HYDROCOLLOIDS - PART 2 Edited by K. Nishinari 2000 Elsevier Science B.V. 211

Effect of chitosan on the gelation of /c -carrageenan under various salt conditions

F.M. Goycoolea^ W. Arguelles-Monal'', C. Peniche^ and I. Higuera-Ciapara^

^ Center of Research for Food and Development, P.O. Box 1735 Hermosillo, Sonora 83000 Mexico. Tel +52-62-800-057; Fax +52-62-800-055. E-mail: fgoyco@cascabel.ciad.mx

^ IMRE, Universidad de La Habana, 10400 Cuba

"" Centro de Biomateriales, Universidad de La Habana, La Habana 10400, Cuba

The behaviour of non-stoichiometric polyelectrolyte complexes of Na^-K-carrageenan with short chitosan segments in presence of NaCl is rationalised in terms of a reduction of the charge density of K-carrageenan chains with a subsequent stabilisation of their ordered conformation leading to a reinforcement of the gel network. Hydrophobic interactions between complexed segments may contribute to a more extensively connected network. By contrast, in KCl, selective cation-driven aggregation of K-carrageenan seems to compete with chitosan for complex formation, thus effectively weakening the carrageenan gel network.

1. INTRODUCTION

Polyelectrolyte complexes (PEC) obtained by mixing two oppositely charged polyelectrolytes, normally lead to an insoluble precipitate due to strong coulombic interactions. However, when the PEC involves two polyelectrolytes of different molecular size, soluble non-stoichiometric heterotypic structures can be formed under controlled conditions, which are regarded as non-stoichiometric polyelectrolyte complexes (NPEC). In such complexes, the larger polymer chain behaves as a 'host' macromolecule to the shorter one (or 'guest') which attaches into stretches of the host chain at random. Furthermore, it has been recognised that the host polymer should be a strong polyelectrolyte in order to retain the interpolymer complex in solution [1]. Recently PECs have received a great deal of attention, due to their practical relevance in chemistry, biotechnology (e.g. microencapsulation, membrane technology, etc.), pharmacy (e.g. controlled drug release devices) and biomaterials engineering (e.g. immobilised pancreas islet cells).

The PEC between chitosan (bearing amine groups) and K-carrageenan (carrying one sulphate group per disaccharide unit) seems to be one of the most interesting because of the behaviour of K-carrageenan as a strong polyelectrolyte and its known gelling capacity. A series of insoluble chitosan/carrageenan PEC systems have recently been described [2], with emphasis on the charge density and the conformation of the carrageenan macromolecule. However, the behaviour of chitosan/K-carrageenan in a soluble NPEC during gelation, in the presence of added counterions, to our knowledge, has not yet been addressed.

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The aim of this study was to investigate the effect of complexing hydrolysed chains of chitosan of 20 or 75 (i-D-glucosamine hydrochloride residues with K-carrageenan in the disordered conformation, to form a soluble NPEC, with respect to the behaviour of K-carrageenan during the sol-gel transition in the presence of sodium or potassium counterions.

2. EXPERIMENTAL

2.1. Materials Chitosan: ^ Source: lobster cephalotorax {Panulirus argus) ^ N-acetyl content (molar fraction of N-acetyl groups, FA): 0.201, ^H-NMR. ^ Mv: 2.3 X 10 , measured at 25°C in 0.3 mol-L" acetic acid/0.2 mol-L' sodium acetate. ^ Controlled hydrolysis with KD^O: 20 and 75 residues of D-glucosamine (respectively

CHI-20 and CHI-75). The final chain length was checked by capillary viscometry. K-Carrageenan: ^ Supplier: Hercules (X6960) ^ Sodium salt was prepared by ionic exchange with Amberlite 200 (Sigma) ^ Content of i-sequences: 8 %.

2.2. Methods The chitosan/K-carrageenan NPECs were prepared by careful dropwise addition of a small

aliquot of 0.039 equiv.-L" chitosan hydrochloride into 0.0103 equiv,*L' carrageenan solution under vigorous mixing at ca. 90°C, in either NaCl or KCl solutions. The hot mixture was directly transferred to the rheometer. The composition of the NPECs, expressed as the molar ratio (Z) of NHs^ to SO3" fixnctional groups, varied in the range between 0.028 and 0.084.

The NPECs formed as above were allowed to gel in a Rheometrics RFSII Fluids Spectrometer fitted with a truncated cone-plate tool (cone angle: 0.0397 rad, diameter: 50 mm, truncation gap: 53 |Lim) and a circulating environmental system for temperature control. The viscoelastic properties were monitored by small deformation oscillatory testing at varying fi-equency (to = 1 to 100 rad-s" ) during cooling and heating programs. Low-amplitude oscillatory measurements were made within the linear viscoelastic region (y = 0.15), as verified by strain sweeps of the gels at 4°C.

