 1994). The high percentage of protein (14%)
can be due to the mixture of species of Enteromorpha in the sample or to the prevalence of
E. intestinalis in both years. The protein content commends Enteromorpha spp. as a nutritive
complement.
Lipids and cholesterol content are very low with respect to milk, fish, and meat (Morales de
! et al., 2000). Lipids are within the range mentioned for seaweed (1–4%) (Chapman and
Leon
Chapman, 1980; Haroon et al., 2000), and are similar to that reported for E. intestinalis (18.5 mg/
g) and Ulva rotundata (19.5 mg/g) (Fleurence et al., 1994). Cholesterol is one of the sterols present
in the seaweed. Its content in green and brown algae (2–76% of total of sterols mixture) is lower
than red algae as mentioned by Gancheva et al. (2003).
The significant difference in average fatty acid content of the two harvests could also be due to
the fatty acid contents of the species collected (Shameel and Khan, 1991). Therefore, there is a
possibility that the predominance of some Enteromorpha species in 1998 contributed to these
differences.
As (n-3, n-6) PUFA have several beneficial biological effects in humans and animals (Sardesai,
1992; Govindan et al., 1993) some important fatty acids were determined; they represent 20% of
the total fatty acid. The fatty acid content of Enteromorpha spp. was high relative to other edible
foods like soybeans and beans. EPA (n-3) and DHA (n-3) is greater than other types of seaweed,
such as Porphyra sp., Laminaria sp., and Ulva sp., and vegetables, such as spinach, and lettuce
(Simopoulos, 1991; Jime! nez-Escrig and Goñi-Cambrodon, ! 1999).
Fleurence et al. (1994) and Khotimchenko et al. (2002) found that the highest concentrations of
fatty acids in E. intestinalis and E. compressa were of 18:3 n-3, 16:4 n-3 and 18:4 n-3, whereas in
Enteromorpha spp. the highest concentrations were of 18:2 n-6, 18:3 n-6 and C 20:5 n-3. The
percentages of the fatty acids evaluated in Enteromorpha spp. were different from the ones
reported by Fleurence et al. (1994) for E. intestinalis. The fatty acid C 18:2 n-6 varied between
6.9% and 9.1%, 18:3 n-3 3.5–6.4% and C 20:5 n-3 2.8–5.7%, whereas for E. intestinalis the first
represented 5.7%, the second 15.5% and the third 1%. All these differences could be due to the
proportion of species present in the samples of Enteromorpha spp.
High protein digestibility shows that the proteins of these algae are easily hydrolyzed for the
enzymes trypsin, chymotrypsin and peptidase (NRC, 1994). The anti-nutritional factors were
scarce to none. Therefore, no risk to the health of algae consumers exists. These factors have no
effect on the digestibility of proteins or other nutriments because the quantities are too low to
affect the different metabolic routes or enzymatic systems that metabolize such nutriments
(Liedner, 1978; Dom!ınguez, 1979).
The cell wall composition of Enteromorpha spp. in 1997 was larger than in 1998, perhaps from
development stadium when the algae were collected. The seaweed was small because ‘‘El Niño’’
delayed the appearance of the algae in the winter of 1998. Although the components of the cell
wall do not contribute to nutrition, they form an important component in diets. During transit in
the intestinal tract, this material absorbs water, metals, and organic substances that are eliminated
in feces (Brownsell and Griffith, 1993). Hemicellulose, besides favoring increase in volume and
weight of feces, increases bile production, which participates in the digestion of fatty acids
(Anderson et al., 1988).
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The values determined for total fiber, soluble fiber and insoluble fiber were high and similar to
those reported by Jime! nez-Escrig and Goñi-Cambrodon ! (1999) in green seaweed.
Enteromorpha spp. is low in energy, which may be determined by the content of dietary
fiber in these algae. The significant difference between the two annual samples was due to the
higher quantity of carbohydrates and ether extract of Enteromorpha spp. from 1998. Energy
content was similar to Nori, Sargassum and Macrocystis, 6.24, 8.8 and 9.2 kJ, respectively, that
are used in human food (Carrillo et al., 1992; Castro et al., 1994; Jime! nez-Escrig and Goñi-
Cambrodon,! 1999).
Mineral content is high relative to other species and to some common vegetables.
Significant differences in the amount of Na, K, and P between collection years is related
to the capacity of algae to accumulate minerals according to environmental conditions,
which, in this work, were quite different in the two years (O’Kelley, 1974; Rodr!ıguez, 1995;
Meza, 1998). Enteromorpha spp. contains 28 times more Ca than spinach, 26 times
more than nopal, and 13 times more than quelite (Morales de Leon ! et al., 2000).
With respect to P, these algae contain more than 30 times the quantity of any of the common
vegetables. Since Ca and P are indispensable minerals for the growth, development, maintenance,
and function of living forms, Enteromorpha spp. is a potentially attractive source of these
minerals.
To obtain a product that can be stored for a long time and diminish the probability of the
bacteria and fungal agents that could alter the quality through decomposition, the content of
moisture is important in the preservation of the algae. With the drying technique used 10% of
moisture was obtained. This is a value considered appropriate for the preservation of this
seaweed.
In spite of drying the samples outdoor, the number of micro-organisms did not increase, even
after an unexpected rain fall. The levels of CF, MA, and Salmonella (S) in Enteromorpha spp.
were below the norm of the Superior Council of Public Hygiene of France for the export of edible
marine algae (Mabeau, 1991). This standard was used because a specific standard for edible algae
does not exist in Mexico.

5. Conclusions

Algae of the genus Enteromorpha can be recommended for human consumption. They are rich
in minerals, mainly Ca and P, and contain acceptable amounts of 9 of 10 essential amino acids as
well as hemicellulose, which enhances digestion and elimination, and increases bile production for
digestion of fatty acids. These algae are low in energy. The anti-nutritional factors measured were
scarce to none and the low level of micro-organisms supports the development of Enteromorpha
spp. for human food. Their collection would contribute to solve a problem of the local tourist
industry and enhance the aesthetic appeal of the local beaches.

Acknowledgements

We especially thank Ira Fogel from CIBNOR for editing the English text.
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