Food Hydrocolloids 31 (2013) 375e382

Contents lists available at SciVerse ScienceDirect

Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Effect of extraction conditions on the yield and purity of ulvan extracted from Ulva
lactuca
Hela Yaich a, **, Haikel Garna b, *, Souhail Besbes a, Michel Paquot c, Christophe Blecker d, Hamadi Attia a
a

Laboratoire Analyses Alimentaires, Ecole Nationale dIngnieurs de Sfax, Route de Soukra, 3038 Sfax, Tunisia
Laboratoire de Biotechnologie et Valorisation des Bio-GoRessources, Institut Suprieur de Biotechnologie de Sidi Thabet, BP-66, 2020 Sidi Thabet, Ariana, Tunisia
c
Unit de Chimie Biologique Industrielle, Universit de Lige, Gembloux Agro, Bio Tech, passage des Dports 2, 5030 Gembloux, Belgium
d
Unit de Valorisation des Bio-ressources, Universit de Lige, Gembloux Agro, Bio Tech, passage des Dports 2, 5030 Gembloux, Belgium
b

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 21 July 2012
Accepted 9 November 2012

A study of the inuence of extraction conditions (pH: 1.5 and 2; temperature: 80
C and 90
C; extraction
time: 1e3 h), on the yield, chemical composition and purity of the sulphated cell wall polysaccharides
ulvan, extracted from the green seaweed Ulva lactuca and precipitated by alcohol is carried out. The
alcohol precipitate yields varied from 21.68% to 32.67% (%dw/dw) depending on the pH. At pH 2, the
alcohol precipitate yields and the uronic acid recovery from extract juice are higher than those obtained
at pH 1.5. Other compounds than ulvan such as cellulose, hemicellulose, proteins and ash are solubilized
from the cell walls of Ulva lactuca at both pH, and they are precipitated with alcohol. The alcohol
precipitate obtained from different extraction conditions has high uronic acid (20.37%e23.60%) and
neutral sugar content (20.09%e29.12%), especially when the conditions (pH, temperature) are drastic. It
contains rhamnose (13.35%e15.59%), glucose (2.90%e10.97%), and xylose (2.36%e2.73%). A decrease in
the molecular weight of ulvan was observed at acid pH, and for long extraction times. The presence of
proteins (1.94%e2.32%) and inorganic material (33.36%e47.15%) in alcohol precipitate prove the lower
purity of ulvan extracted and shows that ulvan precipitation with ethanol is not specic.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Ulvan
Ulva lactuca
Extraction conditions
Alcohol
Yields
Polysaccharide characterization

1. Introduction
Green seaweed Ulva lactuca represents an important biomass
available from proliferating algae in eutrophicated coastal water
between the littoral of Taboulba and Sayada in Tunisia. It is characterized by a very interesting chemical composition and especially
by its high content of cell wall polysaccharides (54% on the dry
weight basis). However, the bioaccumulation of minerals and
more especially the heavy metals (manganese, lead, copper and
cadmium) in this algae, dont allow this seaweed to be used for
human consumption or as an ingredient in some food preparations
(Yaich et al., 2011). For this reason, the valorisation of this alga can
be made only by the extraction of its different components such as
the water soluble Ulvan. This polysaccharide displays physicochemical and biological features of potential interest for diverse
applications (Lahaye & Robic, 2007). Notably it forms unusual soft

* Corresponding author. Tel.: 216 73 260 970; fax: 216 73 260 466.
** Corresponding author.
E-mail addresses: hela_yaich@yahoo.fr (H. Yaich), haikel_1999@yahoo.fr
(H. Garna).
0268-005X/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodhyd.2012.11.013

gels in the presence of divalent cations and borate (Lahaye & Axelos,
1993). Ulvan is mainly built on disaccharides repeating sequences
composed of sulphated rhamnose and glucuronic acid, iduronic
acid or xylose. The two major repeating disaccharides are aldobiuronic acids designated as type A: ulvanobiuronic acid 3-sulphate
(A3S) and type B: ulvanobiuronic acid 3-sulphate (B3S). Partially
sulphated xylose residues at O-2 can also occur in place of uronic
acids. In addition, glucuronic acid can branch at O-2 of rhamnose 3sulphate. Low proportions of galactose, glucose and protein are also
generally found in ulvan (Quemener, Lahaye, & Bobin Dubigeon,
1997; Robic, Rondeau-Mouro, Sassi, Leart, & Lahaye, 2009).
Based on literature, ulvan is extracted by chemical methods
using hot water often containing a calcium chelating agent such as
sodium oxalate (Lahaye & Robic, 2007; Robic, Sassi, Dion, Leart, &
Lahaye, 2009) or acidied ammonium oxalate (Robic, RondeauMouro, et al., 2009) or alkali (Ray & Lahaye, 1995) or sodium
chlorite or DMSO or phenol/acetic acid/water or acid (HCl) in very
determined conditions without studying the inuence of the
variation of the extraction parameters such as pH, time and
temperature in the same extraction conditions on the physicochemical characteristics of ulvan extracted. According to Robic,
Rondeau-Mouro, et al. (2009), Robic, Sassi, et al. (2009), the

376

H. Yaich et al. / Food Hydrocolloids 31 (2013) 375e382

sodium oxalate (50 mM, pH 6.5, 2 h, 85
C), the acidied ammonium oxalate (20 mM, pH 4.6, 1 h, 85
C) and acid extraction
(50 mM HCl, 0.5 h or 1 h at 85
C) are characterized by higher
extraction yields and more ulvan extraction efciency (percentage
recovery of the initial rhamnose content in raw material) than other
extractions using others solvents. Moreover, the uronic acid, the
neutral sugars content and the molecular weight distribution of the
ulvan are depends strongly on the extraction conditions.
In this context and in order to identify the conditions improving
ulvan extraction without degradation, we study the inuence of the
acid extraction conditions by varying the pH (1.5 and 2), the
temperature (80
C and 90
C) and the time (1 h, 2 h and 3 h) on the
extraction yield and on the molecular weight distribution of ulvan
extracted from U. lactuca.

