CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION Sorghum [Sorghum bicolor (L.) Moench] is an important food crop in arid and semi-

arid regions of the world (Sulma et al 1991, Mukuru 1992, Owuama 1997). It is a

major food crop ranking fifth in terms of world grain production. Fermented foods

and beverages constitute a major portion of diets for people in Africa (Sanni 1993,

Oyewole 1997). Cereal grains like sorghum, maize and millet are common

substrates for lactic acid-fermented gruels and beverages (Odunfa and Adeyele

1985). Fermented sorghum or millet-based foods, alcoholic and non-alcoholic drinks

or beverages are prepared in many African countries for human consumption

(Ekundayo 1969, Ahmed et al 1988, Chavan and Kadam 1989, Steinkraus 1996,

Odunfa et al 1996).

Sorghum has been used for centuries to brew opaque beer in South Africa. Being

drought-tolerant and because of its low price, sorghum is suitable for further

development of additional value-added uses. The potential of sorghum as an

alternative substrate for lager beer brewing has been described by Owuama (1997).

A better understanding of grain changes during brewing, for example in starch

degradation, can help in further innovative development in the brewing industry.

Although sorghum is an important staple and commercial crop of many people in

arid and semi-arid regions of the world, there is very little documentation about the

change in starch either quantitatively or qualitatively at each step of the traditional

opaque beer production and the on-going of post-fermentation process. This study

was undertaken to analyze the changes in starch degradation and other related

components at each step of opaque beer production as well as post-fermentation.

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The specific objectives of this study are:

1. To examine the effect of malting to starch degradation in seven varieties of

sorghum grain.

2. To analyze the changes in starch and other components quantitatively at each

step of sorghum opaque beer production.

3. To compare the effect of amylose content at each step of opaque beer

production by substituting the sorghum adjunct with Hi-maize (high amylose)

starch, normal maize starch and waxy maize starch respectively.

4. To investigate the qualitative modification of sorghum starch granules after

malting and at each step of opaque beer production.

5. To trace the changes in starch and other components during the post-

fermentation of opaque beer by using sorghum and maize starch with

different amylose contents as adjunct.

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1.2 REVIEW OF LITERATURE

1.2.1 Grain Sorghum Classification

Sorghum belongs to the grass family Poaceae (or Gramineae), genus Sorghum

Moench. Cultivated sorghums were domesticated from wild Sorghum bicolor around

3000 years ago in the north-east quadrant of Africa (Snowden 1936, Evelyn 1951,

Doggett 1965, 1988). Cultivated sorghums are classified into five basic groups or

races: bicolor, guinea, caudatum, kafir, and durra, and ten hybrid races which

combine the characteristics of any two or more basic races (Harlan and de Wet 1972).

Sorghum bicolor (L.) Moench, is known under various names, e.g. great millet and

guinea corn in West Africa, kafir corn in South Africa, dura in Sudan, mtama in

eastern Africa, jowar in India and kaoliang in China.

The sorghum kernel varies in color from white through shades of red and brown to

pale yellow to deep purple-brown. The most common colors are white, bronze and

brown. Kernels are generally spherical but vary in size and shape. The caryopsis can

be rounded and bluntly pointed, 4 to 8 mm in diameter (Purseglove 1972). The grain

is partially covered with glumes. Sorghum grain that has a testa contains tannin in

varying proportions depending on the variety.

Origin and distribution

Sorghum is an important food crop in arid and semi-arid regions of the world (Sulma

et al 1991, Mukuru 1992, Owuama 1997). Sorghum is among the most drought-

tolerant of cereals, becoming dormant under drought and heat stress and then

resuming growth when conditions improve. It also withstands flooding and is not

influenced much by acid soil.

According to the records of International Crops Research Institute for the Semi-Arid

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Tropics (ICRISAT), sorghum originated in the northeastern quadrant of Africa. From

Ethiopia, it was distributed along trade and shipping routes throughout Africa, and

through the Middle East to India. It reached China via the silk route. Sorghum was

first taken to the America through the slave trade from West Africa. It was

reintroduced in late 19th century for commercial cultivation and has subsequently

been introduced into South America and Australia. Nowadays, sorghum is widely

found in the drier areas of Africa, Asia, the Americas and Australia because of its

adaptation to adverse environments.

Production

Sorghum is the staple food for millions of people in the semi-arid tropics of Africa,

South Asia and Central America. Sorghum is the fifth most important cereal in the

world in terms of production, after wheat, rice, maize and barley. In 2001, total

sorghum production was more than 58 million metric tons from about 42.7 million

hectares and the major producers are Africa with annual production of 19.0 million t

of grain from 22 million ha and the United States with annual production of 13

million t of grain from 3.5 million ha (FAOSTAT 2002).

