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Crystallinity of lyophilised carrot cell wall components
International Journal of Biological Macromolecules 26 (1999) 325 – 331 www.elsevier.com/locate/ijbiomac
Crystallinity of lyophilised carrot cell wall components Dominique M.R. Georget †, Paul Cairns, Andrew C. Smith *, Keith W. Waldron Norwich Laboratory, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK Accepted 26 July 1999
Abstract The aim of this work was to investigate the effect of removal of cell wall components on the crystallinity of cell walls using X-ray diffraction. Various insoluble cell wall residues were prepared following a sequential extraction of carrot cell wall material. X-ray diffraction patterns were typical of cellulose although there was a possible contribution of pectic polysaccharides to the crystallinity. As more amorphous material was removed to produce a cellulose rich residue, the crystallinity index increased from 12 to 16%, larger than that estimated from cellulose alone. For the last residue treated with 4M KOH, a lower value of crystallinity was found (14%) which resulted from the change of some crystalline domains of cellulose into amorphous regions. Pressing conditions (temperature, water content) have been investigated and did not alter the crystallinity index significantly. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Carrot; Cell wall; X-ray; Cellulose; Crystallinity
1. Introduction The cell wall of plant tissue comprises crystalline and amorphous domains [1]. The latter consists of a variety of components such as pectic polysaccharides, hemicellulose, proteins and phenolic derivatives [2]. Some degree of crystallinity has been observed in pectic polysaccharides [3,4] and hemicellulosic polysaccharides [5], although the crystalline regions of the wall consist mainly of cellulose. However, the crystalline and amorphous domains of cellulose/hemicellulose can barely be distinguished using X-ray diffraction, due to the existence of close associations between the two phases [6]. In early work, the crystallinity of carrot phloem cellulose and calcium pectinate produced from citrus [7] was measured and its relevance to the physical properties of dried vegetable tissues suggested. The crystallinity of the cell wall of avocado during
* Corresponding author. Tel.: +44 1603 255000, fax: + 44 1603 507723. E-mail address: [email protected] (A.C. Smith) † Present address: INRA ENSAM, Unite de Technologie des Cereales, 2, place Pierre Viala, 34060 Montpellier Cedex 1, France.
ripening has been assessed and compared with cellulose/pectin model systems [8], showing that the loss of cohesiveness within the cellulose fibril structure and the binding of the cell wall matrix polysaccharides were altered. Major work has been carried out on cellulose for its application in the textile industry [9,10] and cattle feeding [11,12]. Cellulose occurs in several crystalline forms [13] and consequently, when studying cellulose crystallinity in plant material, it is difficult to assign the X-ray diffraction results to those obtained on cellulose of a known lattice type [14,15]. In addition, crystallinity may be reduced by mechanical disruption such as ball milling [16] and increased by heating in an aqueous environment [17]. In the present study, the objectives were to investigate the X-ray diffraction patterns and the crystallinity indices of cell wall material purified from lyophilised carrot in comparison with crystallinity data obtained on proprietary cotton lint cellulose and citrus pectin. The effects of pressing temperature and water content on the degree of crystallinity were investigated and complement studies on the thermomechanical properties of the pressed and extracted cell wall [18].
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2. Materials and methods
2.1. Lyophilised carrot Carrots (Daucus carota cv. Amstrong) were grown locally. Once harvested, the crop was cleaned by brushing and washing. The tissues were then sliced and frozen immediately in liquid nitrogen. The frozen material was freeze-dried (freeze-dryer Model 3.5, Birchover Instruments, Letchworth, Herts, UK). The lyophilised material was then ground to a powder using a mortar and pestle and then stored in a desiccator over silica gel.
2.2. Preparation of carrot cell wall material A slight modification of the method used by Parker and Waldron [19] was carried out. Approximately 20 g of dry, ground lyophilised carrot was mixed with 15 g/l aqueous solution of sodium dodecyl sulphate (SDS) containing 5 mM Na2S2O5 with a Ystral homogeniser (Ystral GmbH, Dottingen, Germany) for 5 min. A few drops of octanol were used to limit the foaming. The homogenate was filtered through a 100 mm nylon mesh (John Stannier and Co., Manchester, UK) and the residue was ball-milled (Pascall, 0.5 l pot) at 0°C in 5 g/l SDS containing 3 mM Na2S2O5 for 2 h at 60 rpm to ensure cell rupture and removal of remaining compounds. After filtering the homogenate through a 75 mm nylon mesh, the residue was resuspended in cold water containing 3 mM Na2S2O5, homogenised for 5 min and refiltered. This procedure was repeated (three times) until the cell wall residue was free of intracellular components and starch granules as assessed by staining with iodine/potassium iodide and by using optical microscopy. The cell wall material was stored as a frozen suspension at − 20°C. This extraction with SDS solubilises mainly intracellular compounds and only very small amounts of cell wall polymers [20] although cell wall enzymes are not fully inactivated. The Huber method [21] was used, modified as follows: buffered phenol solution was prepared by the addition of 700 ml 500 mM Tris[hydroxymethyl]aminomethane, pH= 7.5 to 1.4 kg of phenol. The suspension was stirred and allowed to stand for 8 h. The upper aqueous phase was removed and the phenol phase was used to inactivate the cell wall enzymes by mixing with the thawed cell wall material described above, and stirring for 45 min. The resulting mixture was centrifuged at 400×g for 30 min at room temperature. The buffered phenol-saturated cell wall material was greatly diluted with 95% (v/v) ethanol and filtered through a 75 mm nylon mesh. The residue was washed with 95% (v/v) ethanol, followed by another wash with absolute ethanol and then washed three times with acetone. The remaining residue was left in a beaker at
room temperature allowing acetone to evaporate. The produced material, that is, SDS/phenol buffer insoluble residue (SIR) comprised carrot cell wall material with inactivated enzymes and free from intracellular components.
