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
- Sorghum…………………………………………..…………………………………….1
- Sorghum as a brewing material……..………………….1
- Enhancing the brewing potential of sorghum……………………3
- General outline of the brewing process…………………………………4
- Malting…….……….…………………………..…………….…………….………….4
- Steeping………….…..…………………………….…..………………………………4
- Germination ………..………………………………………………………….………5
- Kilning……….……….….…………………………..…..…………………….………5
- Mashing…….……………..………..………………..……..………………….………6
- Lautering….……………………………………………………..……………………..7
- Fermentation………………………………………………………………….……….7
- 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
- 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
EFFECT OF ALKALINE STEEP AND AIR-REST CYCLE ON THE DEVELOPMENT OF SORGHUM PEROXIDASE ACTIVITY DURING MALTING
