Biomacromolecules 2005, 6, 1871-1876
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Contributions of Intermolecular Interactions between Constitutive Arabinoxylans to the Flaxseeds Mucilage Properties J. Warrand,† P. Michaud,*,† L. Picton,‡ G. Muller,‡ B. Courtois,† R. Ralainirina,§ and J. Courtois† Laboratoire des Glucides - EPMV (CNRS-FRE 2779), IUT d’Amiens (GB), Universite´ de Picardie Jules Verne, Avenue des Faculte´ s, Le Bailly, 80 025 Amiens Cedex 1, France, Laboratoire Polyme` res, Biopolyme` res, Membranes (CNRS-UMR 6522), Universite´ de Rouen, Boulevard Maurice de Broglie, 76 821 Mont Saint-Aignan, France, and Centre de Valorisation des Glucides et des Produits Naturels, 33 Avenue Paul Claudel, 80 000 Amiens, France Received November 27, 2004; Revised Manuscript Received March 18, 2005
The main fraction (about 75%) of the mucilage extracted from seeds of Linum usitatissimum which consists of arabino-xylans (AX) has been studied in dilute and semidilute regimes by SEC/MALLS analysis and rheology, respectively. It has been found that AX contains 3 populations of about 5 000 000 g mol-1 (less than 10%), 1 000 000 g mol-1 (about 40%), and 200 000 g mol-1 (about 50%). We have also observed a great retention of polymer during the filtration procedure, which is much pronounced as the AX concentration increases. This evidences the presence of large aggregates in the solution. The retention can be greatly diminished if the filtration is conducted under higher temperature. Aggregation could result from the establishment of intermolecular associations via hydrogen bonds. This hypothesis seems to be confirmed by the two higher populations in molar masses which present a random coil conformation consistent with a low degree of branching. Rheological measurements, conducted at 20 g L-1, have confirmed the association tendency leading to pseudo gels behavior. Viscoelastic properties have been evidenced by time-temperature master curves of dynamic spectra. Such master curves have also been established with addition of chaotropic (i.e., KSCN) and lyotropic (i.e., NaCl) salts. It has been shown that intermolecular associations are greatly diminished under chaotropic salts influence. This has been also confirmed by SEC/MALLS analysis. These results point out the role of hydrogen bonds in the organization of the AX system. 1. Introduction Among the wide diversity of plant polysaccharides, some of them display characteristic rheological behaviors.1,2 In many cases, these properties have made them especially suitable as additives for several industries.3-5 The simple recuperation of the polymers constitutes frequently a principal key to their future uses. Thus, gums and mucilages represent an easy way to recover a large range of polysaccharides at a low cost.6 If many gums have found industrial applications,7 mucilages stay always less exploited, probably owing to the lack of information. A previous study reports the appearance of a mucilage around the seeds of Linum usitatissimum when they are wetted.8 This mucilage was described to consist of a large part of xylan (75%) as a water-soluble component, associated to pectin-like molecules (25%).9-17 Even if this major biopolymer (xylan) represents the second most abundant hemicellulosic polysaccharide in plant cell walls and grasses, few studies were realized at that time on these polymers in solution.18,19 Then, to bring more information * To whom correspondence should be addressed. Phone: (33) (3) 22 53 40 98. Fax: (33) (3) 22 95 71 17. E-mail:
[email protected]. † Universite ´ de Picardie Jules Verne. ‡ Universite ´ de Rouen. § Centre de Valorisation des Glucides et des Produits Naturels.