2.3.2. Protein content

Total protein was determined by the Kjeldahl method. Protein
was calculated using a nitrogen conversion factor of 6.25 (Ortiz
et al., 2006). Data were expressed as percent of dry weight.
2.3.3. Uronic acid content
Total uronic acid was quantied colorimetrically from the
sulphuric acid hydrolysate method using galacturonic acid as
standard and sulphuric acid (2 M) as blank solution (Englyst,
Quigley, & Hudson, 1994). The amount of uronic acids (in g per
100 g of dry weight sample) was determined by the following
equation:

Uronic acidg=100 gdw

AT  VT  D  C  100
 0:91
AS  MT  DW

(2)

2. Materials and methods

2.1. Materials
The U. lactuca seaweed was collected from the littoral between
the area of Taboulba and Sayada (Monastir e Tunisia), at a weak
courantology and depth of 0.5 m, in July 2007. Fresh plants were
cleaned from epiphytes, rinsed on the spot with seawater, and then
placed in plastic bags. On their arrival at the laboratory, the
seaweed samples were again washed with distilled water and dried
in continuous air ow (35
C, 72 h). The dried alga was then milled
in a mechanical grinder for 5 min, to obtain a ne and homogeneous powder and was stored until use in hermetic bags in a dry,
dark place at room temperature (25
C).
2.2. Ulvan extraction
Different extractions are carried out by heating 60 g of algal
powder in 1 l of HCl solution (pH 1.5 and pH 2) stirred at 250 rpm in
a jacketed stainless steel reactor ask with a thermostatic bath in
a discontinuous process at 80
C and 90
C. At each constant pH and
temperature, samples are extracted after 1 h, 2 h and 3 h.
After extraction, the suspension was ltered through cheesecloth and then was allowed to cool at room temperature. The
ltrate was centrifuged at 10
C for 20 min at 10,000 rpm to remove
solid particles. The supernatant was ltered again through Whatman lter paper N. 1 to remove the impurities and the ltrate obtained was named extract juice.
The pH of this extract juice was adjusted to 3.5 with 1 M NaOH.
For ulvan precipitation, three volumes of 96% w/w ethanol were
added to one volume of the extract juice. The alcohol precipitate
was separated from the supernatants by centrifugation at
5000 rpm, for 20 min at 10
C. Then, the alcohol precipitate was
washed three times with 50%, 75% and 100% ethanol and centrifuged at 5000 rpm for 10 min at 10
C. Finally, it was dried in
a vacuum oven at 40
C to a constant weight and then nely ground
using a centrifugal milling.
The ulvan extraction yield, subject of this study, was calculated
as follows:

Yield %

Dry weight of alcohol precipitate

 100
Dry weight of alga

(1)

2.3. Chemical analysis

2.3.1. Dry matter
The dry matter was ascertained according to the Association of
Ofcial Analytical Chemists (AOAC, 1990).

where AT is the difference in absorbance of the sample; VT is the

total volume of sample solution (in 30 ml); D is the dilution of the
sample solution; C is the concentration of the standard used
(in mg/ml: here 0.1); AS is the difference in absorbance of the
standard (100 mg/ml); MT is the mass of the sample (in mg); DW
is the dry weight of the sample and 0.91 is the factor for converting the experimentally determined monosaccharides to
polysaccharides.
2.3.4. Neutral sugars
Neutral sugars are determined by GLC after chemical hydrolysis
alcohol precipitate and their derivatisation to alditols acetate
according to the procedure of Blakeney, Harris, Henry, and Stone
(1983).
Fifty milligrams of precipitate are hydrolysed with 1 M of H2SO4
(3 ml) at 100
C during 3 h. The reaction medium is then neutralised
with 1 ml N4OH (15 M) and 1 ml of 2-deoxy-D-glucose (1 mg/ml) is
added as internal standard.
A Hewlett Packard HP 6890 Series GC system, tted with
a hydrogen ame ionisation detector is used for neutral sugars
analysis. Alditol acetates derivatives were separated in a high
performance capillary column HP1 (length 30 m, internal diameter
0.5 mm, lm thickness 0.25 mm). The injector (splitless mode) and
detector temperatures were 250
C and 300
C, respectively. The
oven temperature was initially at 120
C programme to rise linearly
at 4
C/min until 220
C after the separation of sugar; then, it
reached 290
C at a rate of 35
C/min in order to condition the
column. The carrier gas was helium.
2.3.5. Ash contents
To remove carbon from alcohol precipitate, samples were
ignited and incinerated in the mufe furnace at 550
C for 16 h
(Lahaye & Jegou, 1993).
2.3.6. Molecular weight distribution
The polymer distribution of the alcohol precipitates obtained
under different conditions was determined by High Performance
Size Exclusion Chromatography (HPSEC). The HPSEC system
consisted of a Waters 2690-HPLC system (Waters Inc., Milford,
MA), equipped with a TSK gel GMPWxL column (300  7.8 mm;
Tosohaas Co. Ltd., Tokyo, Japan) and coupled on-line with
a Waters 2410 differential Refractometer Index detector (RI). This
column has exclusion limits between 1 kDa and 50 kDa using
dextrans.
100 ml of the sample (2 mg/ml) was injected in the chromatographic column. All analyses were carried out at a temperature of
25
C and at a ow rate of 0.7 ml/min with 50 mM sodium nitrate
(NaNO3) solution containing 0.05% sodium azide (NaN3).

