Effect of Aspergillus Niger Amylases on Cassava Starch Hydrolysis

Effect of Aspergillus Niger Amylases on Cassava Starch Hydrolysis.

ABSTRACT  

The enzymatic hydrolysis of cassava starch to glucose has been studied. Aspergillus niger was isolated from deteriorated groundnut seeds and used as the amylase producer source. The effect of various process variables such as culture age, mycelia mass loading, pH, temperature and substrate (cassava starch) concentration were investigated in order to optimize the hydrolysis process.

The A. niger amylases exhibited optimum activity at pH and temperature of 4.8 and 60oC respectively. The cells appear most viable on the fifth day. Results obtained from this study indicated that increase in cassava starch concentration from 0.3 to 1.5 g/l was accompanied with a proportionate increase in glucose concentration from 0.239 to 0.310 g/l and decrease in glucose yield, falling from 74% to 18%.

In addition, increase in mycelia mass loading resulted to increase in both the concentration and yield of glucose. An initial rate method was employed in estimating the kinetic parameters using Michealis-Menten model. The Langmuir form of the Michealis-Menten model gave a high regression coefficient (0.9939) and the values of the maximum reaction velocity (rmax) and Michaelis constant (Km) were found to be 2.42*10-3 g/l.min and 0.221 g/l respectively. 

TABLE OF CONTENTS

Title Page…………………………………………………………………………………i
Declaration……………………………………………………………………………….ii
Certification………………………………………………………….…………………..iii
Dedication……………………………………………………………….………………iv
Acknowledgments……………………………….……………………….………………v
Abstract……………………………………………………………………………………………………………vi
Table of Contents……………………………………………………………….……….vii
List of Tables…………………………………………………………………………………………………….xi
List of Figures…………………………………………………………………………………………………..xii
List of Plates……………………………………………………………………………………………………..xiii
List of Appendices……………………………………………………………………………………………xiv
Abbreviations and Symbols………………………………………………………………………………..xv

1.0 INTRODUCTION………………………………………………………………………………………1
1.1 Background Information ……………………………………………………………………………..1
1.2 Research Problem Statement ………………………………………………………………………2
1.3 Research Aim ……………………………………………………………………………………………..2
1.4 Research Objectives ……………………………………………………………………………………3
1.5 Research Justification …………………………………………………………………………………3
1.6 Scope of the Research ………………………………………………………………………………….3

2.0 LITERATURE REVIEW…………………………………………………………………………….4
2.1 Introduction ………………………………………………………………………………………………..4
2.2 Chemistry of Starch …………………………………………………………………………………….5
2.3 General Characteristics of Starch………………………………….…….…………….7
2.4 Economic Importance of Starch ………………………………………………………………….7
2.5 Modification of Starch ………………………………………………………………………………..8
2.5.1 Chemical treatment ……………………………………………………………………………………9
2.5.2 Physical treatment ……………………………………………………………………………………..9
2.6 Starch Processing Enzymes ……………………………………………………………………….10
2.7 Industrial Starch Hydrolysis ……………………………………………………………………..14
2.7.1 Acid-enzyme liquefaction …………………………………………………………………………15
2.7.2 Single stage enzyme liquefaction ……………………………………………………………….15
2.8 Enzyme Kinetics………………………………………………………………………………………..19
2.9 Evaluation of Steady State Kinetic Parameters ………………………………………….20

