Single crystals of V amylose complexed with glycerol

Biolosical ~leml~ International Journal of BiologicalMacromoleeules 18 (1996) 115-122

ELSEVIER

Single crystals of V amylose complexed with glycerol S.H.D. Hulleman a, W. Helbert h, H. Chanzy *u aAgrotechnological Research Institute (ATO-DLO), P.O. Box 17, 6700 AA Wageningen, The Netherlands bCentre de Recherches sur les Macromol~.cules V~gdtales ( CERMA V-CNRS), B.P. 53, 38041 Grenoble cedex 9, France

Received 10 July 1995; revisionreceived29 August 1995; accepted4 September 1995

Abstract

Lamellar single crystals of amylose V glycerol were grown at 100°C by evaporating water from solutions of amylose in aqueous glycerol. The crystals which were square, with lateral dimensions of several micrometers, gave sharp electron diffraction patterns presenting an orthorhombic symmetry with a probable space group P212121 and unit cell parameters: a = 1.93 ± 0.01 nm, b = 1.86 ± 0.01 nm and c (fiber axis) = 0.83 ± 0.03 nm. The amylose Vo~rol crystal structure which is isomorphous to that of Vv~tso consists of an antiparailel pair of left-handed six-fold amylose helices centered on the two-fold screw axes of the cell and probably separated by glycerol molecules. This packing mode is confirmed by de-solvation experiments where the V~0~rotamylose crystals, annealed in ethanol, could be converted into VH amylose without losing their external appearance. The Voy~eroI amylose crystals could be seeded by cellulose microfibrils to yield a shish-kebab structure where the amylose crystalline lamellae grew perpendicular to the microfibril directions. Keywords: V-amylose; Single crystal; Electron microscopy; Polysaccharides

1. Introduction

Amylose has the unusual property to form crystalline complexes with a variety of small molecules such as iodine, fatty acids, ketones, amines, alcohols, salts, etc [1-5]. All these complexes, known under the generic name of V amylose, have in common a single lefthanded helical conformation of amylose. Beyond this common feature, the V amylose complexes present a large diversity, both in the symmetry of the amylose chains that can adopt 6-8 residues per helical turn [1], and the location of the complexing agent which may be found within the helical cavity, in between the helices, or in both sites [1-5]. The combinations of these possibilities explains why a very large number of V amylose crystalline structures can be obtained. A number of these structures have already been described but it is likely that many more are still to be discovered.

* Correspondingauthor. 0141-8130/96/$15.00 © 1996ElsevierScience B.V. All fights reserved SSDI 0141-8130(95)01069-7

In this work, the focus is o n Voycerol amylose for which almost no data exist. The description of this system presents a great theoretical and practical interest as glycerol is one of the standard, plasticizers used for the manufacturing of thermoplastic starches [6-10]. In these products, the plasticization effect relies on a specific affinity of amylose and amylopectin for the mixture of glycerol and water. To understand this phenomenon, one needs to know more about the interaction between glycerol and amylose. In particular, the following questions should be answered: (i) is glycerol able to induce the helical V structure of amylose and is this structure able to crystallize? (ii) if VoyceroI corresponds to a specific structure, is glycerol localized only within the helical cavity or is it found also in between the amylose helices? (iii) finally, what is the stoichiometry of the V0ycerol amylose complex and in particular, does it requires water to be formed? To our knowledge, there is only one brief preliminary mention about the crystalline complex of amylose and glycerol [11 ]. This short report indicates that the crystals

! 16

S.H.D. Hullemanet al. / InternationalJournal of BiologicalMacromolecules18 (1996) 115-122