3. RESULTS

3.1. Gelation in 0.25 molL^ NaCl The evolution of the storage modulus at o = 1 rad-s" during cooling of a K-carrageenan

solution alone and in combination with CHI-20 in NPECs of varying composition (Z=0.028 to 0.084), at identical carrageenan concentration in 0.25 mol-L" NaCl is presented in Figure 1. Inspection of the individual traces reveals a general elevation of the final G' values of the NPEC at low temperatures as the proportion of complexed chitosan increases. Moreover, the onset temperature of gelation (Tg) of the NPECs is clearly shifted to higher temperature, regardless of the NPEC composition. It should be noted that in two of the complexes (Z = 0.042 and 0.084) the G' moduli registered at temperatures above Tg are greater than the rest of the complexes and K-carrageenan alone.

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The criterion adopted to define the critical gel point (i.e. the percolation threshold at the formation of an incipient continuous network of infinite molecular weight), was to mark where a power-law variation of the dynamic mechanical moduli is obeyed. In other words, it is considered as the point where GXo)) oc G"(®) oc co , hence /a/7 5 = G 7 G ' = const. [3,4].

At this point, individual traces of tan 6 registered at varying frequency versus temperature intersect at a critical value, from which the gelling temperature was calculated. Figure 2 shows that for K-carrageenan and for the NPEC of Z=0.084, at the critical gelation temperature (i.e. the gel point), there is indeed little dependence of the tan 5 values on co, and such critical temperature closely corresponds to that where the tan 5 curves cross over. Similar analysis were conducted for the other NPEC series of CHl-20. Overall similar results to those described above, were

10 20 30 Temperature C C)

Figure 1. Temperature-dependence of G' (1 rads ' ; y = 0.15; PC-min"^) for K-carrageenan in NaCl alone and in complexes with chitosan (CHI-2()) of varying Z (as indicated in label).

observed for NPECs containing CHI-75.

c

0.01

I I I 1111

K-carrageenan

10 ^ CO (rads )

5 10 15 20 25 30 35 40 45 Temperature (°C)

Figure 2. Frequency-dependence of tan 5 (y = 0.15) at vanning temperature in the vicinity of the critical gel point (left frames) and variation of tan 5 with temperature on cooling (PCmin'') at varying frequency of oscillation (right frames) for K-carrageenan alone and for a complex (Z = 0.084) from Figure 1.

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In Figure 3 is shown the variation in the storage modulus at co = 1 rad-s" corresponding to gel formation and melting during respectively cooling and heating scans of K-carrageenan and chitosan/carrageenan NPEC of Z = 0.084. The difference in the cooling and heating traces reflects the thermal hysteresis expected for helix-helix aggregation of K-carrageenan in 0.25 mol-L' NaCl [5]. This thermal hysteresis has a greater magnitude in the presence of chitosan in the NPEC of Z=0.084, and similar results were obtained for the other NPECs.

Figure 4 shows data for G' and G" of the gels formed at 4°C for K-carrageenan alone and NPECs with CHI-20 and CHI-75 as a function of the amount of complexed chitosan. Notice that in both cases, the NPEC with CHI-20 and CHI-75, there is a plateau level in G' values the gels tend to at 4°C as the Z value in the NPEC increases. NTECs with CHI-20 however, seem to level off at Z>0.042, whereas NPECs with CHI-75 reach their plateau at Z>0.028. The mechanical spectra (variation of G', G" and r|* with co) recorded on completion of the cooling scans (results not shown) were typical for a polysaccharide gel network. The presence of chitosan resulted in an overall increase in the viscoelastic moduli, but no significant change in the spectral shape of the gels was observed (i.e. no dependence of G' and G" and linear decrease of rj* on frequency with a slope close to -1).

100 U

NPEC with CHI-20 - •—G' -a~G" NPEC with CHI-75 -A~G' - A - G "

0 10 20 30 40 50 Temperature C*C)

Figure 3. Variation of G' on cooling and heating (as indicated by arrows; I°C-min"') for K-carrageenan alone and for a complex (Z=0.084) from Figure 1.

0.000 0.025 0.050 0.075 0.100

Z ([CHI]/[CAR])

Figure 4. Variation of G" (4°C; 1 rad-s" ; y = 0.15) with Z for complexes of K-carrageenan with chitosan (CHI-20 and CHI-75) in NaCl. The \-alue of G' at Z = 0 corresponds to K-carrageenan alone.

3.2. Gelation in 0.03 mol-L^ KCl The temperature course of gel formation for K-carrageenan complexed with chitosan

(CHI-20) and alone in 0.03 mol-L" KCl, was also monitored by small-deformation oscillatory measurements. The traces of G' at ©=1 rad*s" are drawn in Figure 5. Under these conditions, the final G' moduli values are almost the same within the expected error for the NPECs and for K-carrageenan. Only a very small elevation in the final G' values is observed for the

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NPEC of Z=0.028, as compared to the rest of the gels. It is evident that there is no detectable change in gelation temperature (i.e. the onset of the steep rise in moduli). The sol-gel transition for these systems lied within the range of 32 to 34°C.