2.3. Sequential extraction 2.3.1. CDTA extraction SIR was extracted with 50 mM cyclohexane-trans1,2-diamine-N,N,N’N’ -tetraacetate (CDTA, Na+ salt) pH= 6.5 for 8 h at 20°C. After extraction, the CDTA insoluble residue (CIR) was dialysed exhaustively for 10 days and lyophilised. This procedure solubilises ionically (Ca2 + ) bound pectic polysaccharides [20]. 2.3.2. Na2CO3 extraction Subsequent extraction with 50 mM Na2CO3 containing 20 mM NaBH4 was carried out for over 6 h at 1°C, and then for 2 h at 20°C. The mixture was centrifuged and filtered. After extraction, the resulting residue (NIR) was dialysed for 5 days, neutralised and lyophilised. The Na2CO3 treatment extracts pectic components held into the cell wall directly or indirectly by ester linkages [22]. 2.3.3. KOH extraction After extraction of NIR with 0.5 M KOH containing 20 mM NaBH4 for 2 h, the 0.5M KOH insoluble residue (0.5KIR) was dialysed thoroughly for 3 to 5 days, neutralised and then lyophilised. The final extraction with 4M KOH containing 20mM NaBH4 for 2 h was carried out on 0.5KIR. Again, the latter was dialysed for 3 to 5 days, neutralised then lyophilised to produce the 4M KOH insoluble residue (4KIR).The extractions in alkali initially solubilise pectins and then at the higher concentrations, hemicellulosic polysaccharides [23,24]. All the residues produced above were ground with mortar and pestle and stored in hermetically sealed jars in a desiccator over silica gel. 2.4. Commercial biopolymers Citrus pectin (76% galacturonic acid and 8.6% methoxy), pectic acid (polygalacturonic acid with a low degree of esterification), polygalacturonic acid extracted from orange, galacturonic acid and long fibrous cellulose extracted from cotton lint were supplied by Sigma (Poole, UK).
2.5. Hot pressing Sufficient distilled water was added to the dry cell wall residues to obtain moisture contents of 50, 70 and 80% (wet weight basis). The residues were mixed with
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water, sandwiched between two acetate sheets and then loaded into a press consisting of a rectangular mould ring of sides 28 and 22 mm between two male compaction dies which were temperature controlled by four cartridge heaters and an inner cooling system [25]. A force of 35 kN (corresponding to a pressure of 57 MPa) was applied to the upper die with the use of a hydraulic pump. The whole device was heated up to one of the following temperatures: 30 and 100°C. The sample was then left for 15 min in the press before cold water was circulated in the inner cooling system. After 10 – 15 min cooling, the sample was removed. Approximately, 0.3 – 0.6 g of material gave a rectangular sheet 28 mm long, 22 mm wide and 0.5–1 mm thick. The sheet was then cut in small pieces, lyophilised and the material was ground with a mortar and pestle and then stored in a desiccator.
2.6. X-ray measurement The method followed that described by Singh et al [26]. X-ray measurements were carried out using CuKa1 radiation of wavelength 0.154 nm. The diffractometer was a Philips Scientific PW 1820 vertical goniometer with an Anton Paar TTK camera. The incident beam of X-rays was collimated using a Philips PW 1386/55 automatic divergence slit. With this device the divergence of the X-ray beam from the X-ray tube focus is continuously varied, so that at any angle of the goniometer, the length of sample irradiated remains constant. This avoids the problem of variations in measured intensity, and hence diffraction peak area, with increasing angle encountered when using a fixed divergence slit with a constant width. Data were collected using a proportional detector, then stored and processed on a personal computer using Philips PC-APD (Version 3.6b) automated powder diffraction software. Samples were scanned over the angular range 4.0 – 30.0°, 2u, at a speed of 0.005°, 2u, per second, with a step size of 0.15°, 2u. All measurements were carried out at 20°C and a relative humidity of 44%. The water content was determined by gravimetric drying in a vacuum oven at 70°C at a pressure of less than 5 mmHg over P2O5 desiccant. The moisture content was not determined for 4KIR because of insufficient quantity of material but it would be expected to be similar to that of 0.5KIR. The percentage crystallinity of the samples was calculated from the ratio of the area under the diffraction peaks to the total area under the whole diffraction pattern [27]. Amorphous background patterns were generated, fitted to, and subtracted from each diffraction pattern by means of the PC-APD software.
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Fig. 1. X-ray diffraction patterns of cotton lint cellulose untreated and treated with 4M KOH, polygalacturonic acid, citrus pectin and acetic acid
3. Results and discussion
3.1. X-ray diffraction for cellulose and 6arious pectic polysaccharides Fig. 1 shows the X-ray diffraction patterns of cellulose, and selected pectic polysaccharides. Several peaks were observed at scattering angles, 2u, of approximately 13.6 and 21.6° in citrus pectin, pectic acid and polygalacturonic acid. This is consistent with the findings of Wuhrmann and Pilnik [3] who reported X-ray peaks of pectin and found similar values of the scattering angle at which the peaks occurred. In Table 1, percentages of crystallinity are presented. Polygalacturonic acid and pectic acid with 11 and 12% crystallinity, respectively, had a higher crystallinity percentage than that of citrus pectin (6%) whereas galacturonic acid had a value of 76%. The X-ray diffraction pattern of cotton cellulose showed three distinct peaks at 14.6, 16.5 and 22.7°, 2u, characteristic of cellulose I [13]. It is well documented that the peaks correspond to 101, 101and 002 planes of diffraction [28,29], respectively, also observed in native cellulose. A fourth peak was apparent at 20.3°, 2u, Table 1 Crystallinitya of cotton lint cellulose, citrus pectin, pectic acid, polygalacturonic acid and galacturonic acid Samples
% Crystallinitya
Citrus pectin Pectic acid Polygalacturonic acid Galacturonic acid Cotton lint cellulose 4M KOH treated cotton lint cellulose
6 12 11 76 43 29
a
Standard deviation =1%.
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Fig. 2. X-ray diffraction patterns of SIR pressed at different pressing moisture contents and temperatures
which is characteristic of cellulose II. The percentage of crystallinity for cellulose was found to be 43%, similar to that calculated by Hulleman et al [16] who utilised a comparable technique to determine a crystallinity index of 40–48% in cotton and wood.