H. Yaich et al. / Food Hydrocolloids 31 (2013) 375e382

377

Table 1
Extract juice and alcohol precipitate yields of ulvan obtained after extraction with HCl at varying pH, temperature and time (n 3).
Yields
pH 1.5

pH 2

80 C

Juice
Alcohol
precipitate
% Recovery

g
% (dw/dw)
g
% (dw/dw)

80
C

90 C

90
C

1h

2h

3h

1h

2h

3h

1h

2h

3h

1h

2h

3h

17.37
2.59
10.60
21.68aA
61.02

20.26
3.07
12.40
25.37bA
61.20

20.63
2.82
15.19
31.08cA
73.63

19.70
3.15
13.21
27.02Aa

67.05

22.80
3.43
14.35
29.35bA

62.93

22.75
3.25
15.68
32.09cA
68.92

18.39
2.52
13.02
26.63aB
70.79

19.10
2.69
13.19
26.99aA
69.05

20.72
2.80
14.66
29.98bA
70.75

19.51
2.71
13.75
28.12aA
70.47

20.83
3.10
15.33
31.37bB

73.59

21.97
3.30
15.97
32.67bA

72.69

Means with different small letters are signicantly different (p < 0.05) for the same temperature and pH and at different time of extraction.
Means with different capital letters are signicantly different (p < 0.05) for the same temperature and time and at different pH of extraction.
Means with different symbol are signicantly different (p < 0.05) for the same pH and time and at different temperature of extraction.

2.4. Statistical analysis

Analytical values were determined, using three independent
determinations. Values of different parameters were expressed as
the mean (n 3).
Statistical analyses were carried out using a statistical software
program (SPSS for Windows version 11.0). The data were subjected
to analysis of variance using the general linear model option
(Duncans test) to determine signicant differences between
samples (p < 0.05).
The results of effect of change of pH and of temperature on the
yield of alcohol precipitates and on uronic acid content were
statistically analysed and treated by a Students t-test, to establish
signicance of difference between the samples, at the level of
p < 0.05.
3. Results and discussion
3.1. Effect of extraction conditions on the alcohol precipitate yield
The dry matter content of extract juice before alcohol precipitation and the alcohol precipitate yields under different extraction
conditions are reported in Table 1. According to this table, on
average 3.05% and 2.85% (%dw/dw) of material was extracted
respectively from the U. lactuca seaweed at pH 1.5 and pH 2. After
alcohol precipitation of the acid extract juice, we recovered 65.79%
and 71.22% (%dw/dw) of the material extracted at pH 1.5 and pH 2.
Depending on the extraction conditions, the alcohol precipitate
yield varies from 21.68% to 32.67%. The highest yields were
obtained at pH 2 at 90
C (32.67%), pH 1.5 at 80
C (31.08%) and
90
C (32.09%) for 3 h, whereas the lowest yield (21.68%) results
from 1 h extraction at pH 1.5 and 80
C. This yield is close to the one
obtained (21.5%) by the extraction of ulvan from Ulva rotundata
with 50 mM HCl at 85
C for 30 min (Robic, Rondeau-Mouro, et al.,
2009). However, Lahaye et al., (1993) extracted only 12.2% of

polysaccharides from dried Ulva powder in boiling water for 30 min

after cellulose and protease treatments, whereas Toskas et al.
(2011) extract 24.30% of ulvan in hot water from Ulva rigida. With
sodium oxalate (50 mM, pH 6.5, 85
C, 2 h) 27.5% of the material is
extracted from the alcohol insoluble residue of U. rotundata which
is similar to our result obtained at pH 1.5, 90
C for 1 h (Robic, Sassi,
et al., 2009). On this basis, the extraction yields vary according to
the species and the extraction conditions.
According to Table 1, at constant pH and time, the increase in the
temperature extraction, results in an increase in the yield. This
effect can be observed at pH 1.5 as well as at pH 2. For example, at
pH 1.5 and during 1 h, the yield increased from 21.68% to 27.02%
with temperature. Moreover, at pH 1.5, there are signicant
differences (p < 0.05) between temperature on the yield at different
times 1 h and 2 h, while the yield found after 3 h of extraction did
not show any signicant differences (p > 0.05). We noted also
a signicant difference (p < 0.05) between temperature on the yield
at pH 2, during 2 h and 3 h of extraction, whereas the yield found at
1 h did not show any signicant differences (p > 0.05).
Concerning the inuence of the extraction time, the yield
increases with time. Indeed, at constant pH and temperature, the
yields of alcohol precipitate obtained for 1 h of extraction are lower
than those for 3 h. At pH 1.5 and at 80
C as well as 90
C, the
increase in the extraction time seemed to have a signicant effect
(p < 0.05) on yield. According to Table 1, we noted also a signicant
difference (p < 0.05) on the yield at pH 2, 80
C between 2 h and 3 h
of extraction, whereas the yield found at 1 h and 2 h did not show
any signicant differences (p > 0.05). At pH 2 and at 90
C, significant differences (p < 0.05) in the yield are observed between
different times of extraction, 1 h and 2 h, but not between 2 h and
3 h (p > 0.05).
On the other hand, the pH has a signicant effect (p < 0.05) on
the yield at 80
C during 1 h and at 90
C during 2 h. But in other
various extractions we havent observed any signicant differences
on the yield.

