EFFECT OF ALKALINE STEEP AND AIR-REST CYCLE ON THE DEVELOPMENT OF SORGHUM PEROXIDASE ACTIVITY DURING MALTING

A PROJECT WORK SUBMITTED TO IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE (M.Sc) IN BIOCHEMISTRY (INDUSTRIAL BIOCHEMISTRY AND BIOTECHNOLOGY)

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).

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

The methods used to enhance the brewing potential of sorghum malt include manipulation of steeping sequence (alkaline steep treatment, air-rest cycle, cold and hot water extract and warm water final steep), appropriate cultivar selection, manipulation of germination time, germination temperature, kilning and mashing temperature and addition of exogenous enzymes (Okolo and Ezeogu, 1996; Dewar et al., 1997; Ogbonna et al., 2003; Owuama and Adeyemo, 2009; Ukwuru, 2010). Manipulation of steeping sequence was targeted primarily to increase grain germinability, develop and increase protein and enzyme synthesis, and to reduce polyphenol influence on protein content of malts ( Okolo and Ezeogu ,1996 ; Nwanguma and Eze,1996;Ogbonna et al., 2003). Manipulated malts have improved protein quality characteristics, such as percentage protein, the nitrogen solubility index and the content of the first limiting amino acid, lysine (Dewar, 1997; Ogbonna et al., 2003). It also reduces polyphenol content of sorghum which is known to inhibit the development of enzymes and protein reserves (George et al., 2005). Air-rest cycle when included as part of alkaline steep also helps to increase the enzymic activities of sorghum. Some of this method (alkaline steep and final warm water steep) had rather negative effect on germinative potentials and enzyme development (Okolo and Ezeogu, 1996).

The germination temperature of about 25°C to 30°C seems to favour enzyme development, while Owuama (1997) suggested

that kilning grains in cycles of 45°C to 60°C tend to increase the number of enzymes than at a single temperature treatment. Mashing temperature of 65°C is generally used in mashing barley malt, but when sorghum malt was mashed at the same temperature the result was inadequate gelatinization of the starch and sub-optimal release of sugars even when commercial enzymes were added. However, at a mashing temperature of 85°C and above, sorghum starch was gelatinised effectively and sugars released into the wort was higher than at 65°C, and even higher when commercial enzymes were included at a very low rate. Although higher temperatures and added commercial enzyme preparations used in mashing sorghum malt dramatically increased the sugars released into the wort of sorghum mash, the ratio of glucose to maltose did not change. An industrial exogenous enzyme such as amyloglucosidase contributes more to the release of reducing sugars into the wort during mashing. For more sugar yield in the wort during yeast fermentation industrial amyloglucosidase was recommended as enzyme source (Owuama and Adeyemo , 2009).

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