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.      Sorghum…………………………………………..…………………………………….1
2.  Sorghum as a brewing material……..………………….1
3.      Enhancing the brewing potential of sorghum……………………3
4.      General outline of the brewing process…………………………………4
5. Malting…….……….…………………………..…………….…………….………….4
6. Steeping………….…..…………………………….…..………………………………4
7. Germination ………..………………………………………………………….………5
8. Kilning……….……….….…………………………..…..…………………….………5
9. Mashing…….……………..………..………………..……..………………….………6
10. Lautering….……………………………………………………..……………………..7
11. Fermentation………………………………………………………………….……….7
12. 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. 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).

1.1  Sorghum as brewing material

In Southern Africa, sorghum is used to produce beer, including the local version of Guinness stout. In recent years, sorghum has been used as a substitute for other grains in gluten-free beer. Although the African versions are not “gluten-free”, as malt extract is also used, gluten-free beers are now available using such substitutes as sorghum or buckwheat. Sorghum is used in the same way as barley to produce “malt” that can form the basis of a mash without gliadin or hordein and therefore suitable for coeliacs (Smagalski, 2006).

African sorghum beer is a brownish-pink beverage with a fruity, sour taste. It has an alcohol content that can vary between 1% and 8% (Lermusieau et al., 2001). African sorghum beer is high in protein, which contributes to foam stability, giving it a milk-like head. Because this beer is not filtered, its appearance is cloudy and yeasty, and may also contain bits of grain (Lermusieau et al., 2001).

African sorghum beer is a popular drink primarily amongst the black community. Sorghum beer is known by many different names in various countries across Africa, such as burukutu (Nigeria), pombe (East Africa), bil-bil (Cameroon), bjala in Northern Soweto. In Nigeria as well as other African countries where sorghum is malted commercially, the respective agricultural departments and commercial breeders breed sorghum cultivars with good malting quality for brewing. The primary quality criterion is their potential to produce malt with high diastatic power (amylase activity) (Okolo et al., 2010).

Traditional and commercial sorghum malting process is split into three unit operations: steeping, germination, and drying ( Taylor et al.,2005). Steeping involves immersing the grain in water until it has imbibed sufficient water to initiate the metabolic processes of germination. During germination the moist grain is allowed to grow under controlled cool conditions in the dark with or without any further addition of water (Briggs et al., 2004).

Drying involves reducing the moisture content of the green (moist) sorghum malt to around 10% to produce a shelf-stable product (Arnold, 2005). Drying is generally carried out in a box with a perforated floor, similar to the germination box but with deeper floor. Warm dry air is blown through the green malt. The air temperature should not be more than 50°C, as higher temperatures significantly reduce the amylase activity of the malt. In some outdoor floor malting, the malt is sun-dried by spreading the grain out in thin layer and turning it periodically (Arnold, 2005).

There are many setbacks in brewing with sorghum such as high lipid content, low extract recovery, high polyphenol content, absence of hull etc, which affect the quality of the beer. These problems arising from the use of sorghum to brew beer have been subject of intense research, especially in Africa (Osagie, 1987;Okolo and Ezeogu, 1996;Nwanguma and Eze 1996; Taylor and Dewar, 2001) .

The absence of hull in sorghum was considered a major problem. This is because when brewing with barley malt, the hulls act as a filter bed in lautering, the technology traditionally used to separate the wort (unfermented beer) from the spent grain. In the 1990s, this problem was solved with the development of tangential-flow mash filters with automatic discharge of spent grains. Since then the commercial use of sorghum for clear beer brewing in Africa has become firmly established. Commercial African sorghum beer is packaged in a microbiologically active state. Packaging does not occur in sterile conditions and many microorganisms may contaminate the beer. The use of wild lactic acid bacteria also increases the chances of beer spoilage due to the present of microorganisms. However, the microbiologically active characteristic of the beer also increases the safety of the product by creating competition between organisms. Although aflatoxins from mould were found on sorghum grains, they were not found in industrially produced African sorghum beer (Nakamura et al., 2003).

1.2 Enhancing the brewing potential of sorghum