Table 2
Mean uronic acid content of extract juice and alcohol precipitate obtained after extraction with HCl at varying pH, temperature and time (n 3).
pH 1.5

pH 2

80 C

Juice
Alcohol
precipitate
% Recovery

g
% (dw/dw)
g
% (dw/dw)

80
C

90 C

90
C

1h

2h

3h

1h

2h

3h

1h

2h

3h

1h

2h

3h

2.97
16.68aA
2.49
23.54aA
83.83

3.25
17.81 bA
2.80
22.65abA
86.15

3.50
18.35 bA
3.30
21.74bA
94.28

3.44
17.49 aA
2.97
22.50aA

86.33

3.73
17.52 aA
3.37
23.49bA

90.34

3.92
18.13 aA
3.70
23.60bA

94.38

2.99
16.03 aA
2.86
21.97abB
95.65

3.27
17.35 bA
2.97
22.57aA
90.82

3.15
15.25 aB
3.12
21.34bA
99.04

3.23
16.69a bA
3.02
22.01aA
93.49

3.30
15.93 aB

3.12
20.37aB

94.54

3.83
17.45 bA

3.49
21.86aB

91.12

Means with different small letters are signicantly different (p < 0.05) for the same temperature and pH and at different time of extraction.
Means with different capital letters are signicantly different (p < 0.05) for the same temperature and time and at different pH of extraction.
Means with different symbol are signicantly different (p < 0.05) for the same pH and time and at different temperature of extraction.

378

H. Yaich et al. / Food Hydrocolloids 31 (2013) 375e382

Table 3
Mean neutral sugar composition of extract juice and alcohol precipitate obtained after extraction with HCl at pH 1.5 at 80
C (n 3; % R: % Recovery).
1h

Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
Total

2h

Juice

Alcohol precipitate

mg

mg

% (dw/dw)

1813.38
15.61
312.35
41.64
419.94
69.41
2672.33

1630.65
8.47
289.25
22.26
382.72
56.18
2389.53

15.39
0.08
2.73
0.21
3.61
0.53
22.55

3h

Juice

Alcohol precipitate

Juice

Alcohol precipitate

%R

mg

mg

% (dw/dw)

%R

mg

mg

% (dw/dw)

%R

89.92
54.26
92.60
53.45
91.13
80.93
89.41

2356.47
18.23
421.44
50.65
940.15
131.70
3918.64

1933.48
9.92
331.13
26.04
539.48
63.25
2903.30

15.59
0.08
2.67
0.21
4.35
0.51
23.41

82.04
54.14
78.57
51.41
57.38
48.02
74.08

2661.76
24.70
448.77
59.69
834.15
102.93
4132

2174.27
13.67
358.58
30.38
771.97
68.37
3417.24

14.31
0.09
2.36
0.20
5.49
0.45
22.90

81.68
55.34
79.90
50.89
92.54
66.42
82.70

Table 4
Mean neutral sugar composition of extract juice and alcohol precipitate obtained after extraction with HCl at pH 1.5 at 90
C (n 3; % R: % Recovery).
1h

Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
Total

2h

Juice

Alcohol precipitate

mg

mg

% (dw/dw)

2462.90
19.68
450.84
61.03
869.22
135.84
3999.51

2001.32
11.88
356.67
27.74
584.71
71.33
3053.65

15.15
0.09
2.70
0.21
6.58
0.54
25.27

3h

Juice

Alcohol precipitate

%R

mg

mg

% (dw/dw)

% R.

mg

mg

% (dw/dw)

% R.