on these flaxseed mucilage polysaccharides, a large-scale extraction and purification method has been developed.17 This study allowed the characterization of this major fraction, as polydisperse high molecular weight (HMW) arabinoxylans (Mw ) 1.2 × 106 g mol-1, polydispersity coefficient ) 2.94). As HMW xylans are known to interact easily with each other when they present low ramification levels,20,21 the rheological properties of the mucilage16 could be supposed to be due to putative interactions between several groups. This hypothesis is supported by most of the models of plant cell walls, which are commonly referred to as noncovalent interactions of these polymers with other polysaccharides such as cellulose.22 As the mucilage is already used in some products with regard to their rheological behavior,23-27 the comprehension of the synergistic action of xylans should enhance their industrial developments. In this study, the possible presence of intermolecular interactions between these macromolecules was investigated in dilute and semidilute regimes through SEC/MALLS analysis and rheological measurements. 2. Experimental Section Polysaccharides Extraction and Purification from Mucilage. Yellow species of flaxseeds were gratefully given
10.1021/bm049249p CCC: $30.25 © 2005 American Chemical Society Published on Web 05/07/2005
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by Laboulet (Ets), Airaines (Somme), France. Polysaccharides from flaxseeds mucilage were extracted by an aqueous process (extraction with deionized water) under stirring (1/40 w/V, 2 h, 40 °C). They were purified on large scale and separated using anion exchange chromatography into two fractions, a neutral and an acidic one, following the methods described previously.17 Then, they were dialyzed 24 h against deionized water (14 000 Da). Purified and freeze-dried polymers extracts were used as raw material for SECMALLS investigations and rheological measurements. On Line SEC/MALLS. The average molecular weight and molecular weight distribution were determined by highpressure size exclusion chromatography (HPSEC) with on line multi-angle laser light scattering (MALLS) [DAWNEOS, Wyatt Technology Inc. (Santa Barbara, USA) filled with a K5 cell and a He-Ne laser (λ ) 690 nm)] and differential refractive index (DRI) detectors.28 Columns [OHPAK SB-G guard column, OHPAK SB 804 and 806 HQ columns (Shodex)] were eluted with LiNO3 0.1 M at 0.6 mL min-1. The solvent was filtered through a 0.1 µm filter unit (Millipore), degassed (ERC-413), and filtered through a 0.45 µm filter upstream column. The samples were filtered (0.45 µm) with or without a preheating step (80 °C, 15 min) and injected through a 100 µL full loop. Collected data were analyzed using the Astra V-4-81-05 software package. The concentration of each eluted fraction was determined with the DRI (ERC 7515A) according to the known value of dn/dC (0.15). Rheological Measurements. Rheological determinations were performed with a AR 2000 rheometer (TA instruments, New Castle, Delaware, USA). Measurements, comprehending a Peltier system, were conducted at 25 °C using either a double gap cylinder or steel cone-plate (40 mm radius, 2°) geometries. The double gap cylinder was thermostated with a circulating bath. All of the samples were solubilized at 20 g L-1 in deionized water, in 0.5 M NaCl or in 0.5 M KSCN with stirring until complete solubilization. The shear flow behavior was assessed over shear rates of 0.1-1000 s-1. Oscillatory sweeps were conducted between 0.05 and 100 Hz in the determined linear viscoelactic conditions. 3. Results and Discussion Seeds of Linum usitatissimum present a gel around them when they are wetted.8 This gum, describe as a mucilage, was first characterized to be compose by various constitutive polysaccharides consisting of two distinct groups: rhamnogalacturonans (25%) and arabinoxylans (75%).15-17 Then, after the conception of a large-scale purification procedure,17 further examinations have demonstrated the presence of two families in the pectin-like group and a wide distribution of the arabinoxylans (polydispersity coefficient of 2.94). In this study, the heterogeneity of this major fraction (i.e., arabinoxylans) has been approached by SEC-MALLS analysis in several concentrations from 0.5 to 2 g L-1 (Figure 1). All of these samples were comprised of 3 distinct groups. They were called 1, 2, and 3 respectively, with regard to their molar masses in decreasing order (from the first eluted to the latest). Astoundingly, the whole calculated recovered
Warrand et al.
Figure 1. 90° light scattering and DRI signals of the polymers solutions with different concentrations: 0.5 (full line), 1 (dash-dot line), and 2 g L-1 (dotted line).
