P-STARCH-22 Preparation of microcrystalline cellulose from ... for...

Starch Update 2013: The 7th International Conference on Starch Technology

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P-STARCH-22

Preparation of microcrystalline cellulose from destarched cassava pulp

Sawanya Menggred1, Pathama Chatakanonda2, Kuakoon Piyachomkwan3 and Klanarong Sriroth1

1Departments of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand

2Kasetsart Agro-industrial Product Improvement Institute, Bangokok, Thailand 3Cassava and Starch Technology Research Unit, National Center for Genetic Engineering and Biotechnology,

Pathumthani, Thailand.

Abstract The microcrystalline cellulose (MCC) was prepared from destarched cassava pulp, i.e. cassava pulp being previously hydrolyzed by α-amylase to remove the starch in pulp (% starch in cassava pulp and destarched pulp was 50-60 and 1% dry basis). The process included usual chemical treatment such as alkaline extraction, bleaching and acid hydrolysis. The chemical composition of fibers at different stages was analyzed and the results showed an increase in the cellulose content and a decrease in the lignin content. The Fourier Transform Infrared (FTIR) spectroscopy was applied to confirm the chemical structure of produced fiber and the spectrum suggests that lignin was almost entirely removed during alkali and bleaching treatment. After acid hydrolysis, the X-ray diffraction analysis exhibited an increase of crystallinity index (CrI) of cassava cellulose from 36.84% to 74.39%. The morphology of acid hydrolyzed cellulose by Transmission Electron Microscopy (TEM) demonstrated the rod-like or needle-shaped with the diameter approximately 100-300 nm. Keywords: Microcrystalline cellulose, destarched cassava pulp, morphology 1. Introduction Cassava (Manihot esculenta Crantz) is a root crop largely grown in many tropical countries. It is a starch-rich material, and also containing some proteins, lipids, lignocellulosic fiber and sugars. In Thailand, cassava starch is a large and growing industry of which approximately 10 million tons of fresh cassava roots are used for the production of starch, generating at least 2.5-3.0 million tons of wet pulp annually (Sriroth et al., 2000). The industrial production of cassava starch (CS) involves the elimination of soluble sugars and the separation of fibers, resulting in a purified starch and a solid residue called cassava pulp. The cassava pulp is mainly composed of water (70-80 %wt), residual starch and lignocellulosic fibers. On dry weight basis, the pulp contains starch the most, i.e. more than 50%. In addition, the pulp has cellulose, ranging from 15 to 50 %wt (Teixeira et al., 2009). To improve the utilization of cassava pulp, starch can be reextracted from the pulp, either in a form of starch or hydrolyzed starch, i.e. glucose syrup by enzyme process (Sriroth et al., 2000). This syrup can be exploited as a feedstock for fermentation industry. The residue collected after starch extraction, i.e. destarched cassava pulp is a lignocellulosic material which can be used to produce cellulose micro/nanoparticles. Cellulose is a homopolymer of β-1,4-D-glucose molecules linked in a linear fashion with high degree of polymerization and crystallinity. The preparation of microcrystalline α-cellulose (MCC) from agricultural waste has been reported (Tang et al., 1996; Castro and Bueno, 1996; Chen et al., 1996; Nelson et al., 2000; El-Sakhawy and Hassan, 2007; Teixeira et al., 2009; Rosa et al., 2010; Abraham et al., 2011. Mandal and Chakrabarty, 2011). The process involves various steps including bleaching and synthesis of MCC in different shapes such as sphere-like (Pu et al., 2007;