81.25
60.36
79.11
45.45
67.26
52.51
76.35

2753.10
20.52
465.31
54.74
1418.75
100.36
4812.78

2103.95
11.48
363.09
25.83
1242.85
71.75
3818.95

14.66
0.08
2.53
0.18
8.66
0.50
26.61

76.42
55.94
78.03
47.18
87.60
71.49
79.35

2750.47
22.75
489.12
52.32
1797.25
109.2
5221.11

2326.34
14.11
395.30
29.80
1720.83
81.57
4567.95

14.83
0.09
2.52
0.19
10.97
0.52
29.12

84.57
62.02
80.81
56.95
95.74
74.69
87.49

3.2. Analysis of the carbohydrate composition of juice and alcohol

precipitate

Juice

Alcohol precipitate

The uronic acid content of alcohol precipitate is expressed in

percentage (%dw/dw) in relation to the dry matter of alcohol
precipitate or in quantity (g) contained in the dry precipitate
(Table 2). Depending on the extraction conditions (pH, temperature
and extraction time), the uronic acid content varied between
20.37% and 23.60%. The highest uronic acid content (23.60%) was
observed at pH 1.5 at 90
C after 3 h of extraction, whereas the
lowest content (20.37%) was obtained at pH 2 at 90
C after 2 h.
Uronic acid content found for all alcohol precipitate was higher
than the results obtained by Robic, Sassi, and Lahaye (2008)
(13.6%e19.8%) and by Ray & Lahaye, 1995 (20.9%), except at pH 2,
90
C for 2 h.
However, the variations in the uronic acid content of ulvan could
be attributed to some factors such as climate, species differences,
geography of development of the seaweed, and the method used to
extract ulvan (Robic, Sassi, et al., 2009).
According to Table 2, we noted that at 80
C and at pH 1.5, there
is a signicant difference (p < 0.05) for the quantity of uronic acid
between different extraction times 1 h and 3 h, while the quantity
found at 2 h and 3 h as well as at 1 h and 2 h did not show any
signicant differences (p > 0.05). We noted also a signicant
difference at pH 2 and at 80
C between 2 h and 3 h of extraction,
whereas no signicant difference in the quantity of uronic acid was
observed at 1 h and 2 h or at 1 h and 3 h. Table 2 revealed that at
90
C and at pH 1.5, the increase in the extraction time seemed to

3.2.1. Uronic acid content

Table 2 summarised the uronic acid content of the extract juice
and of the alcohol precipitate obtained in various extraction
conditions. The uronic acid content of extract juice varied from
15.25% to 18.35% (dw/dw). At pH 1.5 and at 80
C, as well as at 90
C,
the uronic acid content of juice increases with extraction time. At
pH 2 and at 80
C, the uronic acid content (in percentage) of juice
increases up to 2 h and decreases after, while at 90
C, this quantity
decreases up to 2 h and increases after. Nevertheless, the statistical
analysis showed that the temperature, pH and time seem to not
have mostly an effect on the quantity of uronic acid. However, the
pH has a signicant effect on the quantity of uronic acid at 80
C
during 3 h and at 90
C during 2 h. Concerning the inuence of the
temperature, we noted only a signicant differences (p < 0.05) in
the uronic acid content at pH 2 and at different time 2 h and 3 h. But
in other various extractions we havent observed any signicant
differences on the uronic acid content. On the other hand, the time
has a signicant effect (p < 0.05) on the amount of uronic acid at
80
C and at pH 1.5 as well as pH 2 between 1 h and 2 h of
extraction. At pH 2 and at 90
C, signicant differences (p < 0.05) in
the quantity of uronic acid are observed between different times of
extraction, 2 h and 3 h.

Table 5
Mean neutral sugar composition of extract juice and alcohol precipitate obtained after extraction with HCl at pH 2 at 80
C (n 3; % R: % Recovery).
1h

Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
Total

2h

Juice

Alcohol precipitate

mg

mg

% (dw/dw)

2253.51
16.55
401.03
53.34
516.92
88.30
3329.65

1857.95
11.71
342.42
23.43
423.15
59.89
2718.55

14.27
0.09
2.63
0.18
3.25
0.46
20.88

3h

Juice

Alcohol precipitate

Juice

Alcohol precipitate

%R

mg

mg

% (dw/dw)

%R

mg

mg

% (dw/dw)

%R

82.44
70.75
85.38
43.92
81.85
67.82
81.64

2284.24
19.09
433.54
49.65
477.47
93.58
3357.57

1888.43
11.87
344.43
23.75
382.70
62.02
2713.20

14.31
0.09
2.61
0.18
2.90
0.47
20.56

82.67
62.17
79.44
47.83
80.15
66.27
80.80

2604.50
20.72
470.34
53.87
549.08
89.09
3787.6

2090.39
11.72
389.93
26.38
425.11
67.43
3010.96

14.26
0.08
2.66
0.18
2.90
0.46
20.54

80.26
56.56
82.90
48.96
77.42
75.68
79.49

H. Yaich et al. / Food Hydrocolloids 31 (2013) 375e382

379

Table 6
Mean neutral sugar composition of extract juice and alcohol precipitate obtained after extraction with HCl at pH 2 at 90
C (n 3; % R: % Recovery).
1h

Rhamnose
Arabinose
Xylose
Mannose
Glucose
Galactose
Total

2h

Juice

Alcohol precipitate

mg

mg

% (dw/dw)

2277.05
15.60
417.55
46.82
565.84
91.70
3414.56

1912.64
11.00
356.12
23.37
489.50
61.87
2854.50

13.91
0.08
2.59
0.17
3.56
0.45
20.76

3h

Juice

Alcohol precipitate

Juice

Alcohol precipitate

%R

mg

mg

% (dw/dw)

%R

mg

mg

% (dw/dw)