Utilization

Sorghum is mainly used for human food and animal feed. Sorghum is commonly

used to make porridges, pancakes, breads, snacks and alcoholic and nonalcoholic

beverages. Sorghum utilized for human consumption in Southern Africa is usually

processed to malt for the production of opaque beer (Beta and Dzama 1997) and up

to 200 000 tonnes of sorghum are processed to malt per annum (Dewar and Taylor

1993).

Carbohydrate

As observed in other cereals, carbohydrates constitute the largest percentage of

sorghum grain, with values ranging from 60 to 80 % of normal kernels (Horan and

Heider 1946, McNeill et al 1975).

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The average starch content of sorghum is 69.5%, ranging from 56 to 73%

(Jambunathan and Subramanian, 1988). Approximately 70 to 80 % of the sorghum

starch is amylopectin and the remaining 20 to 30% is amylose (Deatherage et al

1955, Beta and Corke 2001). Both environmental (Ring et al 1982) and genetic

factors (Beta and Corke 2001) were reported to affect the amylose content of

sorghum. Waxy sorghum varieties are very low in amylose and their starch is

practically 100% amylopectin (Deatherage et al 1955, Ring et al 1982).

According to previous findings, total sugar contents generally lay in the range

between 0.81 to 6.01%. The total sugar contents of sorghum grains were reported to

vary from 1.30 to 5.19% (Subramanian et al 1980), 2.34 to 6.01% (Neucere and

Sumrell 1980) and 0.81 to 1.59% (Edwards and Curtis 1943) respectively. Among

the sugars, stachyose, raffinose, sucrose, glucose and fructose were identified while

no maltose was detected (Subramanian et al 1980). No consensus has been reached

on the predominant sugar in sorghum grain. Sucrose was reported to be the

predominant sugar ranging from 68.7 to 82.7% of soluble sugars in the sorghum

cultivars (Subramanian et al 1980) while glucose and fructose were also reported to

be the major components (Neucere and Sumrell 1980).

Adjunct

In the brewing of opaque beer, adjunct plays an important role to provide extract at a

lower cost as a cheaper form of carbohydrate than is available from malt and impart

the beer its characteristic viscous body (Novellie and de Schaepdrijver 1986).

Adjunct alters the carbohydrate and nitrogen ratio of the wort, as a result, it affects

the formation of by-products, such as esters and higher alcohols (Goldammer 2000a).

If the level of adjunct used is too high, there may be risk of producing wort with

insufficient insoluble nitrogen for yeast growth.

The requirements for an adjunct depend on availability, competitive price, low fat

content and absence of mycotoxins (Daiber and Taylor 1995). Sorghum or maize is

usually used as adjunct in opaque beer production.

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1.2.2 Sorghum malt Sorghum malt is defined as a composite material of partially solubilized,

nutritionally and technologically beneficiated solids containing a spectrum of

enzymes, flavor substances, microorganisms, buffers and other components essential

for brewing (Daiber and Taylor 1995). Sorghum has been malted for centuries and in

Africa, it is used for the production of opaque beers, weaning foods, baby foods and

other traditional alcoholic and nonalcoholic beverages. The malt used in preparation

of opaque beer, a traditional alcoholic beverage in Africa, plays a role in

determining the rate of fermentation and is a source of lactobacilli, yeasts and

essential nutrients (Agu 1995a, Ugboaja et al 1991). Cereal flour, lactic acid bacteria

(LAB) (Spicher and Nierle 1988), and yeasts (Ogrydziak 1993) contain proteinases

and peptidases. Cereal malts are used to initiate spontaneous fermentation in a

number of African indigenous foods (Steinkraus 1996). The proteolytic activities in

cereals increase considerably during germination (Gahlawat and Sehgal 1994,

Anibaba et al 1997), and an improvement in the composition of some of the amino

acids in sorghum has been observed (Mbugua and Njenga 1992).

Malting results in mobilization of hydrolytic enzymes such as amylases and

proteases, which are essential for the solubilization of starch and proteins in the

grains, thus making them susceptible to fermentation (Lorenz and Kulp 1991). It

also brings about the modification of the grain composition and structure. After

malting, a loss of 56-66% and 98-99% in tannin for low and high tannin cultivars

(Elmaki et al 1999) were reported. For starch, a decrease with only 60 and 54% were

remained in 5- and 7-day malted sorghum grain (Von Holdt and Brand 1960). An

increase in hot water extract, diastatic activity and sugar contents (Lasekan et al

1995) in which fermentable mono- and disaccharides are produced depending on the

- and -amylase activities (Hulse et al 1980) were also recorded.

