Physicochemical properties of the cassava root

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HYPOTHESES AND OBJECTIVES

Hypotheses

a) Cellulolytic and hemicellulolytic treatment of cassava cake (wet milled cassava) will result in modification in the structure and composition of cassava cell walls, thereby reducing their water-holding capacity. Bioconversion techniques involving the use of hydrolytic enzymes with cellulolytic and hemicellulolytic activities have been applied to cassava mash in improving starch extraction (Rahman and Rakshit, 2003). By using a multi-enzyme mixture of cellulases, pectinases and hemicellulases (Sriroth et al., 2000), this type of enzymatic treatment of cell wall materials result in the hydrolysis of the soluble cell wall components and fragmentation of the insoluble cell wall components (Demir et al., 2001; Rai et al., 2004).
b) Steeping of milled tannin sorghum grain in dilute sodium hydroxide solution will effectively reduce the negative effects of the tannins in tannin sorghums on the brewing and bioethanol mashing processes. With dilute NaOH steeping of whole tannin sorghum grain which was then milled and used in brewing, tannin were found to still negatively affect wort quality attributes (Adetunji, 2011). According to Gaffet, Bernard, Niepce, Charlot, Gras, Le Caër, Guichard, Delcroix, Mocellin and Tillement (1999), milling process is a means of modifying conditions in which chemical reaction occur by changing reactivity, which enables increase in reaction rates. The effect of modification in grain structure due to germination during malting explains inactivation of tannins in tannin sorghum (Yan et al., 2009).
c) The mechanism of tannin inactivation by NaOH will be due to chemical modification of tannin molecules, resulting from the direct chemical reaction between tannins and NaOH. According to Kiatgrajai, Wellons, Gollob and White (1982), alkaline extraction of condensed tannins is known to result in tannins that are less reactive with aldehydes compared to neutral-solvent extraction. Reaction between tannins and alkali involves C-ring opening and rearrangement via radical reactions in the presence of traces of oxygen (Kennedy et al., 1984). This structural modification of tannins due to the reaction between tannins and alkali can explain reduction in tannin reactivity (Laks, Hemingway and Conner, 1987; Hashida and Ohara, 2002).

Objectives

a) To evaluate the effects of cellulolytic and hemicellulolytic enzyme activities on the structural and compositional properties of cassava cell
b) To evaluate the effects of steeping milled tannin sorghum grains in different concentrations of dilute NaOH solution on tannin content and tannin inhibitory activity.
c) To understand the chemistry of reaction between tannins and NaOH, resulting in tannin inactivation.

Hydrolysis of the fibre material in cassava cake with cellulolytic and hemicellulolytic enzymes to improve wort filtrability

INTRODUCTION

Cassava (Manihot esculenta Crantz L.) is ranked sixth in the world food production for 2013 after sugarcane, maize, rice, wheat and potatoes (FAO, 2015), and serves as a major source of food and dietary calories for many people in the tropical countries of Africa, Asia and Latin America (Pandey, Soccol, Nigam, Soccol, Vandenberghe and Mohan, 2000). Cassava can to grow on impoverished and marginal soils with little technological input (Buschmann, Potter and Beeching, 2002). Cassava production in Africa represents about 57% of the world production in 2013 (FAO, 2014). As reviewed by Pandey et al. (2000), only a low proportion of cassava world production, about 7%, is used commercially in industries such as paper, textile, food and fermentation. Therefore, there is need for increased value addition to cassava in food and beverage product processing, especially in lager beer brewing, due to its high starch content.
Proximate composition of cassava root shows that moisture is about 64% and carbohydrates about 34%, which are the main chemical components (Rickard and Behn, 1987). According to a study by Charles, Sriroth and Huang (2005) on five different genotypes of cassava, carbohydrate and crude fibre composition ranges between 80-86% and 1.5-3.5% dry weight basis, respectively. Among different cultivars, the protein content of cassava root tuber ranges from 0.5 to 1.9 g/100 g dry matter (El-Sharkawy, 2004). In terms of the root cyanogenic quality, cassava roots with levels of hydrocyanic acid below 10 mg/100 g are generally considered sweet cassava (Jennings and Iglesias, 2002). Salvador, Suganuwa, Kitahara, Tanoue, and Ichiki (2000) investigated the cell wall components in cassava root tuber. Fractionation of the cell wall material components identified cellulose (about 48%), hemicellulose (about 22%) and pectin (about 17%) as the major non-starch polysaccharides components of the cell wall materials.
During extraction of starch from cassava, fibre materials are the major solid residue constituting about 15-20% by weight of the cassava tuber processed (Swain and Ray, 2007). As a result of this high residual fibrous material content in cassava root, its application in brewing may constitute considerable problems after starch hydrolysis. This is due to the high water retention capacity of the fibre material (85-90%) (Pandey et al., 2000). In particular, this may constitute a considerable drawback in the  filtration of the wort. As reviewed by Pandey et al. (2000), bioconversion techniques such as enzymatic hydrolysis and solid-state fermentation have been applied in the treatment of cassava fibrous material residues. Therefore, enzymatic hydrolysis could be applied in the treatment of cassava fibre material in order to reduce its negative effect. This study focused on improving the cassava wort filtration processing step through treatment of the cassava with cellulolytic and hemicellulolytic enzymes in order to reduce the amount of fibre material residue left after the mashing (starch hydrolysis) step.