3.2. X-ray diffraction of SIR In Fig. 2, X-ray diffraction patterns of variously ‘hot pressed’ SIR extracted from lyophilised carrot are plotted. Although a major background due to the amorphous material present in the specimens is observed in all the variously pressed cell wall materials, a major peak at 21–22°, 2u, and a second peak at 15°, 2u, were discerned. A similar pattern was described by Ning et al [30] who used X-ray diffraction to examine the crystallinity of corn fiber after extrusion and in combination with chemical treatments. They attributed the fiber crystallinity to the highly ordered cellulose, also in agreement with the findings of Goto and Yokoe [12] who studied the X-ray diffraction of untreated and ammonia-treated barley straw. They assigned the pattern to that of cellulosic materials. In their investigation, two peaks were observed at 2u of 23 and 16°, respectively, the main peak (23°) being an indication of the presence of highly organised ‘crystalline cellulose’, while the second, broader peak (16°) is a measure of a less organised polysaccharide structure. They proposed that hemicellulosic material may contribute to this ‘amorphous’ peak. For the present study, the attribution of X-ray diffraction patterns of SIR to that of cellulose needs some clarification. For this purpose, comparison between Fig. 1 and Fig. 2 leads to the observation that the X-ray diffraction patterns produced from carrot cell wall material might be predominantly related to the highly ordered cellulose with a lesser contribution from
the pectic polysaccharides. By examining the diffraction patterns of cellulose, the width at half-height, the intensity and the peak position were found to be valuable parameters to determine the degree of crystallinity, the dimensions of the crystalline unit cell and the degree of orientation [31]. Depending on these factors, cellulose I, II, III and IV will have typical X-ray diffraction patterns as observed by Marchessault and Sundararajan [13]. In this respect, the X-ray diffraction scan of SIR is similar to that of cellulose IV. The previous authors suggested that cellulose IV is a disordered form of cellulose I as reported by Howsmon and Sisson [28] and when associated with hemicellulose and pectin, a significant amount of cellulose IV can be detected by X-ray diffraction [13]. This is also consistent with the findings of Chanzy et al [9] who studied the electron diffraction from cotton fibers and found that the crystalline structure of the fiber was similar to that of cellulose IV owing to the poor lateral organisation of the network of inter chain hydrogen bonds. The latter might be affected by the presence of hemicellulose and pectin in the case of carrot cell wall. There is a similar observation from Atalla [32] who published the X-ray diffraction pattern of pine kraft pulp which showed a lower degree of order than that of microcrystalline cellulose and suggested the coexistence of cellulose I and cellulose IV. The diffraction pattern of the latter had typical peaks corresponding to the 101 and 101 reflections coincident halfway between their position for cellulose I. In this regard, the X-ray diffraction pattern of carrot cell wall might also be the combination of these of cellulose I and cellulose IV. The occurrence of a background indicating the existence of a high content of amorphous domain, renders the further analysis of the diffraction patterns more difficult. SIR comprises 22% cellulose and 51% pectic polysaccharides [33] which are similar to values of 25% cellulose and 45–50% pectic polysaccharides found by Massiot et al [34]. Since the crystalline structure of hemicellulose has been studied [5] but not quantified, only the crystalline contributions of cellulose and pectic polysaccharides were used in predictions of the crystallinity of the residues. Crystallinity was calculated according to the rule of mixtures using the crystallinities and relative composition of cellulose and pectic polysaccharides assuming crystallinity values for cotton cellulose and citrus pectin from Table 1. The inclusion of the crystallinity of the pectic polysaccharides gives a better estimate of the SIR crystallinity than using the cellulose contribution alone (Table 2).
3.3. X-ray diffraction of CIR, NIR, 0.5KIR and 4 KIR In Fig. 3, the diffraction patterns of the sequentially produced carrot cell wall residues are represented. They have a similar pattern to that of SIR, characteristic of
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Table 2 Crystallinity for residues SIR, CIR, NIR, 0.5KIR and 4KIR Residues
SIR CIR NIR 0.5KIR 4KIR
Water content % (wet weight basis)
6 4 4 5 -
Composition
% Crystallinityc experimental
% Crystallinity predicted
Pectic polysaccharides
Cellulose
On basis of pectic polysaccharidesa and celluloseb
On basis of cellulose aloneb
51 46 40 30 23
22 30 32 42 55
13 16 16 20 24
10 13 14 18 23
12 16 15 16 14
a
Pectic polysaccharides crystallinity = 6% (Table 1, citrus pectin). Cellulose crystallinity = 43% (Table 1, cotton cellulose). c Standard deviation = 1%. b
cellulose as discussed earlier, comparable to that of ‘holocellulose’ (cellulose+hemicellulose) from spear grass [11] and cellulose of a red seaweed [35]. The main diffraction peak at 22.2° 2u increased in intensity when more pectic polysaccharides were removed. This is comparable with results found by Atalla et al [6] who reported a decrease of this peak when cellulose was produced by Acetobacter xylininum in the presence of increasing amount of xyloglucan. In relation to the present investigation, as more amorphous material is extracted during the preparation of the different residues, 0.5KIR will be enriched in the cellulose and hemicellulose (Section 2.3.3) consistent with the increasing values of crystallinity index (Table 2). The results altogether are comparable to the data reported by O’ Donoghue et al [8] who showed that the dimensions of the X-ray diffraction signal peak pertaining to the crystalline cellulose component were dependent on the quantity of cellulose present when studying avicel/ pectin mixtures. The degree of crystallinity of the different cell wall residues was predicted based on the percentages of pectic polysaccharides present in the different extracts together with the cellulose content [33]. The percentages of cellulose in CIR, NIR and 0.5KIR have been calculated from the amount of glucose which originates from cellulose allowing for 6% of the glucose content which is non-cellulosic [33]. For 4KIR, the glucose fully indicates the cellulose content [23]. Crystallinity was calculated as described in Section 3.2. Table 2 shows that the predictions based on cellulose alone are comparable to these obtained from the Xray diffraction patterns, but better agreement with experiment was found when the contribution of pectic polysaccharides crystallinity was included. 4KOH treated material, comprising mainly cellulose (Section 2.3.3), has a value of 14%, due to the severe alkaline extraction which reduces the crystalline domain. This would also explain the discrepancy of 10%
observed between the experimental and the predicted values (Table 2). In order to demonstrate the effect of 4 M KOH on the crystallinity, the chemical treatment was carried out on cotton lint cellulose. The X-ray diffraction pattern of the treated cellulose is shown in Fig. 1. It is noticeable that the area under the peaks is significantly smaller than that for untreated cotton lint cellulose, consistent with a reduction of crystallinity from 43 to 29% (Table 1). Sao et al [36] reported an analogous effect when ramie fiber was treated with 30% (6 M) NaOH which reduced the crystallinity index value by 31%. This is also supported by the findings of Khalifa et al [37] who observed a 11% decrease in crystallinity of Saudi Arabian cotton fiber upon treatment with 25% (5 M) NaOH. The results of the present study together with published data show that a strong alkaline treatment decreases the crystallinity of cellulose.
Fig. 3. X-ray diffraction patterns of SIR, NIR, CIR, 0.5KIR and 4KIR pressed at 70% moisture, 30°C
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Table 3 Crystallinitya of the different cell wall residues unpressed and pressed Cell wall residues
Unpressed
Pressed
Crystallinity
Crystallinity
Pressing conditions (% moisture/T°C)
SIR
12
CIR NIR 0.5KIR
16 15 16
12 12 10 12 13 14 17 18
50/30 50/100 80/30 80/100 70/30 70/30 70/30 70/30
a
Standard deviation =1%.