%R

83.99
70.51
85.28
49.91
86.50
67.47
83.59

2482.01
16.61
465.24
49.84
608.56
101.77
3724.03

2223.78
13.80
418.68
27.60
533.70
75.14
3292.70

14.50
0.09
2.73
0.18
3.48
0.49
21.47

89.59
83.08
89.99
55.37
87.69
73.83
88.41

2923.07
21.94
509.12
54.86
750.51
109.72
4369.22

2132.29
12.77
397.70
25.55
568.61
71.87
3208.79

13.35
0.08
2.49
0.16
3.56
0.45
20.09

72.94
58.20
78.11
46.57
75.76
65.50
73.44

Table 7
Ulvan extraction efciency (%).
pH 1.5

pH 2

80
C

Ulvan extraction
efciency

90
C

80
C

90
C

1h

2h

3h

1h

2h

3h

1h

2h

3h

1h

2h

3h

43.12

51.05

57.40

52.91

55.55

61.37

49.20

50.00

55.29

50.52

58.73

56.34

have a signicant effect (p < 0.05) on the contents of uronic acid,

but at pH 2, the increase in time did not show any signicant
differences on the uronic acid quantity extracted.
On the other hand, we noted a markedly effect of temperature
on the quantity of uronic acid. At pH 1.5, signicant differences
(p < 0.05) in the uronic acid content were observed at different
time 1 h, 2 h and 3 h. We noted also a signicant difference
(p < 0.05) on the quantity of uronic acid at pH 2, during 2 h and 3 h
of extraction, whereas the amount found at 1 h did not show any
signicant differences (p > 0.05).
Concerning the inuence of the pH, it hasnt always an effect on
the quantity of uronic acid. However, the pH has a signicant effect
on the quantity of uronic acid at 80
C during 1 h and at 90
C
during 2 h and 3 h. Nevertheless, in other various extractions we
havent observed any signicant differences on the uronic acid
content.
Otherwise, the recovery of uronic acid from extract juice is
higher at pH 2 than at pH 1.5 whatever the temperature and time of
extraction. This result can be explained by the fact that at the

lowest pH, the extracted ulvan have been degraded to small

molecular weight compounds which have not been precipitated
with the ethanol.
3.2.2. Neutral sugars composition
The monosaccharides composition of the extract juice and
alcohol precipitate under different extraction conditions are presented in Tables 3e6. The juice of different extractions contained
signicant amounts of neutral sugars. Their quantities vary from
15.40% to 22.95% (%dw/dw). Depending on the extraction conditions (pH, temperature and extraction time), the rhamnose was the
most abundant sugar in the juice, followed by glucose and xylose.
Moreover, the composition of each sugar differs from one extraction to another.
In this study, the alcohol precipitate content of total neutral
sugars varies from 20.09% to 29.12%. Similar results have been
obtained for the water-soluble polysaccharides from Ulva species
(Lahaye et al., 1993). However, in this study, the total sugar
content was found to be lower than that of ulvan extracted from

Table 8
Mean proteins contents of extract juice and alcohol precipitate at different extraction conditions (n 3).
pH 1.5

pH 2

80
C

Juice
Alcohol
precipitate
% Recovery

g
g
% (dw/dw)

90
C

80
C

90
C

1h

2h

3h

1h

2h

3h

1h

2h

3h

1h

2h

3h

0.52
0.20
1.94
38.46

0.62
0.28
2.32
45.16

0.66
0.33
2.19
50.00

0.65
0.28
2.12
43.07

0.76
0.32
2.25
42.10

0.83
0.35
2.24
42.16

0.56
0.26
2.07
46.42

0.61
0.27
2.09
44.26

0.65
0.30
2.07
46.15

0.62
0.27
2.03
43.54

0.67
0.32
2.13
47.76

0.73
0.34
2.19
46.57

Table 9
Mean ash contents of extract juice and alcohol precipitate at different extraction conditions (n 3).
pH 1.5

pH 2

80
C

Juice
Alcohol
precipitate
% Recovery

g
g
% (dw/dw)

90
C

80
C

90
C

1h

2h

3h

1h

2h

3h

1h

2h

3h

1h

2h

3h

8.53
4.99
47.15
58.49

8.32
4.84
39.04
58.17

9.34
5.24
34.54
56.10

9.28
4.46
33.80
48.06

8.13
5.17
36.03
63.59

9.33
5.53
35.29
59.27

7.85
4.70
36.14
59.87

8.51
4.56
34.57
53.58

8.94
5.87
40.09
65.65

8.61
6.06
44.12
70.38

8.90
6.25
40.76
70.22

9.40
5.32
33.36
56.59

380

H. Yaich et al. / Food Hydrocolloids 31 (2013) 375e382

U. rotundata with 50 mM sodium oxalate at pH 6.5, 85
C for 2 h

(49.3%) (Robic, Rondeau-Mouro, et al., 2009). The highest amount
of total neutral sugars (29.12%) is obtained at pH 1.5 for 3 h at 90
C,
whereas the lowest content (20.09%) results from 3 h extraction at
pH 2 and 90
C.

Contrary to other factors, the pH has an important effect on sugar

content (Tables 3e6). All the alcohol precipitate extracted at pH 1.5
contained more total neutral sugars than those at pH 2, suggesting
that sugar content of alcohol precipitate increased with decreasing
pH. Indeed, at the same time and at the same temperature of

Fig. 1. HPSEC of alcohol precipitate (ulvan) obtained after acid extraction of Ulva lactuca after 1 h extraction
1.5 at 80
C, (b) at pH 1.5 at 90
C, (c) at pH 2 at 80
C and (d) at pH 2 at 90
C.