MATERIALS AND METHODS

Materials

Cassava cake (wet milled cassava tuber) was obtained from SABMiller (ex. Nampula, Mozambique) and stored at 6-8ºC until analysis. Fresh cassava root tubers from Mozambique were obtained from a retailed outlet in Pretoria. The commercial enzyme preparations used were Viscozyme L and Ultraflo Max (cellulolytic and hemicellulolytic enzymes), and Termamyl SC (Thermal-stable α-amylase enzyme) kindly provided by Novozymes (Benmore, South Africa).

Methods

Pre-weighed fresh cassava root tubers were washed, peeled, chopped and weighed again. The cassava chips were milled using a waring blender, with small quantity of water added to aid the milling process. After milling, the cassava cake obtained was weighed to determine the yield. The cassava cake (wet milled cassava tuber) obtained was stored at 6 °C until required.

Enzymatic treatments

Effect of different levels of Viscozyme treatment

Cassava cake (450 g) was diluted with 450 ml distilled water (50:50) to give a cassava slurry of about 900 g. Viscozyme enzyme was diluted and added to give the following concentrations of the enzyme in the slurry: 0, 250, 500, 700 ppm and overdose of the enzyme (relative to cassava cake solids). The control sample was diluted with distilled water without the addition of enzyme. The cassava slurries were incubated at ambient temperature (about 24ºC) for two weeks. The treatments were performed in duplicate.

Effect of different types of enzyme preparations

Cassava cake (450 g) was weighed into beakers. Nine ml of diluted Viscozyme and Ultraflo enzymes were added to give 250 ppm of the enzyme (relative to cassava cake solids) and mix thoroughly. Combined Viscozyme and Ultraflo enzymes to give 250 ppm of the enzyme was obtained by adding 4.5 ml of diluted Viscozyme and Ultraflo enzymes each and mix thoroughly with the cassava cake. After mixing thoroughly, the cassava cake samples were covered with parafilm to ensure anaerobic condition in order to prevent mould growth. The control sample was mixed with 9 ml distilled water without added enzyme. The cassava cake samples were incubated at ambient temperature (about 24ºC) for two weeks. The treatments were performed in duplicate. The activity of the enzymes in the cassava cake samples for one week incubation period was stopped by storing at -20 ºC and at the end of two weeks incubation the cassava cake samples were also stored frozen to stop the activity of the enzymes until required for analysis. The frozen cassava cake samples after incubation periods were thawed at 24ºC for analysis.

Starch hydrolysis

This was carried out using a BRF mashing bath (Brewing Research Foundation, Nutfield, UK). During mashing, the slurry was stirred manually due to difficulty in stirring the mash by magnetic stirring and the beakers covered with watch glasses. Mashing was performed by quantitatively weighing the treated and the control cassava cake samples into the mashing beaker. The pH was adjusted with 0.1 M NaOH solution to pH 5.0, which is the optimum pH for the α-amylase enzyme used. This was followed by addition of 6 ml diluted Termamyl SC to give 100 ppm of the enzyme in the slurry and cooked for 1 hr. After cooking for 1 hr, one ml of full strength Termamyl SC was added to the mash. Mashing was carried out at 100ºC until the starch was negative by iodine. After mashing, the samples were centrifuge at 470 g for 2 min and the clear supernatant carefully removed. The insoluble solids residue stored by freezing for further analyses. Mashing was performed in duplicate.