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Zhang et al., 2007) and rod-like (Azizi et al., 2005) by using physical means such as shearing, high pressure homogenizing, chemical means such as acid hydrolysis and enzymatic means. Microcrystalline cellulose can be used as a thickening and texturizing agents in food, coloring agents, cosmetics and pharmaceutical products. In addition, the application of cellulose fibers as a reinforcing agent in composite materials has been reported (Habibi et al., 2009). The objective of this work was to prepare and characterize the chemical and physical properties of cassava cellulose from destarched cassava pulp. 2. Materials and Methods Fresh cassava pulp was obtained from Thai cassava starch factory. The α-amylase (Termamyl® from Bacillus licheniformis) was purchased from Novozyme (Novo Nordisk Co., Bagsvaerd, Denmark) while other chemicals were analytical grade obtained from Merck (Darmstadt, Germany). 2.1 Preparation of microcrystallined cellulose (MCC) from destarched cassava pulp The starch was extracted from fresh cassava pulp (20% dwb) by the hydrolysis with α-amylase (Termamyl®, 0.5% w/v; Novo Nordisk Co., Bagsvaerd, Denmark) at 95-100OC, pH 5.5-6 for 2 h. After that, the pulp was washed and dried in a hot air oven at 50OC for overnight. The product was called destarched cassava pulp (DCP). 2.2 Delignification of DCP by alkali treatment The DCP (10 g) were treated with 250 ml of 2% (w/v) NaOH at 120OC, 20 lb for 1 h. After that, the sample was washed until the filtrate was neutral.

2.3 Bleaching of delignified DCP Delignified DCP (10g) was bleached with 125 ml of 0.4% (w/v) sodium chlorite (NaClO2) at pH 5, 80OC for 4 h. The samples were then washed until the filtrate pH was neutral. 2.4 Preparation of microcrystalline cellulose from DCP Microcrystalline cellulose was prepared from delignified, bleached, destarched cassava pulp by acid hydrolysis. The DCP (10g) samples were dispersed in 200 mL of 6.5M sulfuric acid. The reaction was performed at 60OC under vigorous stirring for 40 min (Teixeira et al., 2009) and was immediately terminated by adding two folds of water. The mixture was then neutralized with 40% (w/v) NaOH. The sample was collected by centrifugation at 9,600 rpm for 15 min, washed with distilled water, and then dialyzed against distilled water using a dialysis membrane with 10,000 MW cut-off until the neutral pH was obtained. The resulting suspension was then subjected to an ultrasonic treatment (sonication) for 20 min and stored in a refrigerator. 2.5 Characterization of MCC for DCP Chemical composition The content of the lignin and cellulose in cassava samples was determined by acid detergent fiber (ADF), natural detergent fiber (NDF) and lignin analysis (AACC, 1990). FTIR spectrum The FTIR spectra of the cellulosic samples were recorded on an instrument (Spectrum one, Perkin Elmer) in the range of 400–4,000 cm−1 with a resolution of 4 cm−1. The samples were ground into powder by a fiber microtome and then blended with KBr followed by pressing the mixture into ultra-thin pellets (Mandal and Chakrabarty, 2011).