Production process of sorghum malt

Malting involves steeping, germination, drying and milling of grains.

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Steeping

Steeping refers to the soaking of grain in water in order to hydrate the embryo for

active growth and germination. It has been widely considered as a critical stage in a

malting process (Briggs et al 1981, French and McRuer 1990). Its objective is to

initiate germination such that modification of the endosperm structure will progress

at a rate producing malt of the desired quality.

Alkali treatment, steeping temperature and aeration during steeping process all

contribute to final malt quality. By steeping sorghum grains in dilute NaOH, this can

detoxify high-tannin sorghum cultivars, reduce steeping period and enhance malt

quality in both condensed-tannin free and high-tannin sorghum cultivars due to

increased water uptake and tannin inactivation (Dewar et al 1997a, Beta et al 2000).

Increase in steeping temperature up to 30C can improve malt diastatic power and a steeping temperature of 25C brings both free amino nitrogen and extract content to their maximum levels (Dewar et al 1997b). For aeration, it was also shown to

enhance the extract and free amino nitrogen content of the final malt (Dewar et al

1997b).

Germination

After being steeped, the sorghum grains are then germinated. The main objective of

germination is to trigger the development of hydrolytic enzymes which are absent in

the ungerminated grain during the germination of cereal grain in moist air under

controlled condition. Germination was found to have a highly significant effect on

enzyme activity (Uriyo and Eigel 1999). The presence of hydrolytic enzymes such as

-amylase, protease, -glucanase and pentosanase enzymes in sorghum malt was reported (Aniche and Palmer 1990).

Factors important for germination include adequate moisture content, temperature

and aeration, which play essential roles in production of enzymes and solubilization

of reserve materials in the endosperm (Daiber and Taylor 1995). They also affect

extract yield, diastatic activity and other important malt quality characteristics

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(Morrall et al 1986). It was found that higher germination temperature of 30C when compared with 20C, increased the production of total soluble nitrogen and - and -amylases (Agu and Palmer 1997). Chemical treatment by sprinkling potassium bromate at a concentration of 125 mg/liter on germinating sorghum was found to

increase the diastatic power of sorghum malt significantly after 4-d malting (Agu

1995b).

Drying

Drying ends the germination process by reduction of moisture to 12% or less, which

in turn preserves the malt quality and enzymes during storage (Novellie 1962,

Pathirana et al 1983). Industrially, sorghum malt is dried by forced-draft dryer, the

temperature of which should not be higher than 50C since higher temperatures were reported to cause losses of enzymes especially during the initial stages of drying

(Novellie 1962, Okon and Uwaifo 1985).

Milling

The objective of milling is to split the husk, so as to expose the starchy endosperm.

The dried malt is milled to pass through a 0.5-mm sieve to at least 95% together

with the roots and shoots which are found to be an important source of FAN (Taylor

1983) and flavor (Daiber and Taylor 1995).

1.2.3 Opaque Beer

Description

Opaque beer is a traditional and popular beverage in several countries in Africa. It is

also known as chibuku in Zimbabwe, impeke in Burundi, dolo in Mali and Burkina

Faso and pito in Nigeria (Jambunathan and Subramanian 1995). The beer is usually

brewed for important social and cultural gatherings like weddings, celebrations of

success and traditional religious ceremonies (Madovi 1981, Benhura and Chingombe

1989).

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Opaque beer production involves both lactic acid and alcoholic fermentation stages

and takes about 5 to 7 days to brew depending on ambient temperature (Gadaga et al

1999). It is an opaque, alcoholic, effervescent, pinkish-brown beverage with a sour

flavor resembling yoghurt and the consistency of a thin gruel (Steinkraus 1996). Its

opaque appearance is due to the high content of suspended solids and cells such as

undigested starch residues, yeasts and other microorganisms. Its pinkish-brown color

is due to the solubilization of reddish anthocyanin pigments (Glennie 1983) during

souring and mashing. Its sour flavor is due to the lactic acid fermentation bringing

pH down to 3.2 to 3.9 (Table 1). This beer is distributed and consumed while still

actively fermenting, thus it is held in vented containers so as to allow escape of

carbon dioxide. In contrast to European brewing, opaque beer is not pasteurized and

it has a short shelf life ranging from one to five days depending on how hygienic the

condition is during preparation and fermentation (Daiber and Taylor 1995).