3.4. Effect of hot pressing on the different residues It is noteworthy that the X-ray diffraction patterns of the cell wall materials show similar responses when variously pressed (Fig. 2). This is in agreement with results reported by Artz et al [38] who investigated the extrusion modification of corn fiber using X-ray diffraction They found that extrusion did not affect fiber crystallinity. Although the processing regime used in the present study is less severe in comparison to extrusion, it is interesting to note that the present findings are comparable to the results produced by Artz et al [38]. The crystallinity index of SIR varies from 10 to 13% for pressed specimens (Table 3). Table 3 shows the effect of pressing on the crystallinity index of the different carrot cell wall residues. In general, the crystallinity remains constant when the samples were pressed at 70% moisture at 30°C. These results are in contrast to the findings of Weimer et al [17] who observed an increase in the crystallinity of cellulose upon wetting. During the preparation of the specimens, 70% moisture (wet weight basis) was achieved prior to pressing by adding water to the freeze-dried materials. This might be insufficient to facilitate an increase in the size or distribution of the crystalline lattices or a recrystallisation process [17]. However, when comparing the progressively produced cell wall residues pressed at 70% moisture and 30°C, the crystallinity index increased as more pectic polysaccharides were removed.
4. Conclusion By using X-ray diffraction on the sequentially extracted carrot cell wall residues, the patterns indicated the dominance of the ordered structure of cellulose when compared to other isolated cell wall biopolymers. The assignment for the cell wall residues to a known lattice type such as cellulose I or cellulose IV or the
combination of the two lattice types is suggested. Some evidence for the contribution of pectin to the crystallinity of carrot cell wall was found. A more sophisticated analysis such as multiple least square fitting might be useful in estimating the distribution of the various crystalline and amorphous fractions. When carrot cell wall material underwent various degrees of wetting and heating temperatures during a pressing regime, the crystallinity index was not affected significantly. However when pressed at 70% moisture and heated at 30°C, a small increase in crystallinity was noted for the progressively produced cell wall extracts. The presence of crystalline or at least ordered regions of non-cellulosic polymers indicates that the stability and process properties of cell wall materials may be more complex than might be assumed for a cellulosedominated amorphous matrix.
Acknowledgements The authors wish to thank Dr VJ Morris for helpful discussions and the Biotechnology and Biological Sciences Research Council for funding.
References [1] Carpita NC, Gibeaut DM. Plant J 1993;3:1. [2] Brett C, Waldron K. Physiology and biochemistry of plant cell walls. London: Unwin Hyman, 1996:6. [3] Wuhrman K, Pilnik W. Experientia 1945;1:330. [4] Roelofsen PA, Kreger DR. J Exp Bot 1951;11:332. [5] Yui T, Imada K, Shiuya N, Ogawa K. Biosci Biotechnol Biochem 1995;59:965. [6] Atalla RH, Hackney JM, Uhlin I, Thompson NS. Int J Biol Macromol 1993;15:109. [7] Shimazu F, Sterling C. J Food Sci 1961;26:291. [8] O’Donoghue EM, Huber DJ, Timpa JD, Erdos GW, Brecht JK. Planta 1994;194:573. [9] Chanzy H, Imada K, Vuong R. Protoplasma 1978;94:299. [10] Okano T, Sarko A. J Appl Polym Sci 1985;30:325. [11] Beveridge RJ, Richards GN. Carbohydr Res 1975;43:163.
D.M.R. Georget et al. / International Journal of Biological Macromolecules 26 (1999) 325–331 [12] Goto M, Yokoe Y. Anim Feed Sci Technol 1996;58:239. [13] Marchessault RH, Sundararajan PR. In: Aspinall GO, editor. The Polysaccharides, vol. 2. New York: Academic Press, 1993:44. [14] Cronshaw J, Myers A, Preston RD. Biochim Biophys Acta 1958;27:89. [15] Lahaye M, Jegou D, Buleon A. Carbohydr Res 1994;262:115. [16] Hulleman SHD, van Hazendonk JM, van Dam JEG. Carbohydr Res 1994;261:163. [17] Weimer PJ, Hackney JM, French AD. Biotechnol Bioeng 1995;48:169. [18] Georget DMR, Smith AC, Waldron KW. Thermochim Acta 1998;315:51. [19] Parker ML, Waldron KW. J Sci Food Agric 1995;68:337. [20] Selvendran RR, Ryden P. In: Dey PM, Harbone JB, editors. Methods in Plant Biochemistry, vol. 2. London: Academic Press, 1990:553. [21] Huber DJ. Phytochemistry 1991;42:2900. [22] Jarvis MC, Hall MA, Threlfall DR, Friend J. Planta 1981;152:93. [23] Selvendran RR. J Cell Sci Suppl 1985;2:51. [24] Redgwell RJ, Selvendran RR. Carbohydr Res 1986;157:183. [25] Georget DMR, Smith AC. Carbohydr Polym 1995;28:305. [26] Singh N, Cairns P, Morris VJ, Smith AC. Cereal Chem 1998;75:325.
.
331
[27] Hermans PH, Weidinger A. J Appl Phys 1948;19:491. [28] Howsmon JA, Sisson WA. In: Ott E, Spurlin HM, Graffin MW, editors. Cellulose and Cellulose derivatives, Part I. New York: Interscience Publishers, 1954:239. [29] Preston RD. The Physical Biology of Plant Cell Walls. London: Chapman and Hall, 1974:134. [30] Ning L, Villota R, Artz WE. Cereal Chem 1991;68:632. [31] Uhlin KI. The influence of hemicelluloses on the structure of bacterial cellulose. Doctoral Dissertation. The Institute of Paper Science and Technology: Atlanta, 1990:48. [32] Atalla RH. In: Soltes J, editor. Wood and Agricultural Residues. New York: Academic Press, 1983:65. [33] Georget DMR, Smith AC, Waldron KW. Dynamic mechanical thermal analysis of cell wall polysaccharides extracted from lyophilised Daucus carota (Submitted). [34] Massiot P, Rouau X, Thibault JF. Carbohydr Res 1988;172:217. [35] Hoffman RA, Gidley MJ, Cooke D, Frith W. Food Hydrocoll 1995;9:281. [36] Sao KP, Samantaray BK, Bhattacherjee S. J Appl Polym Sci 1994;52:1687. [37] Khalifa BA, Abdel-Zaber N, Shoukr FS. Text Res J 1991;61:602. [38] Artz WE, Warren C, Villota R. J Food Sci 1990;55:746.