; after 2 h extraction

; after 3 h extraction

: (a) at pH

H. Yaich et al. / Food Hydrocolloids 31 (2013) 375e382

extraction, the averages content of total neutral sugars found at pH

1.5 were higher than those quantities found at pH 2.
Tables 3e6 indicated that the sugars composition of alcohol
precipitate was mainly represented by rhamnose, glucose and
xylose. Similar results were reported by Toskas et al. (2011) on the
composition of ulvan extracted from U. rigida. In this study, rhamnose content ranged from 13.35% to 15.59% of the extract dry
weight. Robic et al. (2008) obtain between 23.6% and 29.3% of
rhamnose for the ulvan extracted from U. rotundata after different
treatments of stabilisation. These variations in rhamnose content
can be attributed to the species and to the period of collection of
algae and also to the extraction conditions (Robic, Sassi, et al., 2009).
The efciency of ulvan extraction can be expressed as the
percentage recovery of the initial rhamnose content in the fresh
algae, since this sugar is the main neutral sugar constituent in ulvan
(Robic et al., 2008). The rhamnose yield varied between 43.12% and
61.37% of the initial rhamnose, according to the different extraction
conditions (Table 7). In the study of Robic et al. (2008), this yield
varied between 25% and 60%, according to the stabilization and
storage conditions of U. rotundata. The best yield was obtained at
pH 1.5, 90
C during 3 h but the lowest yield was obtained at the
same pH, 80
C during 1 h.
The glucose and xylose content in the alcohol precipitate vary
between 2.90% and 10.97% and from 2.36% to 2.73%, respectively.
Tables 3e6 showed that the glucose content was predominantly
inuenced by the pH. The residues extracted at pH 1.5 contained
more glucose than those at pH 2. Therefore, the glucose content
increased when the pH decreased. The highest glucose content
(10.97%) was obtained at pH 1.5 and at 90
C for 3 h of extraction.
The values were higher than those obtained for ulvan extracted
from U. rotundata (Robic et al., 2008). According to the bibliography
the xylose is referred to as part of ulvan structure in ulvanobioses
moieties and glucose is frequently associated with glucans or
amorphous cellulose co-extracted with ulvan (Lahaye, Jegou, &
Buleon, 1994; Lahaye & Ray, 1996; Lahaye & Robic, 2007.
These extracts were characterized also by the presence of traces
of other sugars such as galactose, mannose and arabinose in the
alcohol precipitate. The presence of these sugars except for the
galactose was conrmed with previous results obtained by Toskas
et al. (2011).
3.3. Non-carbohydrate components of the extract juice and alcohol
precipitate
Tables 8 and 9 describes the proteins contents and the ash
contents determined for the extract juice and for the alcohol
precipitate at different extraction conditions, respectively.
The protein content found in the present study was relatively
low. In fact, this content varies from 1.94% to 2.32% of the extract
dry weight (Table 8). This amount was in conformity with the value
reported by others for this seaweed species (Costa et al., 2012).
However, this quantity was found to be lower than that of others
Ulva species (9.4%e9.9%) (Robic, Sassi, et al., 2009) and higher than
that of U. rigida species (0.2%) (Toskas et al., 2011).
Otherwise, the alcohol precipitate obtained under different
extraction conditions was contaminated by proteins. These polymers have already been described as potential contaminants of cell
wall polysaccharides, mostly because they are part of the structure
of cell walls and closely associated with polysaccharides (Robic
et al., 2008).
The ash content of the juice and of the alcohol precipitates at
different extraction conditions are illustrated in Table 9. Unfortunately, we noted a high percentage of ash recovery from extract
juice, varying from 48.06% to 70.38%. All samples have high ash
content (33.36%e47.15% of dry extract). These results are in

381

accordance with the results of Costa et al. (2012). The richness of

alcohol precipitate in ashes can be attributed to the high ash content
(w20%) of U. lactuca (Yaich et al., 2011). Indeed, this seaweed was
characterized by its high bioaccumulation of minerals in the water.
The presence of a high level of ash in all fractions showed again
the lower purity levels of polysaccharide extracted.
3.4. Distribution of molecular weight
The distribution of molecular weights of different fractions of
alcohol precipitate is presented in Fig. 1.
Based on the RI proles, extracts contained one to two major
macromolecular populations. The rst population, referred to as A
eluted from 8.40 to 10.60 min and the second, referred to as B, from
10.60 to 13.00 min. A third minor population C consisted of smaller
molecular weight molecules (sugars, acids, ions..). It eluted from
13.00 to 14.40 min. However, the presence of three populations for
all precipitates was in agreement with the results reported by
others for Ulva armoricana (Robic, Sassi, et al., 2009).
With respect to the inuence of the time of extraction, the
degradation increased with the time of extraction. Indeed, at
constant pH and temperature, the degradation of precipitate obtained for 1 h of extraction was lower than those for 3 h. Moreover,
whatever the extraction conditions, we observed that ulvan was
degraded partially after 1 h of extraction. This is conrmed by the
displacement of the rst peak, high molecular weight, towards
lower molecular weights.
The molecular distribution of different ulvan populations was
also markedly affected by the pH of extraction. Indeed, the degradation of all precipitates was more important at pH 1.5 than pH 2.
At the lowest pH 1.5, the extracted ulvan was characteriszd by the
presence of high amount of small molecular weight compounds
than ulvan extracted at pH 2. These observations were also supported by Robic, Rondeau-Mouro, et al. (2009).
4. Conclusion
The effect of pH (1.5 and 2), time of extraction (1 h, 2 h and 3 h)
and temperature (80
C and 90
C) on the chemical characteristics
of acid-extracted ulvan from U. lactuca was shown. The pH was the
most signicant factor which affects the alcohol precipitate yield,
the monosaccharide composition and the macromolecular characteristics of ulvan extracted. At pH 1.5 the alcohol precipitate
yields and the recovery of uronic acids from extract juice are lower
than at pH 2, whereas the ulvan extraction efciency and the sugars
content of alcohol precipitate are higher at pH 1.5. Moreover, at
lowest pH of extraction, there is a presence of high quantity of small
molecular weight components in the alcohol precipitate which
originates from the degradation of high molecular components.
This phenomenon of degradation is more important when
increasing the temperature and the extraction time.
Furthermore, the presence of high level of ash in all fractions
showed the lower purity levels of polysaccharide extracted. The
purication of the extracts by the use of other techniques before
alcohol precipitation (ultraltration or dialyse) allow us to avoid
the presence of high content of inorganic material in the nal
product and to study the biological and technofunctional properties of ulvan extracted.
References
AOAC. (1990). Ofcial methods of analyses. Washington, DC: Association of Ofcial
Analytical Chemist.
Blakeney, A. B., Harris, P. J., Henry, R. J., & Stone, B. A. (1983). A simple and rapid
preparation of alditol acetates for monosaccharide analysis. Carbohydrate
Research, 113, 291e299.

