- Email: [email protected]
Effect of extraction conditions on the yield and purity of ulvan extracted from Ulva lactuca
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 d’Ingénieurs de Sfax, Route de Soukra, 3038 Sfax, Tunisia Laboratoire de Biotechnologie et Valorisation des Bio-GéoRessources, Institut Supérieur de Biotechnologie de Sidi Thabet, BP-66, 2020 Sidi Thabet, Ariana, Tunisia c Unité de Chimie Biologique Industrielle, Université de Liège, Gembloux Agro, Bio Tech, passage des Déportés 2, 5030 Gembloux, Belgium d Unité de Valorisation des Bio-ressources, Université de Liège, Gembloux Agro, Bio Tech, passage des Déportés 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 influence 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 specific. Ó 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, don’t 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.: þ216 73 260 970; fax: þ216 73 260 466. ** Corresponding author. E-mail addresses: [email protected] (H. Yaich), [email protected] (H. Garna). 0268-005X/$ e see front matter Ó 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 acidified 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 influence 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 acidified 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 efficiency (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 influence 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 quantified 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 acidðg=100 gdwÞ ¼
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 flow (35 C, 72 h). The dried alga was then milled in a mechanical grinder for 5 min, to obtain a fine 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 flask 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 filtered through cheesecloth and then was allowed to cool at room temperature. The filtrate was centrifuged at 10 C for 20 min at 10,000 rpm to remove solid particles. The supernatant was filtered again through Whatman filter paper N. 1 to remove the impurities and the filtrate 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 finely ground using a centrifugal milling. The ulvan extraction yield, subject of this study, was calculated as follows:
Yield ð%Þ ¼
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 Official 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, fitted with a hydrogen flame 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, film 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 muffle 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 flow 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 ¼ 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 significantly different (p < 0.05) for the same temperature and pH and at different time of extraction. Means with different capital letters are significantly different (p < 0.05) for the same temperature and time and at different pH of extraction. Means with different symbol are significantly 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 ¼ 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 (Duncan’s test) to determine significant 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 Student’s t-test, to establish significance 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 significant 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 significant differences (p > 0.05). We noted also a significant 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 significant differences (p > 0.05). Concerning the influence 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 significant effect (p < 0.05) on yield. According to Table 1, we noted also a significant 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 significant 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 significant 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 haven’t observed any significant 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 ¼ 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 significantly different (p < 0.05) for the same temperature and pH and at different time of extraction. Means with different capital letters are significantly different (p < 0.05) for the same temperature and time and at different pH of extraction. Means with different symbol are significantly 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 ¼ 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 ¼ 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 significant 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 significant differences (p > 0.05). We noted also a significant difference at pH 2 and at 80 C between 2 h and 3 h of extraction, whereas no significant 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 significant effect on the quantity of uronic acid at 80 C during 3 h and at 90 C during 2 h. Concerning the influence of the temperature, we noted only a significant 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 haven’t observed any significant differences on the uronic acid content. On the other hand, the time has a significant 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, significant 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 ¼ 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 ¼ 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 efficiency (%). pH 1.5
pH 2
80 C
Ulvan extraction efficiency
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 significant effect (p < 0.05) on the contents of uronic acid, but at pH 2, the increase in time did not show any significant 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, significant 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 significant 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 significant differences (p > 0.05). Concerning the influence of the pH, it hasn’t always an effect on the quantity of uronic acid. However, the pH has a significant 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 haven’t observed any significant 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 significant 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 ¼ 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 ¼ 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 efficiency 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 influenced 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 confirmed 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 profiles, extracts contained one to two major macromolecular populations. The first 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 influence 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 confirmed by the displacement of the first 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 significant 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 efficiency 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 purification of the extracts by the use of other techniques before alcohol precipitation (ultrafiltration or dialyse) allow us to avoid the presence of high content of inorganic material in the final product and to study the biological and technofunctional properties of ulvan extracted. References AOAC. (1990). Official methods of analyses. Washington, DC: Association of Official 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 fibre 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 fibres 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 fiber, 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). Nanofibers 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.