mass ratio for each sample was increased inversely to the concentration, with 7, 15, and 40% respectively for the 2, 1, and 0.5 g L-1 solutions (Table 1). Those observed losses could suggest the existence of very aggregated structures, called macroaggregates, retained during the filtration procedure. This result seems to indicate an organization level of aggregates depending of the polymers concentrations. As light scattering is proportional to both molar masses and concentration, the obtained result for 2 g L-1 can be considered as nonrepresentative according to the poor precision of the light scattering response due to the very large losses of polymer (i.e., low resultant analysis concentration). Nevertheless, the molar masses obtained for 0.5 and 1 g L-1 are nearby. Yet, one can consider that these losses did not modify sensibly the distribution of the three populations. It can be see that molar masses are obtained with non-negligible uncertainty which decreases logically with the increase of the recovered mass. Thus, paradoxically, the precision is improved for the lower concentration and consequently, the most representative result has been obtained for 0.5 g L-1. The higher molar masses fraction (i.e., 1st population) about 5 500 000 g‚mol-1 represents less than 10% of the sample. The 2nd population (about 1 000 000 g mol-1) and the 3rd population (about 200 000 g mol-1) concern respectively 40% and 52% of the sample. We notice that the best precision is obtained for the second population because of the greater concentration and molar mass. At the concentration of 2 g L-1, a preheating treatment (80 °C, 15 min) was applied before the filtration. Results of both 2 g L-1 with and without preheating are compared each other in Figure 2. The quality of analysis is largely improved with a preheating as supported by both DRI and light scattering (LS) signals, which are clearly increased. At the same sample injected concentration, the Figure 2 shows that the recovered mass after filtration is larger with a preheating, in the benefit of the HMW population. This is fully confirmed by the results of Table 1 where the 2 g L-1
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Table 1. SEC-MALLS Analysis as a Function of AX Concentration and Preheating Filtration Conditions
filtration procedure without preheating
conc. (g L-1) 2
1
0.5 with preheating
a
2
population number
each population molar masses (Mw) (g mol-1)
relative proportion (%)
1 2 3 1 2 3 1 2 3 1 2 3
5 000 000 (( 38%) 1 000 000 (( 15%) 180 000 (( 43%) 7 000 000 (( 21%) 1 000 000 (( 10%) 170 000 (( 28%) 6 000 000 (( 12%) 900 000 (( 6%) 200 000 (( 22%) 5 500 000 (( 7%) 950 000 (( 4%) 235 000 (( 8%)
5 33 63 6 38 56 7 41 52 10 28 61
whole sample molar mass (Mw) (g mol-1)
recovered massa (%)
735 000 (( 27%)
7
900 000 (( 17%)
15
930 000 (( 10%)
40
1 000 000 (( 7%)
35
Ration of Initial Mass/Detected Mass (by DRI).
Figure 2. 90° light scattering (full line) and DRI signals (dashed line) and molar masses distributions versus elution volume of 2 g L-1 AX solutions, with (b) or without (O) preheating treatment.
Figure 3. Bilogarithmic plots and slopes of root-mean-square radius versus molar mass for populations 1, 2, and 3.
solution recovered mass raises at 35% with preheating, instead of 7% without a heating protocol. The obtained molar masses and the proportions of the three fractions for 2 g L-1 with preheating appears not so far from that of 0.5 and 1 g L-1 filtered without preheating (Table 1). This result points out the disaggregating role of the temperature. As a consequence, heated filtration, which offers a clear decrease of the losses (i.e., the best precision in the results), has been used for the following SEC-MALLS analysis. The slope of the bi-logarithmic plot of root-mean-square radius (Rg) versus molar masses allows us to give an indication of the molecular conformation of a polymer (Figure 3). The 1st and 2nd populations, the largest in molar masses, indicate a slope close to 0.5 (from 0.55 to 0.46 for 1st and 2nd populations, respectively), theoretically characteristic of a random coil conformation. Nevertheless, when comparing the data slopes of the three populations, we observe a tendency to decrease together with the average molecular weight. This becomes really significant for the 3rd population that exhibits a slop of about 0.35, which is characteristic of a compact structure and/or of branched molecules. In fact, branched molecules may have smaller slopes than the typical random coil value. Hence, these slopes could be a possible indicator of branching, which seems to increase as the molar masses of the arabino-xylans polymers decrease. This result should be of interest even though the branching conformation (3rd population) is not favorable to polymer interactions. On the contrary, random coil confor-
mation (1st and 2nd populations) should indicate high level of interactions. These hypotheses are legitimate to envisage interesting rheological properties of the flaxseed mucilage neutral fraction. From then on, our study was naturally headed for the confirmation research of the putative associations in the studied systems in the concentrated regime. Consequently, rheological studies have been undertaken. The steady shear flow curves of the flaxseed mucilage neutral fraction were performed on four polymer solutions (5, 10, 15, and 20 g L-1) in pure water (Figure 4). As a first result, it appears that the zero shear viscosity [evaluated by fitting the data with the Cross model (Table 2)] is greatly increased with rising concentration (more than 2 decades between 5 and 20 g L-1). Furthermore, the obtained flow curves brought to the fore a strong shear thinning behavior (apparent viscosity decreases with the increase of the shear rate) for all solutions over the entire range of the shear rate. This behavior could be correlated to possible interactions between the HMW arabinoxylans. The viscosities observed here were different than those displayed in the previous study16 for system close to ours. In this referenced study, when compared to our results, a viscosity that was approximately 200 times lower was itemized, with also a widely weaker shear-thinning behavior (decrease less pronounced). This result was probably due to a stronger extraction condition (90 °C, 3 h). Such a difference with the literature seems to corroborate our results about the filtration procedure with preheating, where
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Figure 4. Flow curves of AV solutions at different sample concentrations: 5 (b), 10 (1), 15 (0), and 20 (#) g L-1 (25 °C).