382

H. Yaich et al. / Food Hydrocolloids 31 (2013) 375e382

Costa, C., Alves, A., Pinto, P. R., Sousa, R. A., Borges da Silva, E. A., Reis, R. L., et al.
(2012). Characterization of ulvan extracts to assess the effect of different steps
in the extraction procedure. Carbohydrate Polymers, 88, 537e546.
Englyst, H. N., Quigley, M. E., & Hudson, G. J. (1994). Determination of dietary bre
as non starch polysaccharides with gas liquid chromatography or spectrophotometric measurement of constituent sugars. Analyst, 119, 1497e1509.
Lahaye, M., & Axelos, M. A. V. (1993). Gelling properties of water-soluble polysaccharides from proliferating marine green seaweeds (Ulva spp.). Carbohydrate
Polymers, 22, 261e265.
Lahaye, M., & Jegou, D. (1993). Chemical and physicalechemical characteristics of
dietary bres from Ulva lactuca (L) Thuret and Enteromorpha compressa (L) Grev.
Journal of Applied Phycology, 5, 195e200.
Lahaye, M., Jegou, D., & Buleon, A. (1994). Chemical characteristics of insoluble
glucans from the cell wall of the marine green alga Ulva lactuca (L.) Thuret.
Carbohydrate Research, 262, 115e125.
Lahaye, M., & Ray, B. (1996). Cell-wall polysaccharides from the marine green alga
Ulva rigida (Ulvales, Chlorophyta) e NMR analysis of ulvan oligosaccharides.
Carbohydrate Research, 283, 161e173.
Lahaye, M., & Robic, A. (2007). Structure and functional properties of ulvan,
a polysaccharide from green seaweeds. Biomacromolecules, 8, 1765e1774.
Ortiz, J., Romero, N., Robert, P., Araya, J., Lopez-Hernandez, J., Bozzo, C., et al. (2006).
Dietary ber, amino acid, fatty acid and tocopherol contents of the edible
seaweeds Ulva lactuca and Durvillaea antarctica. Food Chemistry, 99, 98e104.

Quemener, B., Lahaye, M., & Bobin Dubigeon, C. J. (1997). Sugar determination in
ulvans by a chemical-enzymatic method coupled to high performance anion
exchange chromatography. Journal of Applied Psychology, 9, 179e188.
Ray, B., & Lahaye, M. (1995). Cell-wall polysaccharides from the marine green alga
Ulva rigida (Ulvales, Chlorophyta). Extraction and chemical composition.
Carbohydrate Research, 274, 251e261.
Robic, A., Sassi, J. F., & Lahaye, M. (2008). Impact of stabilization treatments of the
green seaweed ulva rotundata (chlorophyta) on the extraction yield, the
physico-chemical and rheological properties of ulvan. Carbohydrate Polymers,
74, 344e352.
Robic, A., Rondeau-Mouro, C., Sassi, J. F., Lerat, Y., & Lahaye, M. (2009). Structure and
interactions of ulvan in the cell wall of the marine green algae Ulva rotundata
(Ulvales Chlorophyceae). Carbohydrate Polymers, 77, 206e216.
Robic, A., Sassi, J. F., Dion, P., Lerat, Y., & Lahaye, M. (2009). Seasonal variability of physico-chemical and rheological properties of ulvan from two
Ulva species (Chlorophyta) of Brittany coast. Journal of Phycology, 45,
962e973.
Toskas, G., Hund, R. D., Laourine, E., Cherif, C., Smyrniotopoulos, V., & Roussis, V.
(2011). Nanobers based on polysaccharides from the green seaweed Ulva
Rigida. Carbohydrate Polymers, 84, 1093e1102.
Yaich, H., Garna, H., Besbes, S., Paquot, M., Blecker, C., & Attia, H. (2011). Chemical
composition and functional properties of Ulva lactuca seaweed collected in
Tunisia. Food Chemistry, 128, 895e901.