of Voy,=roI appear to be isomorphous with those of VVMSO[12] as well as with those of Vethylcncdiamine [13]. If this could be confirmed, V£yccrol should consist of left-handed, six-fold helices crystallized into a pseudotetragonal symmetry. In addition, there would be glycerol both within the helical cavity as well as in between the amylose helices. X-ray analysis on stretched fibers, combined with electron diffraction data on single crystals appear to be the best experimental method to resolve the molecular structure of the VoyceroI complexes. However, the preparation of amylose VoyceroI fibers suitable for X-ray studies has not been possible so far. Unlike amylose in DMSO, amylose in glycerol does not make strong films by evaporation [11]. These films are crystalline but they present the peculiarity to be crystallized with the amylose chain axis perpendicular to the plane of the film. Thus these films behave more as mats of single crystals rather than continuous films easily stretchable to yield crystalline fibers. This unusual behavior indicates that at least the preparation of single crystals of amylose Voy~ro~suitable for electron diffraction analysis could be attempted with a reasonable chance of success. The present report describes the preparation and characterization of such crystals. It also gives information on their specific properties such as nucleation, swelling and de-swelling behaviour. 2. Exl~rimeat~ 2.1. Materials Potato starch amylose from AVEBE was recrysta]lized prior to its use. It was first dispersed at a concentration of 2% (w/v) into dimethyl sulfoxide (DMSO) and then to.tally dissolved by raising the temperature to 50°C for 30 rain. The solution was then cooled to room temperature. After 3 h, the solution was filtered on a fritted disk funnel of porosity 4 (pore size between 5 and 15 /~m). An equal volume of ethanol was added to the filtrate to induce the precipitation of amylose. After 24 h, the amylose precipitate was filtered off on a fritted disk funnel of porosity 4. The precipitate was then redispersed three times in ethanol, three times in acetone and three times in diethyl ether with a filtration step between each redi~aersion. After the last filtration, the amylose precipitate was air dried and stored. 2.2. Preparation of the amylose crystals complexed with glycerol An aqueous solution (0;5%o, w/v) of recrystallized amyiose was prepared by heating a mixture of amylose and water at 140°C in a sealed autoclave for 10 rain. After solubilization, the solution was cooled to 90°C and nine parts of glycerol at 90°C were mixed with one part of the amylose solution. The mixture was heated to 150°C and kept at this temperature for 1 h in an open vessel. During this treatment a slow nitrogen purge was

applied in order to remove most of the water from the solution and to minimize the amylose thermal degradation. Still under nitrogen purge, the amylose solution was cooled to 100°C and kept at this temperature. After 10 h, a turbidity indicated that crystallization had begun. The solution was kept for another 16 h at 100°C after which crystallization was complete and the solution was brought back to room temperature. At this stage, the water content of the crystallization solution, determined by Karl-Fisher coulometric measurement, was lower than 0.2% (w/v). The crystals were washed by centrifugation, using anhydrous glycerol that had been dried by heating at 150°C for 1 h in a open vessel and under dry nitrogen purging. After the last washing, the crystals were resuspended in glycerol and the suspensions were stored in a desiccator over dried silica gel. 2.3. Treatment of the crystals with ethanol Some crystals were transferred to ethanol by successive centrifugations into mixtures of glycerol and ethanol of increasing concentration to finally reach pure ethanol. Part of these crystals was stored as a suspension in ethanol. The other part was annealed at 130°C for 2 h inside a pressure vessel. It was then cooled to room temperature and stored as a suspension in ethanol. 2.4. Oriented growth of Vgtyce,oI amylose crystals on cellulose microfibrils Crystallization experiments similar to the above procedure were achieved except that the amylose concentration was five times higher and that strips of delaminated cell wall fragments of Valonia ventricosa were added to the crystallization solutions. Under these conditions, most of the crystals grew as decoration on the Valonia fragments. These fragments were carefully removed from the solution and gently washed several times in fresh anhydrous glycerol before mounting for electron microscopy. 2.5. X-ray diffraction analysis Suspensions of amylose crystals in glycerol were dried at 70°C in vacuum to yield mats of crystals that were stacked and transferred into thin-walled X-ray capillary tubes. These tubes were sealed quickly and examined by X-ray with a Warhus flat-film vacuum camera mounted on a Philips PWI720 X-ray generator operated with Ni filtered CuKtx radiation at 30 kV and 20 mA. Diffraction diagrams were recorded with the film surface perpendicular and parallel to the X-ray beam. Calibration was achieved by dusting the specimen with CaCO3 and using the strong ring at d=0.3035 nm as a standard. 2.6. Optical microscopy In the case of specimens crystallized on Valonia cellulose, the samples were stained purple with a solution of

S.H.D. Huilemanet al./ InternationalJournal of BiologicalMacromolecules18 (1996) 115-122