Figure 6. Frequency dependence of the storage modulus G′ (b), loss modulus G′′ (O) and dynamic viscosity ([) of the AX system (20 g L-1, 25 °C). Table 3. Influence of the Supplementation by KSCN or NaCl on the Activation Energies (Ea) of the AX System (20 g L-1, 25 °C)
Figure 5. Evolution of the storage modulus G′ (b) and the loss modulus G′′ (O) versus oscillation stress (20 g L-1, 25 °C). Table 2. Low Shear Plateau Viscosity (η0) Determined by the Cross Model for Various Concentrated AX Solutions (25 °C) solution conc. (% w/v)
0.5
1.0
1.5
2.0
η0 (Pa s)
1.5
56
360
700
the high temperature gives rise to a destruction of a part of the aggregates. As a consequence, the hypothesis of the presence of weak physical interactions acting in the studied system, like hydrogen bonds, can be suggested to explain these peculiar rheological properties. The 20 g L-1 solution, which displayed the higher viscosity, was used for the dynamic oscillatory measurements. In the first part, the linearity of viscoelastic behavior was verified in pure water with a stress sweep at a frequency of 0.1 Hz (Figure 5). For applied stresses below 5 Pa, the viscoelastic response appears linear. Thus, we have chosen to work at 0.2 Pa for further experiments. The drop in linearity (about 10 Pa) put in evidence an apparent yield stress beyond which the solution begins to flow. The dynamic rheological spectra of the solution was then examined (Figure 5). The behavior is preponderantly elastic, with G′ always exceeding G′′ over the whole frequency range investigated (10-2 to 101 Hz). Even if the evolution of the moduli are faintly frequency dependent, the ratio between the storage modulus G′ and the loss modulus G′′ was stable and continuously superior to 5. Hence, the polymer solution can
solution type
Ea (KJ mol-1)
complete neutral fraction + KSCN (O.5 M) + NaCl (O.5 M)
100 65 115
be considered as exhibiting typical weak gel properties. This slightly frequency dependence can possibly generate, under longer solicitation times, the presence of a crossing point characteristic of relaxation times (i.e., entangled polymers systems). In order to examine this possibility, curves need to be shifted through longer solicitation times by the William-Landel-Ferry method.29 Thus, the same oscillation experiment was carried out at 0.2 Pa (it ensures always the linearity response of viscoelastic behavior) with various temperatures, from 10 to 70 °C. Master curves were realized by shifting the curves between themselves on the G′ variable, with 30 °C as reference (Figure 7a). Although a strong tendency to bring G′ closer to G′′ around 10-3 Hz, no crossing point appears and the system stays always mainly elastic. Nevertheless, the obtained master curve indicates that the system is typical of Maxwell fluid characteristic of entangled polymer solution. Additional investigations were done with the adjunction of KSCN or NaCl (both at 0.5 M; Figure 7, parts b and c). KSCN is a chaotropic salt, which decreases the internal organization of liquid water, increases its mobility and therefore its entropy. As a consequence, the hydrogen bonds between dissolved molecules are reduced. As expected (Figure 7b), the master curve pointed out a clear crossing point located close to 10-2 Hz. The observed shift of master curves with KSCN, as compared to the master curve in pure water, indicates that the dynamic of the entangled system has been accelerated with the chaotropic salt. This result should demonstrate that a part of the hydrogen bonds, acting as stickers interactions, has disappeared. KSCN treatment acted and confirmed the major occurrence of hydrogen bonds as preponderant macromolecular linkages. These results were confirmed by the NaCl treatment, where the pseudo-gelbehavior appeared to be reinforced (Figure 7c). Indeed, this lyotropic salt is an antagonism of KSCN by structuring the
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Table 4. SEC-MALLS Analysis as a Function of Solvent Condition (Pure Water, Chaotropic (KSCN) and Lyotropic (NaCl) Salts); 40 °C Preheating Filtration
filtration procedure with preheating 40 °C
solvent condition pure water
0.5 M KSCN
0.5 M NaCl
population number
each population molar mass (Mw) (g mol-1)
relative proportion (%)
1 2 3 1 2 3 1 2 3