Crystallinity of lyophilised carrot cell wall components Dominique M.R. Georget †, Paul Cairns, Andrew C. Smith *, Keith W. Waldron Norwich Laboratory, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK Accepted 26 July 1999
Abstract The aim of this work was to investigate the effect of removal of cell wall components on the crystallinity of cell walls using X-ray diffraction. Various insoluble cell wall residues were prepared following a sequential extraction of carrot cell wall material. X-ray diffraction patterns were typical of cellulose although there was a possible contribution of pectic polysaccharides to the crystallinity. As more amorphous material was removed to produce a cellulose rich residue, the crystallinity index increased from 12 to 16%, larger than that estimated from cellulose alone. For the last residue treated with 4M KOH, a lower value of crystallinity was found (14%) which resulted from the change of some crystalline domains of cellulose into amorphous regions. Pressing conditions (temperature, water content) have been investigated and did not alter the crystallinity index significantly. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Carrot; Cell wall; X-ray; Cellulose; Crystallinity
1. Introduction The cell wall of plant tissue comprises crystalline and amorphous domains [1]. The latter consists of a variety of components such as pectic polysaccharides, hemicellulose, proteins and phenolic derivatives [2]. Some degree of crystallinity has been observed in pectic polysaccharides [3,4] and hemicellulosic polysaccharides [5], although the crystalline regions of the wall consist mainly of cellulose. However, the crystalline and amorphous domains of cellulose/hemicellulose can barely be distinguished using X-ray diffraction, due to the existence of close associations between the two phases [6]. In early work, the crystallinity of carrot phloem cellulose and calcium pectinate produced from citrus [7] was measured and its relevance to the physical properties of dried vegetable tissues suggested. The crystallinity of the cell wall of avocado during
* Corresponding author. Tel.: +44 1603 255000, fax: + 44 1603 507723. E-mail address: [email protected] (A.C. Smith) † Present address: INRA ENSAM, Unite de Technologie des Cereales, 2, place Pierre Viala, 34060 Montpellier Cedex 1, France.
ripening has been assessed and compared with cellulose/pectin model systems [8], showing that the loss of cohesiveness within the cellulose fibril structure and the binding of the cell wall matrix polysaccharides were altered. Major work has been carried out on cellulose for its application in the textile industry [9,10] and cattle feeding [11,12]. Cellulose occurs in several crystalline forms [13] and consequently, when studying cellulose crystallinity in plant material, it is difficult to assign the X-ray diffraction results to those obtained on cellulose of a known lattice type [14,15]. In addition, crystallinity may be reduced by mechanical disruption such as ball milling [16] and increased by heating in an aqueous environment [17]. In the present study, the objectives were to investigate the X-ray diffraction patterns and the crystallinity indices of cell wall material purified from lyophilised carrot in comparison with crystallinity data obtained on proprietary cotton lint cellulose and citrus pectin. The effects of pressing temperature and water content on the degree of crystallinity were investigated and complement studies on the thermomechanical properties of the pressed and extracted cell wall [18].
0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 4 1 - 8 1 3 0 ( 9 9 ) 0 0 1 0 2 - 6
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2. Materials and methods
2.1. Lyophilised carrot Carrots (Daucus carota cv. Amstrong) were grown locally. Once harvested, the crop was cleaned by brushing and washing. The tissues were then sliced and frozen immediately in liquid nitrogen. The frozen material was freeze-dried (freeze-dryer Model 3.5, Birchover Instruments, Letchworth, Herts, UK). The lyophilised material was then ground to a powder using a mortar and pestle and then stored in a desiccator over silica gel.
2.2. Preparation of carrot cell wall material A slight modification of the method used by Parker and Waldron [19] was carried out. Approximately 20 g of dry, ground lyophilised carrot was mixed with 15 g/l aqueous solution of sodium dodecyl sulphate (SDS) containing 5 mM Na2S2O5 with a Ystral homogeniser (Ystral GmbH, Dottingen, Germany) for 5 min. A few drops of octanol were used to limit the foaming. The homogenate was filtered through a 100 mm nylon mesh (John Stannier and Co., Manchester, UK) and the residue was ball-milled (Pascall, 0.5 l pot) at 0°C in 5 g/l SDS containing 3 mM Na2S2O5 for 2 h at 60 rpm to ensure cell rupture and removal of remaining compounds. After filtering the homogenate through a 75 mm nylon mesh, the residue was resuspended in cold water containing 3 mM Na2S2O5, homogenised for 5 min and refiltered. This procedure was repeated (three times) until the cell wall residue was free of intracellular components and starch granules as assessed by staining with iodine/potassium iodide and by using optical microscopy. The cell wall material was stored as a frozen suspension at − 20°C. This extraction with SDS solubilises mainly intracellular compounds and only very small amounts of cell wall polymers [20] although cell wall enzymes are not fully inactivated. The Huber method [21] was used, modified as follows: buffered phenol solution was prepared by the addition of 700 ml 500 mM Tris[hydroxymethyl]aminomethane, pH= 7.5 to 1.4 kg of phenol. The suspension was stirred and allowed to stand for 8 h. The upper aqueous phase was removed and the phenol phase was used to inactivate the cell wall enzymes by mixing with the thawed cell wall material described above, and stirring for 45 min. The resulting mixture was centrifuged at 400×g for 30 min at room temperature. The buffered phenol-saturated cell wall material was greatly diluted with 95% (v/v) ethanol and filtered through a 75 mm nylon mesh. The residue was washed with 95% (v/v) ethanol, followed by another wash with absolute ethanol and then washed three times with acetone. The remaining residue was left in a beaker at
room temperature allowing acetone to evaporate. The produced material, that is, SDS/phenol buffer insoluble residue (SIR) comprised carrot cell wall material with inactivated enzymes and free from intracellular components.