117

2 mg 12 and 20 mg KI per ml of anhydrous glycerol. The decorated Valonia cell wall fragments, still in glycerol, were mounted on glass slides and observed with a Zeiss Universal polarizing optical microscope operated with crossed nicols. 2. 7. Transmission electron microscopy For the crystals in glycerol, small drops of the crystal suspensions were deposited on carbon-coated electron microscopy grids that were allowed to dry at 70°C under vacuum for several hours. For the samples in ethanol, the same procedure was applied but the samples were allowed to dry at room temperature without vacuum. Some grids were shadowed with W/Ta at an angle of 30°. The grids were observed with a Philips CM200 CRYO transmission electron microscope operated at 200 kV for low dose imaging and electron diffraction and 80 kV for the imaging of shadowed crystals. For electron diffraction the grids were mounted on a rotation holder that allowed to align one of the main cell axes of a given crystal with the tilt axis of the microscope. Some of the electron diffractograms were calibrated by depositing the amylose crystals on carboncoated grids on which a thin layer of gold had been deposited by evaporation. 3. Results 3.1. Crystals of Vglycerot amylose and their diffraction diagrams A typical lamellar crystal of V0yceroI amylose is shown in Fig. 1. This crystal has a contour that is roughly square with lateral dimensions of approximately 2 ~m, It consists of a stack of square-shaped layers organized in spiral growths centered on screw dislocations. Its individual layer thicknesses, measured from the shadow cast angle are of the order of 10 nm. When

Fig. 2. Low dose imageof a singlecrystalof Voycerot amylose.Insert: electron diffractiondiagramwithproper orientationcorrespondingto the circled area.

probed by electron diffraction, each crystal yields a sharp electron diffraction pattern that extends to a resolution of around 0.3 nm. A representative electron diffractogram properly oriented with respect to the crystal is shown in Fig. 2. At first glance, this diagram can be indexed along a tetragonal net and seems to present a symmetry that can be described as 4ram. However, a careful calibration indicates that there is a small difference between the value of a* and b*. In addition, weak spots indexed as 510 and 600 are not mirrored to 150 and 060. The diagram in Fig. 2 is thus indexed along two perpendicular symmetry axes a* and b*. Along a* the diffraction spots with indices h00 with h odd are systematically absent. Similarly, there are also systematic absences along b*, with 0k0 odd. The diagram in Fig. 2 has therefore the symmetry Pgg, even though at first approximation it could have been defined as 4mm.

.] Fig. 1. Electronmicrographafter shadowingwith W/Ta of a preparation of singlecrystalsof Voyceroiamylose.

m/

Fig. 3. Typicalelectrondiffractogramsrecordedon singlecrystals of V0ycerot amyloserotated about b* by 29, 37 and 47°.

S.H.D. Hulleman et al. / International Journal of Biological Macromolecules 18 (1996) 115-122

118

The symmetry of the three dimensional structure was established by rotating the amylose crystals about the two axes a* and b*. Patterns rotated by a given angle about a* bad the same aspect as those rotated by the same angle about b*. In addition, patterns rotated clockwise by a given angle were identical to those rotated anticlockwise by the same angle. Thus the third axis c* is perpendicular to the base plane defined by a'b* and the orthorhombic symmetry is confirmed. Specific diagrams obtained by rotating about b* by 29 °, 37 ° and 47 ° are shown in Fig. 3. Diagrams obtained by rotating about a* by the same angles had nearly equivalent appearance and are not shown here. After proper indexation and calibration, the unit cell of the amylose Vgly~rol crystals can be refined to a = 1.93 ± 0.01 nm, b = 1.87 ± 0.01 nm and c = 0.83 ± 0.03 nm. The analysis of these data indicates that the space group is either P21212t or P21212. In view of the diffractogram presented in Fig. 2, a body centered space group of the

Table I List of the observed and calculated d-spacings in the electron diffractograms recorded on tilted and un-tilted Vglycero I amylose crystals, Comparison with the X-ray data given by French [1 I]

hkO

d-spacings (nm) a (obs)

d-spacings Intensities a'b d-spacings (nm) a (calc) (obs) (nm) b'c

Layer line zero 1I0 200 020 220 310 130 400 040 330 420 240 510 600 610

1.34 0.958 0.934 0.670 0.611 0.595 0.480 0.467 0.447 0.429 0.421 0.381 0.324

1.339 0.965 0.930 0.669 0.607 0.590 0.482 0.465 0.446 0.428 0.418 0.377 0.321

M S S M M M VS VS VS VS VS W W

0.971 (VS) 0.686 (MS) 0.615 (M)

Fig. 4. X-ray diagrams recorded with the beam parallel to the surface of a mat of single crystals of Vglycero I amylose.