3.0 MATERIALS AND METHODS……………………………………………………………….22
3.1 Materials ………………………………………………………………………………………………….22
3.1.1 Sample collection ……………………………………………………………………………………22
3.1.2 Chemicals………………………………………………………………………………………………..22
3.1.3 Equipment/apparatus ……………………………………………………………………………….22
3.2 Methods ………………………………………………………………………………………….……….23
3.2.1 Media preparation ……………………………………………………………………………………23
3.2.2 Plate preparation………………………………………………………………………………………23
3.2.3 Isolation of A. niger …………………………………………………………………….…………..24
3.2.4 Lactophenol cotton blue staining of A. niger……………………………………………….24
3.2.5 Sub-culturing of A. niger…………………………………………………………………………..25
3.3 Proximate Analysis of Cassava Starch……………………………………………………….25
3.3.1 Determination of moisture content………………………………………………………………25
3.3.2 Determination of ash…………………………………………………………………………………26
3.3.3 Determination of ether extract (oil)……………………………………………………………..27
3.3.4 Determination of crude protein……………………………………………………………………27
3.3.5 Determination of crude fibre……………………………………………………………………….29
3.3.6 Determination of nitrogen free extract………………………………………………………….30
3.4 Acetate Buffer Preparation…………………………………………………………………………30
3.5 Sample (cassava starch) Slurry Preparation………………………………………………..31
3.6 Enzymatic Treatment of Cassava Starch…………………………..………………………..31
3.7 Assays for Glucose Concentration ……………………………………………………………..32
3.8 Optimisation of Process Conditions…………………………………………………………….32
3.8.1 Effect of culture age on cassava starch hydrolysis…………………………………….…..33
3.8.2 Effect of mycelia mass loading on cassava starch hydrolysis………………. …………33
3.8.3 Effect of pH on A. niger amylases activity ……………………………………………..…..33
3.8.4 Effect of temperature on A. niger amylases activity………………………………………34
3.8.5 Effect of cassava starch concentration on A. niger amylases activity …….………34

4.0 RESULTS AND DISCUSSION………………………………………………………………….35
4.1 Macroscopic Observation of A. niger……………………………………………………………35
4.2 Microscopic Observation of A. niger……………………………………………………………35
4.3 Proximate Composition of Cassava Starch…………………………………………………..39
4.4 Optimisation of Process Conditions……………………………………………………………..39
4.4.1 Effect of culture age on cassava starch hydrolysis……………………….….……….39
4.4.2 Effect of mycelia mass loading on cassava starch hydrolysis…………………………..39
4.4.3 Effect of pH on A. niger amylases activity……………………………………………………43
4.4.4 Effect of temperature on A. niger amylases activity………………………………….43
4.4.5 Effect of cassava starch concentration on A. niger amylases activity……………….44
4.5 Evaluation of Cassava Starch Enzymatic Hydrolysis Kinetic Parameters Based
on Michaelis-Menten Kinetic Model…………………………………………………………….49
4.6 Estimation of Km and rmax under Optimal Condition by the Linear Methods…50

5.0 CONCLUSION AND RECOMMENDATIONS………………………………………….59
5.1 Conclusion…………………………………………………………………………………………………59
5.2 Recommendations………………………………………………………………………………………59

REFERENCES………………………………………………………………………………………………..60
APPENDICES…………………………………………………………………………………………………66

INTRODUCTION 

There has been a great emphasis on plant biomass as a source of fermentable sugars. Starch is considered as one of the world most abundant polymer, believed to be the renewable energy source that can provide liquid fuel and other bulk chemicals on a sustainable basis (Gomez et al., 2008). The world cassava production was estimated to be 230 million tons in 2010 (Leen et al., 2007; Hermiati et al., 2012; FAO, 2012). Nigeria has been recognised as the largest producer of cassava, a viable source of starch with an aggregate annual production rate of about 49 million tonnes (Aderi et al., 2010).

However, despite its abundance in Nigeria, it has remained relatively underutilized. A large proportion of this tuber starch is lost yearly due to inadequate and ineffective storage facilities (Omemu et al., 2005; Zainab et al., 2011), non-availability of appropriate processing technology and industry to convert these raw materials into various value-added products such as glucose syrup, maltose syrup, high fructose syrup and maltodextrins, which are industrial products of economic relevance.

Processing starch into finished products at industrial scale has involved both chemical and enzymatic approaches. Owing, to the short comings associated with the chemical method of starch hydrolysis such as formation of undesirable coloured and offflavoured by-products, which consequently corrode processing equipment (Shambe et al., 1989). In addition, the process appears to be totally random which is not influenced 2 by the presence of α-1,6 glucosidic linkages (Haki and Rakshit, 2003; Zainab et al., 20011).

The use of enzyme has become a method of choice to industrialists and others alike. Conventionally, conversion of starch to glucose is accomplished on a large scale with the use of two commercially purified enzymes such as α-amylase and amyloglucosidase (glucoamylase) from different sources (Kyriakides et al., 2001; Azmi et al., 2012). With the view of minimising the cost of operation, this study is aimed at using a microorganism that can secret the two enzymes under favourable conditions, thereby hoping to mitigate the high cost of glucose production.

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StudentsandScholarship Team.