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Crystallinity index The X-ray diffraction patterns of cellulosic samples were measured by an X-ray diffractometer (AXS Model DE Discover, Bruker) using Cu Ka radiation at 40 kV and 30 mA. Scattered radiation was detected in the range 2θ = 5–40o, at a speed of 2o/min. The crystallinity (Ic) was estimated by means of Eq. Icrytalline = (I200-Iam)/I200 x 100 using the height of the 200 peak (I200, 2θ = 22.6o) and the minimum between the 200 and 110 peaks (Iam, 2θ = 18o). I200 represents both crystalline and amorphous material while Iam represents amorphous material (Teixeira et al., 2009). Transmission Electron Microscopy (TEM) Transmission electron microscopy (Jeol JEM 1220) was used to investigate the morphology of acid hydrolysed cellulose. A droplet of a diluted suspension was deposited on a Cu grid covered with a thin carbon film. To enhance the contrast, the acid hydrolysed cellulose were negatively stained with 2 %wt uranyl acetate solution in deionized water for 1 min and then dried at room temperature (Deepa et al., 2010). 3. Results and Discussion When lignocellulosic materials are subjected to alkali treatment, the hemicellulose is partially hydrolyzed while the lignin is depolymerized, giving rise to sugars and phenolic compounds that are soluble in water (Abraham et al., 2011; Deepa et al., 2010). Generally, the alkali treatment results in the hydrolysis of glycosidic bonds in the hemicelluloses and also leads to a cleavage of the α-ether linkages between lignin and hemicelluloses (Deepa et al., 2010). As a result, the purity of cellulose increased (the ratio of cellulose to lignin = 3.8 to 5.6; Table 1). Yet, the lignin may not be completely removed during alkali treatment. Bleaching further helps remove the majority of the lignin component as suggested by a higher content of cellulose and lower content of lignin, resulting in a greater ratio of cellulose to lignin to 11.8 (Table 1). This is caused by the reaction of lignin and NaClO2, which dissolves lignin out as lignin chloride (Abraham et al., 2011) or lignin-chlorine complex. Furthermore, this lignin-chlorine complex is highly soluble in sodium sulfite solution (Mandal and Chakrabarty, 2011). The Fourier transform infrared (FTIR) spectroscopy was applied to confirm the chemical structure of produced fiber and the spectrum suggests that lignin was almost entirely removed during alkali and bleaching treatment which are shown in Figure 1. DCP has a characteristic peak in between 1700-1730 cm-1 which was attributed to the C=O stretching vibration of the acetyl and uronic ester groups from pectin, hemicellulose or the ester linkage of carboxylic group of ferulic and p-coumaric acids of lignin or hemicelluloses (Mandal and Chakrabarty, 2011). The peak at 1245 cm-1 presents C-O out of plane stretching vibration of the aryl group in lignin. All these peaks were disappeared completely in the spectra of alkali-treated, bleached and acid-hydrolyzed cellulose. In addition, an increase in the peak at 890-900 cm-1, representing the β–glycosidic linkages of glucose ring of cellulose (Mandal and Chakrabarty, 2011; Sain and Panthapulakkal, 2006) was noticeably pronounced in acid hydrolyzed cellulose comparable to destarched cassava pulp, indicating higher purity of cellulose in cassava cellulose products. The X-ray diffraction patterns are shown in Figure 2. After acid hydrolysis, the crystallinity index of DCP was increased from 36.84% to 74.39% as the acid removed the amorphous region in the microfibrillar cellulose. Figure 3 shows the TEM micrograph of a very dilute suspension of acid hydrolysed cellulose from DCP. It was the rod-like or needle-shaped with the diameter approximately 100-300 nm. Figure 4 presents the physical appearance of cassava cellulose.


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Table 1 Chemical composition of destarched cassava pulp (DCP), delignified, bleached

cassava pulp with sodium chlorite (NaOCl2) Chemical Composition (% dry basis)

Sample Cellulose Lignin

Destarched cassava pulp (DCP) 45.07 ± 0.15 11.97 ± 0.05 Alkali-treate cassava pulp 73.49 ± 0.43 13.10 ± 0.03 NaClO2-bleached cassava pulp 83.26 ± 0.39 7.07 ± 0.20

Figure 1 Fourier transform infrared (FTIR) spectra of DCP, alkali-treated, NaOCl2-bleached

and acid-hydrolyzed cassava cellulose.


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Figure 2 X-ray diffraction patterns and crystallinity index of cassava pulp samples including destarched cassava pulp, acid-hydrolyzed cassava cellulose and commercial microcrystalline cellulose (Sigma cellulose).

Figure 3 Transmission electron microscopic picture of acid hydrolyzed cassava cellulose From destarched cassava pulp.


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Figure 4 Physical appearance of acid-hydrolyzed cassava pulp. 4. Conclusion Microcrystalline cellulose (MCC) from destarched cassava pulp has been success-fully prepared by a series of process including alkali treatment, bleaching and acid hydrolysis. The produced cassava cellulose is white, having rod-like or needle-shaped with the diameter approximately 100-300 nm. It also has higher purity and crystallinity index. In addition, the MCC suspension is thick. A further study on rheological properties of cassava MCC will be investigated. Acknowledgements This work was supported by European Union FP7 commissioners under the project title “Gains from Losses of Root and Tuber Crops: Gratitude” (http://www.fp7-gratitude.eu) and Ministry of Science and Technology. References Abraham, E.; Deepa, B.; Pothan, L.A.; Jacob, M.; Thomas, S.; Cvelbar, U. and Anandjiwala,

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