I type could have been proposed. However, it has to be rejected because of the occurrence of several reflections where h + k + l = 2 n + ! on the first layer. The observed electron diffraction spots, their indexations, dspacing values and intensities are listed in Table 1 which presents also the list of the calculated d-spacing values, deduced from the above unit cell. For comparison, Table 1 presents also the list of the diffraction data reported by French [11]. The electron diffraction data were confirmed by Xray analysis on mats of single crystals that were X-rayed with the beam either parallel or perpendicular to the surface of the mat. Fig. 4 shows the diagram with the beam parallel. The diagram is a pseudo fiber diagram holding most of the hk0 reflections that were observed in the

0.486 (VS) 0.460 (MS) 0.431 (VS) 0.382 (MW) 0.320 (VW)

Table 2 List of the observed intensities in the X-ray diagrams recorded on mats of Vglycero I amylose crystals Indices

d-spacings (nm) (obs)

Intensities a

110 200. 220 011, 310, 400, 330, 420, 430, 510, 600

1.37 0.943 0.668 0.75/0.67 0.601 0.476 0.446 0,425 0.399 0.376 0.339

W S M Sb M S VS VS M W W

First layer line 201 211 221 301 311 131 321 231 141 241

0.616 0.573 0.508 0.503 0.484 0.481 0.443 0.440 0.404 0.379

0.629 0.596 0.521 0.508 0.490 0.481 0.446 0.441 0.396 0.374

M M W M S S M M M W

aThis work. bVS, very strong; S, strong; M, medium; W, weak; VW, very weak. The reflections having intensities lower than the background or belonging to systematic absences have not been listed. CFrom Ref. [Ill.

020 101, I I I 130 040, 311, 131 32 I, 321 240 340

aVS, very strong: S, strong: M, medium; W, weak. The reflections having intensities lower than the background or belonging to systematic absences have not been listed. bBroad.

S.H.D. Hulleman et al./.International Journal of Biological Macromolecules 18 (1996) 115-122

119

tograms were however modified (insert B in Fig. 5). This diagram can still be indexed along a pseudo tetragonal unit cell but with parameters that are substantially larger: a = b = 2.74 nm. Upon annealing in ethanol, the morphology of the crystals was not modified. However a de-solvation occurred that resulted in a shrinkage of the unit cell that reverted to that of VH amylose. This behaviour is illustrated in the diagram shown as insert C in Fig. 5 This pattern can be resolved to the superposition of two single crystal VH electron diffractograms rotated by 90° with respect to one another.

Fig. 5. A: Low dose image of a crystal that was exchanged in pure ethanol. B: Typicalelectron diffractiondiagram correspondingwith proper orientationto a crystal as in A. C: Sameas in B but after high temperature annealing in ethanol. This diagram corresponds to the superposition of two single crystal VH patterns, rotated by 90° with respect to one another.

electron diffraction diagrams of the base plane. On the meridian, there is only a strong broad arc centered at 0.67 nm, that corresponds to the superposition of the strong diffraction spots 011,101 and 111 located on the first layer line. A list of the observed reflections together with their indexation and intensity is given in Table 2. 3.2. Exchange of the crystals in ethanol When the Vglycero I amylose crystals were exchanged in ethanol at room temperature, they kept their external shape and feature a seen in Fig. 5. Their electron diffrac-

3.3. Oriented crystallization When the crystallization solutions of amylose were seeded with cellulose microfibrils from Valonia cell walls, crystals of Vglycero I amylose grew attached to the microfibriis. This phenomenon is illustrated in the optical micrograph shown in Fig. 6A where the sample is observed in its mother liquor between crossed nicols. Due to the staining with iodine, the crystals that are seen edged on, are highly birefringent. Most of the crystals are seen as shish-kebab decoration on the cellulose microfibriis and only a few are un-attached. The density of the decoration is not very high, as on a given microfibrils, the crystals are frequently separated from one another by several microns. The same sample is observed by transmission electron microscopy in Fig. 6B after adequate washing in ethanol, to remove the excess glycerol. This picture confirms that all the amylose crystals are attached to the cellulose microfibrils. However, most of them are observed flat, due to the drying effect. Nevertheless, when the crystals are close enough to prevent collapsing, their initial edged-on position is preserved, as seen in several areas in this figure.