In Figure 6 is shown the variation in the storage modulus corresponding to gel formation and mehing during respectively, cooling and heating scans of K-carrageenan and the chitosan/carrageenan NPEC of Z=0.028. The difference in the cooling and heating evolution of G' values reflects again thermal hysteresis, expected for helix-helix aggregation of K-carrageenan in 0.03 mol*L" KCl [5]. In this case, the ion-driven transition is promoted at significantly lower ionic strength than in the systems prepared in the presence of NaCl. This is due to the well known specific binding of K' cations to the double-helical form of K-carrageenan, thus effectively promoting conformational ordering and aggregation at significantly lower concentrations than do non-specific ions, acting solely by charge screening [6]. In contrast with NaCl gels, this thermal hysteresis is unaffected by the presence of chitosan in the NTEC of Z=0.028, and similar results were obtained for the other NPECs.

1000

100

CO Q L

ID 10

,1 1 I i I I I I 11 I I 1 ' i " " i " " i ' "

- D -- O -- A -

i - V -- ^

K-carrageenan | Z = Z =

z = z =

•• 0.028 1 : 0.042 1 : 0.056 1 •' 0.084 1

r i 1 M i I 1 1 11 M I I i I

1000

5 10 15 20 25 30 35 Temperature (*'C)

Figure 5. Temperature-dependence of G' (1 rad-s"; y = 0.15; l°C'min'^) for K-carrageenan in KCl alone and in complexes with chitosan (CHI-20) of var\ing Z (as indicated in label).

4. DISCUSSION

100

- 1 — I — I — I — I — r

NPEC Z=0.028 J

K-carrageenan

^ 10 Q.

o 1 fc-

0.1

10 20 30 40 50

Temperature ( X )

Figure 6. Variation of G' on cooling and heating (as indicated by arrows; l C-min" ) for K-carrageenan alone and for a complex (Z = 0.028) from Figure 5.

Chitosan, one of the few cationic industrial polysaccharides, has deserved special attention in the formation of PECs. Carrageenans in turn, are strong polyanions, whose composition and charge density, vary with botanical source and method of isolation. One of the key features of carrageenans, namely of K - or i-carrageenan, is their ability to undergo a coil-to-double helix conformational transition leading to helix-helix aggregation and the development

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of a gel network. This process is sensitive to the type and amount of external counterions. In this study we have investigated the effect of complexing small stoichiometric amounts of short chitosan sequences of P-D-glucosamine into K-carrageenan chains in the disordered coil state. The complexation was carried out under ionic conditions known to promote conformational ordering and gel formation of the carrageenan macromolecules. We have gathered evidence to indicate that chitosan binds to K—carrageenan even in the presence of a high concentration of non-specific counterions (Na^ and CI"). Under such ionic conditions charge screening is known to stabilise the coil-to-helix transition and hence to induce aggregation of K-carrageenan, leading to gel formation, provided the concentration of carrageenan is above the critical gelling concentration (Co). The complexation of chitosan with K-carrageenan coils leads to a reduction of charge density and hence to the stabilisation of the ordered form of this gelling biopolymer. However, the obtained gel network is stronger than that of K-carrageenan at the same equivalent concentration. This effect is rationalised in terms of the formation of smaller helical junction zones, therefore greater in number, thus effectively leading to a denser gel network. It seems reasonable to argue that the complexation of small amounts of chitosan onto K-carrageenan in the coil state, leads to the formation of hydrophobic regions along the carrageenan chain, which self-associate [7] at temperatures well above Tg (Figurel). The formation of such hydrophobic PEC junctions seems to reinforce the carrageenan network. This would also be in agreement with wider thermal hysteresis between setting and melting processes (Figure 3), as well as with greater G' values recorded for the CHI-20 than for the CHI-75 NPECs as the Z value increases as illustrated in Figure 4 (i.e. since the number of CHI-20 chain species introduced into the complex is larger than that of CHI-75 at identical concentration). These differences are a logical consequence of the cooperative nature of the complex formation. By contrast, in the presence of K^ counterions -firmly established to bind specifically to the carrageenan helix- the addition of chitosan leads to the development of a slightly weaker gel network as the amount of chitosan in the complex increases. This is rationalised as a consequence of a competition for charged sites, thus effectively interfering with the overall degree of K^-driven aggregation in the K-carrageenan network [8].

ACKNOWLEDGMENTS W.A.M. wishes to recognise the financial support from CONACYT, Mexico ("Programa de Colaboracion Mexico-Cuba"), and his colleagues from CIAD for their kind invitation to collaboration. We also thank Prof M. Rinaudo and Prof E.R. Morris for helpfiil discussions.

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