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 d’Ingénieurs de Sfax, Route de Soukra, 3038 Sfax, Tunisia Laboratoire de Biotechnologie et Valorisation des Bio-GéoRessources, Institut Supérieur de Biotechnologie de Sidi Thabet, BP-66, 2020 Sidi Thabet, Ariana, Tunisia c Unité de Chimie Biologique Industrielle, Université de Liège, Gembloux Agro, Bio Tech, passage des Déportés 2, 5030 Gembloux, Belgium d Unité de Valorisation des Bio-ressources, Université de Liège, Gembloux Agro, Bio Tech, passage des Déportés 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 influence 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 specific. Ó 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, don’t 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.: þ216 73 260 970; fax: þ216 73 260 466. ** Corresponding author. E-mail addresses: [email protected] (H. Yaich), [email protected] (H. Garna). 0268-005X/$ e see front matter Ó 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 acidified 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 influence 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 acidified 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 efficiency (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 influence 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 quantified 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 acidðg=100 gdwÞ ¼
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 flow (35 C, 72 h). The dried alga was then milled in a mechanical grinder for 5 min, to obtain a fine 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 flask 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 filtered through cheesecloth and then was allowed to cool at room temperature. The filtrate was centrifuged at 10 C for 20 min at 10,000 rpm to remove solid particles. The supernatant was filtered again through Whatman filter paper N. 1 to remove the impurities and the filtrate 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 finely ground using a centrifugal milling. The ulvan extraction yield, subject of this study, was calculated as follows:
Yield ð%Þ ¼
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 Official 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, fitted with a hydrogen flame 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, film 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 muffle 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 flow 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 ¼ 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 significantly different (p < 0.05) for the same temperature and pH and at different time of extraction. Means with different capital letters are significantly different (p < 0.05) for the same temperature and time and at different pH of extraction. Means with different symbol are significantly 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 ¼ 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 (Duncan’s test) to determine significant 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 Student’s t-test, to establish significance 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 significant 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 significant differences (p > 0.05). We noted also a significant 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 significant differences (p > 0.05). Concerning the influence 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 significant effect (p < 0.05) on yield. According to Table 1, we noted also a significant 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 significant 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 significant 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 haven’t observed any significant 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 ¼ 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 significantly different (p < 0.05) for the same temperature and pH and at different time of extraction. Means with different capital letters are significantly different (p < 0.05) for the same temperature and time and at different pH of extraction. Means with different symbol are significantly 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 ¼ 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 ¼ 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 significant 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 significant differences (p > 0.05). We noted also a significant difference at pH 2 and at 80 C between 2 h and 3 h of extraction, whereas no significant 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 significant effect on the quantity of uronic acid at 80 C during 3 h and at 90 C during 2 h. Concerning the influence of the temperature, we noted only a significant 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 haven’t observed any significant differences on the uronic acid content. On the other hand, the time has a significant 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, significant 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 ¼ 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 ¼ 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 efficiency (%). pH 1.5
pH 2
80 C
Ulvan extraction efficiency
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 significant effect (p < 0.05) on the contents of uronic acid, but at pH 2, the increase in time did not show any significant 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, significant 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 significant 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 significant differences (p > 0.05). Concerning the influence of the pH, it hasn’t always an effect on the quantity of uronic acid. However, the pH has a significant 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 haven’t observed any significant 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 significant 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 ¼ 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 ¼ 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 efficiency 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 influenced 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 confirmed 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 profiles, extracts contained one to two major macromolecular populations. The first 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 influence 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 confirmed by the displacement of the first 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 significant 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 efficiency 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 purification of the extracts by the use of other techniques before alcohol precipitation (ultrafiltration or dialyse) allow us to avoid the presence of high content of inorganic material in the final product and to study the biological and technofunctional properties of ulvan extracted. References AOAC. (1990). Official methods of analyses. Washington, DC: Association of Official 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 fibre 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 fibres 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 fiber, 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). Nanofibers 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.