4. Discussion

Fig. 6. A: Optical micrographin polarized light with crossed nicolsof a typicalshish kebab structure of VslyceroI amylosecrystalsgrown on cellulosemicrofibrilspulled out from Valoniacell wall. The sample is in glyceroland was stained with iodine. B: Electronmicrographof the same sample after washing with ethanol.

To our knowledge, the results presented in this study are, we believed, the first report on single crystals and electron diffraction analysis of Vglycero I amylose. The present work confirms the preliminary X-ray data given by French I1 l] on such complexes. As already noted by this author, the Vglycero I amylose crystals appear to be nearly isomorphous to those of amylose VDMSO. This is confirmed by comparing the list of the diffraction data presented in Tables l and 2 with those of amylose VDMSO reported by French and Zobel [12]. This comparison indicates that there is a close match between our and their d-spacings of the equatorial and first layer line. In addition, our intensities as well as our unit cell are in good agreement with their observations. Thus the packing of amylose helices in the Vglyeerolcomplex should resemble that of VDMSO described by French and Zobel [12]. This packing which is schematically drawn in Fig. 7 describes the structure of VglyccroI in terms of two lefthanded six fold helices packed in a pseudo tetragonal

120

$.H.D. Hulleman et aL / International Journal o f Biological Macromolecules 18 (1996) 115-122

b*

a* v

Fig. 7. Schematic diagram of the molecular organization of the amyiose molecules in the Vo~ro I amylose crystals: ab projection.

system along a P212121 symmetry, with helices centered on the two fold screw axes. In such organization, there is an alternation of up and down chains and therefore the single crystals of amylose V0ycerolmust be crystallized in the chain folded arraagement that is classical for polymer single crystals. In Fig. 7, if the diameter of a typical VH amylose helix is assumed to be 1.36 nm, the total inter-helical volume accessible to the solvent is of the order of 0.580 nm 3 per unit cell or roughly 20% of the unit cell volume. As we could not obtain reliable density data on these crystals, it is difficult to establish the stoichiometry of the complex. A rough estimate with the reasonable amount of 4 inter-helical molecules of glycerol - - each of them occupying 0.12 nm 3 - - per unit cell and without accounting for the intra-helical solvent would lead to a calculated density of 1.29 g/cc. This value remains to be ascertained. In two papers subsequent to that of French and Zobel [12], Winter and Sarko [14,15] have obtained superior X-ray diagrams of amylose VVMSOcomplexes. The high resolution data of their patterns indicate without ambiguity that the c parameter of their crystalline complex is 2.439 um instead of the 0.812 nm of French and Zobel. Thus, in the improved structure of Winter and Sarko, there are three turns of amylose helix per crystallographic repeat. Our diffraction data do not have the quality of those of Winter and Sarko who took great care in their diffraction set up to preserve the complexing solvent. For experimental reason, as much solvent as possible had to be removed for the above electron microscopy experiments. This may explain why no additional layer lines could be detected in our rotated electron diffraction patterns. As for the X-ray diagrams on crystal mats, they were not resolved enough to show any detail leading to the tripling of the c parameter.

Thus, at this stage it is not possible to conclude without ambiguity whether the c parameter of V0~ro I amylose is 0.83 ~ 0.03 nm as we propose here or three times higher, as given by Winter and Sarko for amylose VDMSo [14,15]. The occurrence of glycerol within the helical cavity of the V~ycerolamylose crystals cannot be demonstrated by our experimental approach as our diffraction data are not resolved enough to be able to resolve water or glycerol inside the amylose helix, even if this complexant was located at a specific crystallographic position. As the crystals can be readily stained with iodine that is known to occupy the amylose cavity [16], we can state that the intra-helical complexant must be fairly mobile and therefore not positioned in crystallographic position. The staining of V amylose complexes by iodine is not specific of the VglyceroI. In fact, it seems to be a general feature of the six-fold amylose helices: it has been demonstrated in the case of Vn.bu~oI [17] as well as VH and VthymoI [18]. On the other hand, the eight-fold helices occurring in Vct.naphthol o r Vquinoline cannot be stained by iodine [18]. The intra-crystalline swelling of the Voy~rol amylose crystals is another interesting behaviour of these com-

®

®

1

Fig. 8. A: As in Fig. 7 but after exchanging the crystals against ethanol. B: As in A, but after annealing the crystals in ethanol for I h at 130°C.