Purification of enzyme treated cassava cake and mashed solid materials

After incubation, 25 g of the samples were weighed and diluted to 50 g with distilled water in 100 ml glass centrifuge tubes. This was followed by centrifugation at 470 g for 2 min and the clear supernatant carefully removed. The samples were then re- suspended in distilled water and re-centrifuged in order to completely wash out the soluble solids. The purified residual solid materials of both the control and enzyme treated cassava cake samples before and after starch hydrolysis by α-amylase enzyme were analysed for the following: total solids, starch, soluble and insoluble fibre contents, particle size (sieving) and light microscopy. Part of the purified residual insoluble cell walls materials were freeze dried and analysed for compositional and structural properties by gas chromatography.

Analyses

Titratable acidity and pH determination

Titratable acidity of the cassava cake was determined according to GEA Niro analytical method A 19 a (GEA Niro, 2006), with slight modification. Samples were prepared by weighing 10 g cassava samples and diluted with 20 ml distilled water. The pH of the cassava cake was determined using pH meter.

Viscosity determination

Cassava cake samples viscosity in terms of flowability was determined using a Bostwick viscometer. The samples were allowed to equilibrate to ambient  temperature (24ºC) before determination of viscosity. Cassava cake (100 g) was poured into the Bostwick cell with the bridge closed. The bridge was opened completely to allow the sample to flow down the trough section for 30 seconds and the length covered was recorded in mm.

Total solids content determination

Total solids content of the cassava cake was determined based on dry matter remaining, by drying at 103ºC for 3 hr. The total solids content of the treated samples were determined after washing and centrifuging to remove the soluble solids. The results were expressed in percentage (w/w) wet basis.

Total starch content determination

Total starch content was determined using the Megazyme Total Starch Assay Procedure (Amyloglucosidase/α-Amylase Method) (Megazyme International, 2011). The starch content of the total solids in the cake was expressed in percentage (w/w) dry basis.
 
DECLARATION
DEDICATION
ACKNOWLEDGEMENTS 
ABSTRACT
LIST OF TABLES 
LIST OF FIGURE
1. INTRODUCTION AND PROBLEM STATEMENT
2. LITERATURE REVIEW.
2.1 Cassava
2.1.1 Physicochemical properties of the cassava root
2.1.2 Developments in processing of cassava to improve starch yield
2.2 Sorghum
2.2.1 Physicochemical properties of sorghum grain
2.2.2 Sorghum phenolic compounds and their chemical properties
2.2.3 Developments in condensed tannin inactivation and their limitations
2.3 Analytical techniques for cell wall and condensed tannin determination
2.3.1 Cassava cell wall materials
2.3.2 Sorghum phenolics
2.4 Conclusions
3. HYPOTHESES AND OBJECTIVES 
3.1 Hypotheses
3.2 Objectives
4.1 RESEARCH CHAPTER
ABSTRACT
4.1.1 INTRODUCTION
4.1.2 MATERIALS AND METHODS
4.1.3 RESULTS AND DISCUSSION
4.1.4 CONCLUSIONS
4.1.5 REFERENCES
4.2 RESEARCH CHAPTER
ABSTRACT
4.2.1 INTRODUCTION
4.2.2 MATERIALS AND METHODS
4.2.3 RESULTS AND DISCUSSION
4.2.4 CONCLUSIONS
4.2.5 REFERENCES
4.3 RESEARCH CHAPTER
ABSTRACT
4.3.1 INTRODUCTION
4.3.2 MATERIALS AND METHODS
4.3.3 RESULTS AND DISCUSSION
4.3.4 CONCLUSIONS
4.3.5 REFERENCES
5. GENERAL DISCUSSION
5.1 Methodology: Critical review
5.2 Research findings
5.3 Potential application of cell wall and condensed tannin pre-treatments in cassava root tuber and tannin sorghum utilisation
5.4 Future work
6. CONCLUSIONS
7. REFERENCES
8. APPENDIXE
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