5 200 000 (( 11%) 950 000 (( 8%) 325 000 (( 18%) 5 500 000 (( 8%) 1 200 000 (( 4%) 650 000 (( 16%) 5 700 000 (( 12%) 950 000 (( 10%) 270 000 (( 22%)
11 39 50 20 44 36 10 40 50
whole sample molar mass (Mw) (g mol-1)
recovered mass (%)
920 000 (( 7%)
30
1 500 000 (( 6%)
75
930 000 (( 10%)
25
Figure 8. Arrhenius plots of the X-shift factor obtained from master curves of dynamic spectra (between 10 and 70 °C) of the AX system (20 g L-1) in the different solvent conditions: pure water ([), with addition of KSCN (0.5 M) (O) or NaCl (0.5 M) (4).
Figure 7. Master curves of the AX system at 20 g L-1 in pure water (a), with addition of KSCN (0.5 M) (b), or NaCl (0.5 M) (c). log G′ (b) and log G′′ (O).
water and strengthening the hydrogen bonds. The activation energy (Ea) of the system, obtained by fitting the previous shift (aT) data in an Arrhenius type curve log aT ) (Ea/2.303 R) (1/T - 1/T0) (Figure 8),30 illustrated clearly the action of all treatments. With KSCN, the Ea decreased widely (Table 3). To verify the impact of those treatments on the three distinct groups of polymers, the molar mass distributions were investigated (at 1 g L-1 after 40 °C filtration in pure water, 0.5 M KSCN and 0.5 M NaCl) with SEC-MALLS analysis (Table 4). The presence of KSCN leads to a large diminution of losses during the filtration procedure (75% recovered), whereas significant losses have been obtained in pure water (30% recovered) and worthy in 0.5 M NaCl (25% recovered). This let us foresee their potential for aggregation in solution, impeaching afterward their passes through a 0.45 µm filter. According to the chaotropic role of KSCN, the large increase of the recovered sample in such a solvent (much pronounced than the temperature effect) confirms the implication of hydrogen bonds in the aggregates formation. This treatment has no impact on the number of populations (with still three distinct populations), and this appears to be a real overview of the sample with regard to the recovering yield. As a result, it also appears that KSCN leads to a significant increase of the whole sample molar mass (about 1.5 M g mol-1) which concerns mainly the second and the third populations (the lower in mass). Therefore, both of those populations are mainly on the basis of the constitution of the aggregates. When KSCN is added, those aggregates are disassociated allowing the populations
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to pass through the filter and to be further analyzed. This hypothesis of a network based principally on hydrogen bonds is confirmed by the action of NaCl (antagonism of KSCN) that leads to a strong decreasing of the recovered yield. 4. Conclusion Flaxseed mucilage, through a biochemical approach, has been previously demonstrated to be composed of three different arabinoxylans with variable molecular weights and branched chain ratios (structural heterogeneity).31 This present study, focused on physicochemical properties, confirmed those results (three distinct populations). Furthermore, we show that those high molecular weight molecules have the tendency to form macrostructures (aggregates) in solution. Different treatments by addition of salts (chaotropic and lyotropic) bring to the fore that this network is mainly based on weak linkage (hydrogen bonds), furnishing further this interesting rheological behavior (gel formation). The random coil conformation of the polymers favor probably the constitution of intermolecular interactions with a resulting characteristic shear-thinning behavior. The response on heattreatment (decrease of viscosity) can led us to envisage the loss of those rheological properties for the water extract coming from an oil industry byproduct (a possible cheap source of arabinoxylans): the flaxseed cake. In addition, the network can be formed only on the basis of a part of the 3 AX (one or two). Those experiments are actually underway in our laboratory. Acknowledgment. This work was supported by the European social fund and the region of Picardie (France). References and Notes (1) Aspinall, G. O. AdV. Carbohydr. Chem. Biochem. 1969, 24, 333379. (2) Towle, G. A.; Whistler, R. L. Phytochemistry 1973, 1, 198-248. (3) Verbeken, D.; Dierckx, S.; Dewettinck, K. Appl. Microbiol. Biotechnol. 2003, 63, 10-21.