S. H. D. Hulleman et al. / International Journal of Biological Macromolecules 18 (1996) 115-122

plexes. Upon immersion in ethanol the external appearance of the crystals does not change but their electron diffractogram is modified (Fig. 5). This diagram can still be indexed along a pseudo-tetragonal 4mm system, but the symmetry axes are now oriented along the sides of the square crystals as opposed to the diagonal in the crystals before swelling. The unit cell of the swollen crystals appears to be orthorhombic with new a and b lattice constants larger than those of the initial parameters to which they are related by a factor that is slightly above a square root of two. The two unit cells before and after swelling are schematically drawn in Fig. 8A which shows the organization of the amylose helices inside an Vglycero I amylose crystal. In this drawing, the shaded area corresponds to the pockets of inter-helical solvent. The unit cell of the Vglycero I amylose crystals appear to depend only on the packing of the amylose helices. As aforementioned, this unit cell contains two helices: one 'up' at the cell corner and one 'down' at the center. Our data indicate that when ethanol is added, the unit cell content goes from two to four chains. This increase indicates that the ethanol molecules which are replacing the glycerol must be positioned at specific crystallographic locations. From this new situation, it follows that the unit cell of the ethanol-treated crystals must encompass the whole interhelical solvent pocket as opposed to only one half in the initial crystals. The conversion of the ethanol-treated Vglycero t amylose crystals into VH is another property of these crystals. This de-swelling which corresponds to a shrinkage of about 13% of the unit cell content does not affect the morphology of the crystals which appear to keep their initial integrity. On the other hand their diffraction diagram (insert C in Fig. 5) is drastically modified as it corresponds now to a superposition of two arced amylose VH diagrams rotated by 90° from one another. At the molecular level, the transformation into VH indicates that the amylose helices must slide past one another to yield the hexagonal close packed arrangement. As illustrated in the schematic drawing in Fig. 8B, this sliding may occur in two perpendicular directions with equal probability. Thus, at the molecular level, the deswollen crystals must consist of a mosaic of amylose V H domains organized in two families where the unit cell parameters are roughly perpendicular to one another (Fig. 8B). The arcing of the diagram (insert C in Fig. 5) confirms the fragmentation and the slight misalignment of the crystalline blocks within each family. It is interesting to notice that the conversion of the VglyceroI amylose crystals to VH amylose via the swelling/de-swelling presented here, does not induce any cracking of the crystals despite the 13% shrinkage of the unit cell content. In similar case and even for a unit cell shrinkage of only 10%, the rectangular amylose nbutanol crystals became cracked [19,20]. Depending of the de-swelling conditions, these cracks occurred either

121

parallel or perpendicular to the long dimension of the crystals. Similar cracks resulted also from the 20% shrinkage of the rectangular Visopropanolinto VH amylose [21]. The absence of cracking in the de-swelling of the VoyceroI amyiose crystals must be due to the pseudo tetragonal symmetry of this crystalline complex. With such crystals, the shrinkage is essentially bi-dimensional, with the consequence of a breakdown of the crystals into several VH crystalline blocks. These domains that are organized at right angles are not large enough to induce the major cracking that is observed in the n-butanol or isopropanol complexes where the shrinking is essentially unidirectional. The oriented crystallization of the Vglycero I amylose crystals on cellulose microfibrils is another property of these crystals. This transcrystallization phenomenon which was observed already in the case of Vn.butano I amylose [22] cannot be rationalized in terms of an epitactic overgrowth as the repeat distance of cellulose is 1.034 nm as opposed to that of V amylose that is of the order of 0.8 nm. In fact, cellulose is able to induce the nucleation of crystals of a number of other polysaccharide crystals as well as those of polypropylene [23]. In this case, Gray has suggested that it was the geometrical property of fibrillar cellulose and especially the grooves that are located in between the microfibrils that were responsible for the nucleating power of these systems. According to this suggestion, during a seeded crystallization experiment, the first amylose glycerol complexes to precipitate should do so by aligning themselves in the elongated interstices located in between the microfibrils. It is then likely that these first deposited chains will then act as nuclei for the growth of the crystals which will develop perpendicularly to the cellulose microfibrillar direction into a classical shish kebab morphology. In Fig. 6, the density of the lamellar overgrowth is not very high as the amylose crystals appear to be separated from one another by distances greater than 1 #m. This density which is much less than in the case of Vn.butano I crystals must reflect only a moderate affinity of V0ycerol amyiose for the cellulose microfibrils. The property of cellulose microfibrils to nucleate and orient the growth of V0ycerolamylose could lead to interesting developments in the field of thermoplastic starches as glycerol is one of the standard plasticizer that is used to process starch. Normally, the mechanical properties of these starch-based products correspond to those of average commodity plastics such as branched polyethylene or polypropylene [24]. It is expected that the reinforcement of thermoplastic starch by high modulus fibers or microfibrils such as those of cellulose should lead to superior starch products. With these reinforced products, it should be interesting to see whether an in situ shish-kebab morphology similar to that in Fig. 6 could be induced by transcrystallization. If yes, the