2.3. Sequential extraction 2.3.1. CDTA extraction SIR was extracted with 50 mM cyclohexane-trans1,2-diamine-N,N,N’N’ -tetraacetate (CDTA, Na+ salt) pH= 6.5 for 8 h at 20°C. After extraction, the CDTA insoluble residue (CIR) was dialysed exhaustively for 10 days and lyophilised. This procedure solubilises ionically (Ca2 + ) bound pectic polysaccharides [20]. 2.3.2. Na2CO3 extraction Subsequent extraction with 50 mM Na2CO3 containing 20 mM NaBH4 was carried out for over 6 h at 1°C, and then for 2 h at 20°C. The mixture was centrifuged and filtered. After extraction, the resulting residue (NIR) was dialysed for 5 days, neutralised and lyophilised. The Na2CO3 treatment extracts pectic components held into the cell wall directly or indirectly by ester linkages [22]. 2.3.3. KOH extraction After extraction of NIR with 0.5 M KOH containing 20 mM NaBH4 for 2 h, the 0.5M KOH insoluble residue (0.5KIR) was dialysed thoroughly for 3 to 5 days, neutralised and then lyophilised. The final extraction with 4M KOH containing 20mM NaBH4 for 2 h was carried out on 0.5KIR. Again, the latter was dialysed for 3 to 5 days, neutralised then lyophilised to produce the 4M KOH insoluble residue (4KIR).The extractions in alkali initially solubilise pectins and then at the higher concentrations, hemicellulosic polysaccharides [23,24]. All the residues produced above were ground with mortar and pestle and stored in hermetically sealed jars in a desiccator over silica gel. 2.4. Commercial biopolymers Citrus pectin (76% galacturonic acid and 8.6% methoxy), pectic acid (polygalacturonic acid with a low degree of esterification), polygalacturonic acid extracted from orange, galacturonic acid and long fibrous cellulose extracted from cotton lint were supplied by Sigma (Poole, UK).
2.5. Hot pressing Sufficient distilled water was added to the dry cell wall residues to obtain moisture contents of 50, 70 and 80% (wet weight basis). The residues were mixed with
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water, sandwiched between two acetate sheets and then loaded into a press consisting of a rectangular mould ring of sides 28 and 22 mm between two male compaction dies which were temperature controlled by four cartridge heaters and an inner cooling system [25]. A force of 35 kN (corresponding to a pressure of 57 MPa) was applied to the upper die with the use of a hydraulic pump. The whole device was heated up to one of the following temperatures: 30 and 100°C. The sample was then left for 15 min in the press before cold water was circulated in the inner cooling system. After 10 – 15 min cooling, the sample was removed. Approximately, 0.3 – 0.6 g of material gave a rectangular sheet 28 mm long, 22 mm wide and 0.5–1 mm thick. The sheet was then cut in small pieces, lyophilised and the material was ground with a mortar and pestle and then stored in a desiccator.
2.6. X-ray measurement The method followed that described by Singh et al [26]. X-ray measurements were carried out using CuKa1 radiation of wavelength 0.154 nm. The diffractometer was a Philips Scientific PW 1820 vertical goniometer with an Anton Paar TTK camera. The incident beam of X-rays was collimated using a Philips PW 1386/55 automatic divergence slit. With this device the divergence of the X-ray beam from the X-ray tube focus is continuously varied, so that at any angle of the goniometer, the length of sample irradiated remains constant. This avoids the problem of variations in measured intensity, and hence diffraction peak area, with increasing angle encountered when using a fixed divergence slit with a constant width. Data were collected using a proportional detector, then stored and processed on a personal computer using Philips PC-APD (Version 3.6b) automated powder diffraction software. Samples were scanned over the angular range 4.0 – 30.0°, 2u, at a speed of 0.005°, 2u, per second, with a step size of 0.15°, 2u. All measurements were carried out at 20°C and a relative humidity of 44%. The water content was determined by gravimetric drying in a vacuum oven at 70°C at a pressure of less than 5 mmHg over P2O5 desiccant. The moisture content was not determined for 4KIR because of insufficient quantity of material but it would be expected to be similar to that of 0.5KIR. The percentage crystallinity of the samples was calculated from the ratio of the area under the diffraction peaks to the total area under the whole diffraction pattern [27]. Amorphous background patterns were generated, fitted to, and subtracted from each diffraction pattern by means of the PC-APD software.
327
Fig. 1. X-ray diffraction patterns of cotton lint cellulose untreated and treated with 4M KOH, polygalacturonic acid, citrus pectin and acetic acid
3. Results and discussion
3.1. X-ray diffraction for cellulose and 6arious pectic polysaccharides Fig. 1 shows the X-ray diffraction patterns of cellulose, and selected pectic polysaccharides. Several peaks were observed at scattering angles, 2u, of approximately 13.6 and 21.6° in citrus pectin, pectic acid and polygalacturonic acid. This is consistent with the findings of Wuhrmann and Pilnik [3] who reported X-ray peaks of pectin and found similar values of the scattering angle at which the peaks occurred. In Table 1, percentages of crystallinity are presented. Polygalacturonic acid and pectic acid with 11 and 12% crystallinity, respectively, had a higher crystallinity percentage than that of citrus pectin (6%) whereas galacturonic acid had a value of 76%. The X-ray diffraction pattern of cotton cellulose showed three distinct peaks at 14.6, 16.5 and 22.7°, 2u, characteristic of cellulose I [13]. It is well documented that the peaks correspond to 101, 101and 002 planes of diffraction [28,29], respectively, also observed in native cellulose. A fourth peak was apparent at 20.3°, 2u, Table 1 Crystallinitya of cotton lint cellulose, citrus pectin, pectic acid, polygalacturonic acid and galacturonic acid Samples
% Crystallinitya
Citrus pectin Pectic acid Polygalacturonic acid Galacturonic acid Cotton lint cellulose 4M KOH treated cotton lint cellulose
6 12 11 76 43 29
a
Standard deviation =1%.
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Fig. 2. X-ray diffraction patterns of SIR pressed at different pressing moisture contents and temperatures
which is characteristic of cellulose II. The percentage of crystallinity for cellulose was found to be 43%, similar to that calculated by Hulleman et al [16] who utilised a comparable technique to determine a crystallinity index of 40–48% in cotton and wood.
