Abstract
The effect of alkaline steep and air-rest cycle on the development of peroxidase activity during malting was investigated in sorghum variety, KSV8. Preliminary experiment showed that alkaline steep (test) and the distilled water steep (control) had germinative energy of 92± 2.87 % and 89± 0.57 % respectively. In regime II (sorghum grains steeped in distilled water for 24h), both the test experiment and the control had germinative energy of 95± 1.41 %. Germinative capacity was high in both regimes. The two regimes were not water sensitive, however malting loss were high in alkaline (20.1± 0.93 %) and in distilled water steep (20.0± 1.28 %).Malting loss for distilled water in regime II (sorghum grains steeped in distilled water for 24h) was 5.6± 1.28 % and it is relatively comparable to that of barley. Malting loss was also high in distilled water steep (control) (15.84± 0.19 %). From the results, there was an appreciable increase in peroxidase activity from day 1 through day 3 of germination for distilled water steep in regime I (control) when compared to the test with regression in peroxidase activity. There was a positive gradual increase in peroxidase activity influence by air-rest cycle from day 1 through day 3 in regime II (distilled water steep for 24h). At the end of kilning at 60 ͦ C for 7 h, peroxidase activity dropped sharply in both regimes. Consequently, the introduction of air-rest cycle as malting condition will be beneficiary to brewers. It reduces malting loss associated with sorghum beers, increases the germinative energy and the defensive role of peroxidase against lipid peroxidation during malting. Conversely, the alkaline steep with final warm steep had an inhibitory effect on the development of peroxidase during malting.
TABLE OF CONTENTS
Title page………..………………………………………………..…………………………..i
Approval page…….…………………………………………………………..……………..ii
Dedication……………..………………………………………………………………….…iii
Abstract………………..……………………….……………………………………………iv
Table of contents……………………………………………………………………………..v
Acknowledgement…………….…………………………………….……………………….vi
List of tables…………………….…………………………………………………………..vii
List of figures……………………….………………………………………………………viii
CHAPTER ONE
INTRODUCTION AND LITERATURE REVIEW
1.0 Sorghum…………………………………………..…………………………………….1
1.1 Sorghum as a brewing material……..………………………………………………….1
1.2 Enhancing the brewing potential of sorghum……………….…………………………3
1.3 General outline of the brewing process………….……….……………………………4
1.3.1 Malting…….……….…………………………..…………….…………….………….4
1.3.2 Steeping………….…..…………………………….…..………………………………4
1.3.3 Germination ………..………………………………………………………….………5
1.3.4 Kilning……….……….….…………………………..…..…………………….………5
1.3.5 Mashing…….……………..………..………………..……..………………….………6
1.3.6 Lautering….……………………………………………………..……………………..7
1.3.7 Fermentation………………………………………………………………….……….7
1.3.8 Conditioning………………………………………………………………….……….8
1.3.9 Filtration…………………………………………………………………………..8
1.3.10 Packaging……………………………………………………..…………………..8
1.4 Role of lipids in brewing …………………………………………………………8
1.4.1 Beneficial role of lipid in brewing…..……….………………………………..…..9
1.4.2 Non-Beneficial role of lipid in brewing……………………………………….…..9
1.4.3 Lipid oxidation in brewing…………..………………….………………….……10
1.4.4 Enzymatic oxidation of lipid in brewing……………………..……………..……11
1.4.5 Non- enzymatic oxidation of lipid in brewing…………….….……….……..…..12
1.4.6 Control of lipid oxidation during brewing………….…………..……..……..…..14
1.4.7 Role of anti-oxidant enzymes in beer stability………………………………….…16
1.4.8 Mechanism of action of peroxidase…………….……………………………..…17
1.4.9 Importance of peroxidase in brewing……………..…………………………..…20
1.4.10 Aim and objectives ……..…………………………………….………..……..…21
CHAPTER TWO
MATERIALS AND METHODS
2.1 Materials………………………..………………………………………………………22
2.2 Equipment …………………………………………………………………….….22
2. 3 Chemicals/Reagents ……………………………………………….………………..22
2. 4 Methods……………………………………………………………….……….….23
2. 5 Malting ……………………………………………………………….………….. .23
2. 5.1 Steeping methods ………………………………………….………….……………23
2.5. 2 Determination of germinative energy ……………………………………………23
2.5. 3 Determination of water sensitivity ……………………………….………………24
2.5. 4 Determination of average root length …………………………….………………24