Warrand et al. (4) Glicksman, M. Food Hydrocolloids 1983, 2, 7-29. (5) Ward, F. M.; Andon, S. A. Cereal Foods World 2002, 47, 52-55. (6) Stephen, A. M.; Churms, S. C. In Food Science and Technology; Stephen, A. M., Ed.; Marcel Dekker: New York, 1995; Vol. 67, pp 377-440. (7) Osman, M. E.; Williams, P. A.; Menzies, A. R.; Phillips, G. O. J. Agric. Food Chem. 1993, 41, 71-77. (8) Fauconnet, L. Pharm. Acta HelV. 1948, 23, 101-108. (9) Anderson, E.; Lowe, H. J. J. Biol. Chem. 1947, 168, 289-297. (10) Easterby, D. G.; Jones, J. K. N. Nature 1950, 165, 614. (11) Erskine, A. J.; Jones, J. K. N. Can. J. Chem. 1957, 35, 1174-1182. (12) Hunt, K.; Jones, J. K. N. Can. J. Chem. 1962, 40, 1266-1279. (13) Fedeniuk, R. W.; Biliaderis, C. G. J. Agric. Food Chem. 1994, 42, 240-247. (14) Muralikrishna, G.; Salimath, P. V.; Tharanathan, R. N. Carbohydr. Res. 1987, 161, 265-271. (15) Fedeniuk, R. W.; Biliaderis, C. G. J. Agric. Food Chem. 1994, 42, 240-247. (16) Cui, W.; Mazza, G.; Biliaderis, C. G. J. Agric. Food Chem. 1994, 42, 1891-1895. (17) Warrand, J.; Michaud, P.; Picton, L.; Muller, G.; Courtois, B.; Ralainirina, R.; Courtois, J. Chromatographia 2003, 58, 331-335. (18) Susheelamma, N. S. J. Food Sci. Technol. 1987, 24, 103-106. (19) Cui, W.; Kenaschuk, E.; Mazza, G. Food Hydrocolloids 1996, 10, 221-227. (20) Sandhu, J. S.; Hudson, G. J.; Kennedy, J. F. Carbohydr. Res. 1981, 93, 247-259. (21) Roubroeks, J. P.; Andersson, R.; A° man, P. Carbohydr. Polym. 2000, 42, 3-11. (22) Joseleau, J. P.; Comtat, J.; Ruel, K. In Xylans and xylanases; Visser, J., Ed.; Elsevier Science Publishers B. V.: Amsterdam, 1992; pp 1-15. (23) O’Mullane, J. E.; Hayter, I. P. patent WO 9,316,707, 1993. (24) Minkov, E.; Ovcharov, R.; Bogdanova, S.; Kassarova, M. Pharm. Ind. 1975, 37, 836-839. (25) Collin, N. patent US 6,491,931, 2002. (26) Collin, N. patent FR 2,817,743, 2000. (27) Chevalier, V.; Collette, A. patent FR 2,846,556, 2002. (28) Capron, I.; Grisel, M.; Muller, G. Int. J. Polym. Anal. Chem. 1995, 2, 9-20. (29) Williams, M. L.; Landel, R. F.; Ferry, J. D. J. Am. Chem. Soc. 1955, 77, 3701. (30) Van Wazer, J. R.; Lyons, J. W.; Kim, K. Y.; Colwell, R. E. Viscosity and flow measurements - A laboratory handbook of rheology; Interscience: New York, 1963. (31) Warrand, J.; Michaud, P.; Picton, L.; Muller, G.; Courtois, B.; Ralainirina, R.; Courtois, J. Int. J. Biol. Macromol. 2005, 35, 121-125.
BM049249P