3.2. X-ray diffraction of SIR In Fig. 2, X-ray diffraction patterns of variously ‘hot pressed’ SIR extracted from lyophilised carrot are plotted. Although a major background due to the amorphous material present in the specimens is observed in all the variously pressed cell wall materials, a major peak at 21–22°, 2u, and a second peak at 15°, 2u, were discerned. A similar pattern was described by Ning et al [30] who used X-ray diffraction to examine the crystallinity of corn fiber after extrusion and in combination with chemical treatments. They attributed the fiber crystallinity to the highly ordered cellulose, also in agreement with the findings of Goto and Yokoe [12] who studied the X-ray diffraction of untreated and ammonia-treated barley straw. They assigned the pattern to that of cellulosic materials. In their investigation, two peaks were observed at 2u of 23 and 16°, respectively, the main peak (23°) being an indication of the presence of highly organised ‘crystalline cellulose’, while the second, broader peak (16°) is a measure of a less organised polysaccharide structure. They proposed that hemicellulosic material may contribute to this ‘amorphous’ peak. For the present study, the attribution of X-ray diffraction patterns of SIR to that of cellulose needs some clarification. For this purpose, comparison between Fig. 1 and Fig. 2 leads to the observation that the X-ray diffraction patterns produced from carrot cell wall material might be predominantly related to the highly ordered cellulose with a lesser contribution from
the pectic polysaccharides. By examining the diffraction patterns of cellulose, the width at half-height, the intensity and the peak position were found to be valuable parameters to determine the degree of crystallinity, the dimensions of the crystalline unit cell and the degree of orientation [31]. Depending on these factors, cellulose I, II, III and IV will have typical X-ray diffraction patterns as observed by Marchessault and Sundararajan [13]. In this respect, the X-ray diffraction scan of SIR is similar to that of cellulose IV. The previous authors suggested that cellulose IV is a disordered form of cellulose I as reported by Howsmon and Sisson [28] and when associated with hemicellulose and pectin, a significant amount of cellulose IV can be detected by X-ray diffraction [13]. This is also consistent with the findings of Chanzy et al [9] who studied the electron diffraction from cotton fibers and found that the crystalline structure of the fiber was similar to that of cellulose IV owing to the poor lateral organisation of the network of inter chain hydrogen bonds. The latter might be affected by the presence of hemicellulose and pectin in the case of carrot cell wall. There is a similar observation from Atalla [32] who published the X-ray diffraction pattern of pine kraft pulp which showed a lower degree of order than that of microcrystalline cellulose and suggested the coexistence of cellulose I and cellulose IV. The diffraction pattern of the latter had typical peaks corresponding to the 101 and 101 reflections coincident halfway between their position for cellulose I. In this regard, the X-ray diffraction pattern of carrot cell wall might also be the combination of these of cellulose I and cellulose IV. The occurrence of a background indicating the existence of a high content of amorphous domain, renders the further analysis of the diffraction patterns more difficult. SIR comprises 22% cellulose and 51% pectic polysaccharides [33] which are similar to values of 25% cellulose and 45–50% pectic polysaccharides found by Massiot et al [34]. Since the crystalline structure of hemicellulose has been studied [5] but not quantified, only the crystalline contributions of cellulose and pectic polysaccharides were used in predictions of the crystallinity of the residues. Crystallinity was calculated according to the rule of mixtures using the crystallinities and relative composition of cellulose and pectic polysaccharides assuming crystallinity values for cotton cellulose and citrus pectin from Table 1. The inclusion of the crystallinity of the pectic polysaccharides gives a better estimate of the SIR crystallinity than using the cellulose contribution alone (Table 2).
3.3. X-ray diffraction of CIR, NIR, 0.5KIR and 4 KIR In Fig. 3, the diffraction patterns of the sequentially produced carrot cell wall residues are represented. They have a similar pattern to that of SIR, characteristic of
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Table 2 Crystallinity for residues SIR, CIR, NIR, 0.5KIR and 4KIR Residues
SIR CIR NIR 0.5KIR 4KIR
Water content % (wet weight basis)
6 4 4 5 -
Composition
% Crystallinityc experimental
% Crystallinity predicted
Pectic polysaccharides
Cellulose
On basis of pectic polysaccharidesa and celluloseb
On basis of cellulose aloneb
51 46 40 30 23
22 30 32 42 55
13 16 16 20 24
10 13 14 18 23
12 16 15 16 14
a
Pectic polysaccharides crystallinity = 6% (Table 1, citrus pectin). Cellulose crystallinity = 43% (Table 1, cotton cellulose). c Standard deviation = 1%. b
cellulose as discussed earlier, comparable to that of ‘holocellulose’ (cellulose+hemicellulose) from spear grass [11] and cellulose of a red seaweed [35]. The main diffraction peak at 22.2° 2u increased in intensity when more pectic polysaccharides were removed. This is comparable with results found by Atalla et al [6] who reported a decrease of this peak when cellulose was produced by Acetobacter xylininum in the presence of increasing amount of xyloglucan. In relation to the present investigation, as more amorphous material is extracted during the preparation of the different residues, 0.5KIR will be enriched in the cellulose and hemicellulose (Section 2.3.3) consistent with the increasing values of crystallinity index (Table 2). The results altogether are comparable to the data reported by O’ Donoghue et al [8] who showed that the dimensions of the X-ray diffraction signal peak pertaining to the crystalline cellulose component were dependent on the quantity of cellulose present when studying avicel/ pectin mixtures. The degree of crystallinity of the different cell wall residues was predicted based on the percentages of pectic polysaccharides present in the different extracts together with the cellulose content [33]. The percentages of cellulose in CIR, NIR and 0.5KIR have been calculated from the amount of glucose which originates from cellulose allowing for 6% of the glucose content which is non-cellulosic [33]. For 4KIR, the glucose fully indicates the cellulose content [23]. Crystallinity was calculated as described in Section 3.2. Table 2 shows that the predictions based on cellulose alone are comparable to these obtained from the Xray diffraction patterns, but better agreement with experiment was found when the contribution of pectic polysaccharides crystallinity was included. 4KOH treated material, comprising mainly cellulose (Section 2.3.3), has a value of 14%, due to the severe alkaline extraction which reduces the crystalline domain. This would also explain the discrepancy of 10%
observed between the experimental and the predicted values (Table 2). In order to demonstrate the effect of 4 M KOH on the crystallinity, the chemical treatment was carried out on cotton lint cellulose. The X-ray diffraction pattern of the treated cellulose is shown in Fig. 1. It is noticeable that the area under the peaks is significantly smaller than that for untreated cotton lint cellulose, consistent with a reduction of crystallinity from 43 to 29% (Table 1). Sao et al [36] reported an analogous effect when ramie fiber was treated with 30% (6 M) NaOH which reduced the crystallinity index value by 31%. This is also supported by the findings of Khalifa et al [37] who observed a 11% decrease in crystallinity of Saudi Arabian cotton fiber upon treatment with 25% (5 M) NaOH. The results of the present study together with published data show that a strong alkaline treatment decreases the crystallinity of cellulose.
Fig. 3. X-ray diffraction patterns of SIR, NIR, CIR, 0.5KIR and 4KIR pressed at 70% moisture, 30°C
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Table 3 Crystallinitya of the different cell wall residues unpressed and pressed Cell wall residues
Unpressed
Pressed
Crystallinity
Crystallinity
Pressing conditions (% moisture/T°C)
SIR
12
CIR NIR 0.5KIR
16 15 16
12 12 10 12 13 14 17 18
50/30 50/100 80/30 80/100 70/30 70/30 70/30 70/30
a
Standard deviation =1%.