2.5. 5 Determination of malting loss ……………..……………………….…………24
2. 6 Preparation of reagents ……………..………………………..…………………24
2. 6.1 Preparation of phosphate buffer Solution…………..……………….………25
2. 6.2 Preparation of o-dianisidine substrate……..……………………..…………..25
2. 6.3 Preparation of stock hydrogen peroxide solution………………..………….25
2.6.4 Preparation of reagent for protein determination……………………………26
2.7 Extraction of peroxidase from sorghum ……………………………………26
2.8 Assay of sorghum in peroxidase …………….………………………………26
2.9 Kilning method……………………………………………………………….26
2.9.1 Statistical Method……………………………………………………………26
CHAPTER THREE ……………………………………………………….……………27
RESULTS
3.0 Results……………………………………………………….……… ………27
CHAPTER FOUR
4.0 Discussion………………….…………………………………………………39
4.1 Conclusion………….……………………………………….…………….….43
4.2 Recommendation/ Suggestion for further studies ………….…………….…44
4.3 References……………………………………………………………………..45
4.4 Appendix…………………………………………………………………………..52
LIST OF FIGURES
Figure 1.1: Lipid peroxidation of fatty acids……………………………………………….….10
Figure 1.2: Specific oxidation of linoleic acid…………………………………….. …..……….12
Figure 1.3: The general mechanism of horseradish peroxidase ……………………….….…..19
Figure 1.4 : Complete peroxidases catalytic cycle……………………………..……………….20
Figure 1.5: The catalytic cycle of horseradish peroxidase (HRP C)……………………….…21
Figure 3.1: Peroxidase activity for alkaline and warm steep at 40°c for regime I ……………..29
Figure 3.2: Peroxidase activity at the end of 24h germination for regime I ……………………..30
Figure 3.3: Peroxidase activity at the end of 48h germination for regime I ………………….31
Figure 3.4: Peroxidase activity at the end of 72h germination for regime I ……….…………32
Figure 3.5: Peroxidase activity at the end of kilning for regime I ……..…..………..…………33
Figure 3.6: Peroxidase activity at the end of 24 h of steep for regime II …….…..….……..….34
Figure 3.7: Peroxidase activity at the end of 24 h of germination for regime II …..…..………..35
Figure 3.8: Peroxidase activity at the end of 48h germination for regime II ……….…………..36
Figure 3.9: Peroxidase activity at the end of 72 h germination for regime II ……………….…37
Figure 3.10: Peroxidase activity at the end of kilning for 7 h for regime II ……..……………38
Figure 4.11: Graph of standard protein curve using BSA………………………..………….….63
LIST OF TABLES
Table 3.1: Germinative properties of KSV8 …………………………..………..……………27
Table 4.0: Peroxidase activity for regime 1 at the end of 40 h steep…………………..….…50
Table 4.1: Peroxidase activity at the end of 24 h germination for regime I …………………51
Table 4.2: Peroxidase activity at the end of 48 h germination for regime I ………..….……52
Table 4.3: Peroxidase activity at the end of 72h germination for regime I ……….……..….53
Table 4.4: Peroxidase activity at the end of kilning for regime I …………….………..……54
Table 4.5: Peroxidase activity at the end of 24 h steep for regime II …………………….…55
Table 4.6: Peroxidase activity at the end of 24 h germination for regime II ……..………….56
Table 4.7: Peroxidase activity at the end of 42h germination regime II ……………………..57
Table 4.8: Peroxidase activity at the end of 72 h germination regime II………………….58
Table 4.9: Peroxidase activity at the end of kilning for regime II ……….….……….……….59
Table 4.10: Peroxidase activity of both dry KSV8 and kilned KSV8 ………..……….……60
Table 4.11: Protocol for Protein Standard Curve…………..………………..……….……61
Table 4.12 : Total protein concentration of the crude enzyme (mg/ml)……..……….…62
CHAPTER ONE
INTRODUCTION
1.0 Sorghum (Sorghum bicolor (L.) Moench)
Sorghum (Sorghum bicolor (L.) Moench) is the grain of choice to produce traditional cloudy and opaque beers throughout sub-saharan Africa. The key ingredient of these beers is sorghum malt, which provides hydrolytic enzymes (especially amylases) to ferment sugars into ethanol and carbon dioxide. Sorghum is used for food, fodder, and the production of alcoholic beverages. It is both drought and heat tolerant, and is especially important in arid regions. Sorghum ranks fifth in the world cereal production, and as of 2008 the world annual sorghum production stood at 65.5 million tones (Akintayo and Sedgo,2001). It is an important food crop in Africa, Central America, and South Asia (Akintayo and Sedgo,2001).