122

S.H.D. Hulleman et al. /International Journal of Biological Macromolecules 18 (1996) 115-122

physical properties of the corresponding reinforced materials should be interestingly different from those of the samples with no transcrystallization.

Acknowledgements The authors thank W.T. Winter and A.D. French for valuable suggestions during this work. References [ll French, A.D. and Murphy, V.G. Cereal Foods Worm 1977; 22: 61 [2l French, A.D. and Murphy, V.G. Polymer 1977; 18:489 [3l Sarko, A. In: Structure of Fibrous Biopolymers, Colston Papers No. 26 (E.D.T. Atkins and A. Keller, eds.) Butterworths, London, 1975, p. 335 [4l Sarko, A. and Zugenmaier, P. In: Fiber Diffraction Methods (A.D. French and K.C. Gardner, eds.), ACS Symposium Series 1980; 141:459 [5l French, D. In: Starch, Chemistry and Technology, 2nd Ed. (R.L. Whistler, J.N. BeMiller and E.F. Paschall, eds.), Academic Press, New York, 1984, p. 203 [6] Wolff, I.A., Davis, H.A., Cluskey, J.E., Gundrum, L.J. and Rist, C.E. Ind. Eng. Chem. 1951; 43:915

[7] Muetgeert, J. and Hiemstra, P. U.S. Patent No. 2-822-581, 1958 [8] Protzman, T.F., Wagoner, J.A. and Young, A.H.U.S. Patent No. 3-344-,216, 1967 [9] Sala, R.M. and Tomka, I. Angew. Makromol. Chem. 1992; 199: 45 [10] Van Socst, J.J.G., de Wit, D., Tournois, H. and Vliegenthart, J.F.G. Polymer 1994; 35: 4722. [11] French, A.D. PhD Thesis, Arizona State University, 1971 [12] French, A.D. and Zobel, H.F. Biopolymers 1967; 5:457 [13] Simpson, T.D. Biopolymers 1970; 9:1039 [14] Winter, W. and Sarko, A. Biopolymers 1972; 11:849 [15] Winter, W. and Sarko, A. Biopolymers 1974; 13:1461 [16] Bluhm, T.L. and Zugenmaier, P. Carbohydr. Res. 1981; 89: I [17] Rundle, R.E. and Edwards, F.C.J. Am. Chem. Soc. 1943; 65: 2200 [18] Helbert, W. Doctoral Dissertation, Joseph Fourier University of Grenoble, France, 1994. [19] Manley, R. St John, J. Polym. Sci. [A] 1964; 2:4503 [20] Yamashita, Y., Ryugo, J. and Monobe, K. J. Electron Microsc. 1973; 22: 19. [21] Bul6on, A., Delage, M.M., Brisson, J. and Chanzy, H. Int. J. Biol. MacromoL 1990; 12:25 [22] Helbert, W. and Chanzy, H. Carbohydr. Po/ym. 1994; 24:119 [23] Gray, D.G. Polym Lett 1974; 12:509 [241 Schroeter, J. and Endres, H. Kunststoffe 1992; 82:11