3.4. Effect of hot pressing on the different residues It is noteworthy that the X-ray diffraction patterns of the cell wall materials show similar responses when variously pressed (Fig. 2). This is in agreement with results reported by Artz et al [38] who investigated the extrusion modification of corn fiber using X-ray diffraction They found that extrusion did not affect fiber crystallinity. Although the processing regime used in the present study is less severe in comparison to extrusion, it is interesting to note that the present findings are comparable to the results produced by Artz et al [38]. The crystallinity index of SIR varies from 10 to 13% for pressed specimens (Table 3). Table 3 shows the effect of pressing on the crystallinity index of the different carrot cell wall residues. In general, the crystallinity remains constant when the samples were pressed at 70% moisture at 30°C. These results are in contrast to the findings of Weimer et al [17] who observed an increase in the crystallinity of cellulose upon wetting. During the preparation of the specimens, 70% moisture (wet weight basis) was achieved prior to pressing by adding water to the freeze-dried materials. This might be insufficient to facilitate an increase in the size or distribution of the crystalline lattices or a recrystallisation process [17]. However, when comparing the progressively produced cell wall residues pressed at 70% moisture and 30°C, the crystallinity index increased as more pectic polysaccharides were removed.
4. Conclusion By using X-ray diffraction on the sequentially extracted carrot cell wall residues, the patterns indicated the dominance of the ordered structure of cellulose when compared to other isolated cell wall biopolymers. The assignment for the cell wall residues to a known lattice type such as cellulose I or cellulose IV or the
combination of the two lattice types is suggested. Some evidence for the contribution of pectin to the crystallinity of carrot cell wall was found. A more sophisticated analysis such as multiple least square fitting might be useful in estimating the distribution of the various crystalline and amorphous fractions. When carrot cell wall material underwent various degrees of wetting and heating temperatures during a pressing regime, the crystallinity index was not affected significantly. However when pressed at 70% moisture and heated at 30°C, a small increase in crystallinity was noted for the progressively produced cell wall extracts. The presence of crystalline or at least ordered regions of non-cellulosic polymers indicates that the stability and process properties of cell wall materials may be more complex than might be assumed for a cellulosedominated amorphous matrix.
Acknowledgements The authors wish to thank Dr VJ Morris for helpful discussions and the Biotechnology and Biological Sciences Research Council for funding.
References [1] Carpita NC, Gibeaut DM. Plant J 1993;3:1. [2] Brett C, Waldron K. Physiology and biochemistry of plant cell walls. London: Unwin Hyman, 1996:6. [3] Wuhrman K, Pilnik W. Experientia 1945;1:330. [4] Roelofsen PA, Kreger DR. J Exp Bot 1951;11:332. [5] Yui T, Imada K, Shiuya N, Ogawa K. Biosci Biotechnol Biochem 1995;59:965. [6] Atalla RH, Hackney JM, Uhlin I, Thompson NS. Int J Biol Macromol 1993;15:109. [7] Shimazu F, Sterling C. J Food Sci 1961;26:291. [8] O’Donoghue EM, Huber DJ, Timpa JD, Erdos GW, Brecht JK. Planta 1994;194:573. [9] Chanzy H, Imada K, Vuong R. Protoplasma 1978;94:299. [10] Okano T, Sarko A. J Appl Polym Sci 1985;30:325. [11] Beveridge RJ, Richards GN. Carbohydr Res 1975;43:163.
D.M.R. Georget et al. / International Journal of Biological Macromolecules 26 (1999) 325–331 [12] Goto M, Yokoe Y. Anim Feed Sci Technol 1996;58:239. [13] Marchessault RH, Sundararajan PR. In: Aspinall GO, editor. The Polysaccharides, vol. 2. New York: Academic Press, 1993:44. [14] Cronshaw J, Myers A, Preston RD. Biochim Biophys Acta 1958;27:89. [15] Lahaye M, Jegou D, Buleon A. Carbohydr Res 1994;262:115. [16] Hulleman SHD, van Hazendonk JM, van Dam JEG. Carbohydr Res 1994;261:163. [17] Weimer PJ, Hackney JM, French AD. Biotechnol Bioeng 1995;48:169. [18] Georget DMR, Smith AC, Waldron KW. Thermochim Acta 1998;315:51. [19] Parker ML, Waldron KW. J Sci Food Agric 1995;68:337. [20] Selvendran RR, Ryden P. In: Dey PM, Harbone JB, editors. Methods in Plant Biochemistry, vol. 2. London: Academic Press, 1990:553. [21] Huber DJ. Phytochemistry 1991;42:2900. [22] Jarvis MC, Hall MA, Threlfall DR, Friend J. Planta 1981;152:93. [23] Selvendran RR. J Cell Sci Suppl 1985;2:51. [24] Redgwell RJ, Selvendran RR. Carbohydr Res 1986;157:183. [25] Georget DMR, Smith AC. Carbohydr Polym 1995;28:305. [26] Singh N, Cairns P, Morris VJ, Smith AC. Cereal Chem 1998;75:325.
.
331
[27] Hermans PH, Weidinger A. J Appl Phys 1948;19:491. [28] Howsmon JA, Sisson WA. In: Ott E, Spurlin HM, Graffin MW, editors. Cellulose and Cellulose derivatives, Part I. New York: Interscience Publishers, 1954:239. [29] Preston RD. The Physical Biology of Plant Cell Walls. London: Chapman and Hall, 1974:134. [30] Ning L, Villota R, Artz WE. Cereal Chem 1991;68:632. [31] Uhlin KI. The influence of hemicelluloses on the structure of bacterial cellulose. Doctoral Dissertation. The Institute of Paper Science and Technology: Atlanta, 1990:48. [32] Atalla RH. In: Soltes J, editor. Wood and Agricultural Residues. New York: Academic Press, 1983:65. [33] Georget DMR, Smith AC, Waldron KW. Dynamic mechanical thermal analysis of cell wall polysaccharides extracted from lyophilised Daucus carota (Submitted). [34] Massiot P, Rouau X, Thibault JF. Carbohydr Res 1988;172:217. [35] Hoffman RA, Gidley MJ, Cooke D, Frith W. Food Hydrocoll 1995;9:281. [36] Sao KP, Samantaray BK, Bhattacherjee S. J Appl Polym Sci 1994;52:1687. [37] Khalifa BA, Abdel-Zaber N, Shoukr FS. Text Res J 1991;61:602. [38] Artz WE, Warren C, Villota R. J Food Sci 1990;55:746.