Composition/Nutritive Value

Opaque beer is more a food than a beverage as shown by the important role it plays

in the nutrition of Bantu tribesmen who work at the diamond and gold mines. The

beer is rich in carbohydrate with an alcohol content ranging from 1-4% and

consumption of 1-L opaque beer can supply 13.1% daily food energy requirements

of an adult (Table 2). The beer contains a considerable amount of protein and 1-L

opaque beer can satisfy 9.6% recommended daily dietary allowance for protein

(Table 2). It also contains plenty of B vitamins, including thiamin, riboflavin and

niacin (Table 2). It was reported that pellagra, which is relatively common in people

subsisting on maize diets, was never found in people consuming usual amounts of

opaque beer (Platt 1964). Opaque beer contains various kinds of mineral such as

copper, iron, zinc, manganese, magnesium and phosphorus. It was found that by

using sorghum as adjunct rather than maize grits, this increases the nutritional value

of opaque beer in terms of vitamins and minerals (Van Heerden 1989a). When

compared with barley beer, opaque beer has a higher protein, thiamin, riboflavin and

mineral content, but a lower alcohol and niacin content (Van Heerden 1988).

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Opaque Beer Production

Souring

Souring refers to lactic acid fermentation, which involves the growth of lactic acid

bacteria on slurry of approximately 8-10% sorghum malt in water inoculated with a

pure culture of thermophilic, homofermentative Lactobacillus delbrueckii

(leichmanni). The temperature during souring should be strictly kept at 48-50C so as to prevent the growth of mesophilic organisms and favour the growth of

thermophilic, homofermentative Lactobacillus delbrueckii (leichmanni), which

produces solely lactic acid (Van der Walt 1956) from maltose or glucose under

anaerobic conditions. It is kept for up to 2 days until pH is about 3.0 3.3 and with a

lactic acid content of about 0.8-1.0% (Briggs 1998d).

Lactic acid fermentation can be classified into two types, spontaneous or inoculated.

Spontaneous fermentation, though a simple way to sour, is rather a hit or miss

process depending on the presence of the lactic acid bacteria which form part of the

natural microflora of the sorghum malt (Daiber and Taylor 1995). It involves a

complex microbial process (Daeschel et al 1987) and may result in a product of

variable quality (Novellie and de Schaepdrijver 1986, Kingamkono et al 1995).

Inoculated fermentation can be done either by means of 10% by volume of the

previous sour or isolated pure strains of Lactobacillus. This is normally adopted in

industrial brewing and in many home brewing processes (Daiber and Taylor 1995).

Souring serves three functions. The lactic acid produced imparts the beer with its

characteristic sour taste and lowers the pH of the beer, thus slows down the rate of

microbial spoilage and inhibits the growth of pathogenic organisms. This low pH

also prevents the complete hydrolysis of starch into sugars during mashing by

retarding the rate of enzyme activity (Novellie 1966, Taylor 1989) because the

resulting residual starch in the beer is important for giving the beer with its opaque

and viscous character (Daiber and Taylor 1995).

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Cooking

Cooking involves the gelatinization of starch in the adjunct. When the correct level

of acidity is obtained after souring, unmalted cereal, known as adjunct, and more

water are added and the mixture is boiled for 2 hr. The starch in the mixture, mainly

from the adjunct then undergoes gelatinization, thus making it readily hydrolyzable

during mashing by the diastatic enzymes of the malt because ungelatinized (raw)

starch is very resistant to amylolytic digestion and is only slowly attacked by the

malt -amylase and not at all by the -amylase (Hyun and Zekius 1985, Daiber and Taylor 1995). The gelatinized starch also serves to give the beer its characteristic

creamy body and keeps the particles of grain and malt in suspension (Novellie and

Schutte 1961). Both gelatinized and ungelatinized starches are present in

considerable amounts in the end product (Novellie and Schutte 1961, Novellie 1966)

and both starches provide calories to the consumer.

Sorghum starch granules are tightly enclosed by endosperm protein which creates a

barrier to starch gelatinization (Chandrashekar and Kirleis 1988). This can be solved

by the softening effect of lactic acid, which is produced in previous souring step,

thus allowing more rapid water uptake by the starch granules and speeds up

gelatinization (Novellie 1968).

Cooking and mashing cannot be carried out simultaneously because the temperature

at which the starch of adjuncts (maize, sorghum or millets) is gelatinized falls in the

range 62 to 75C (Briggs et al 1981). This gelatinization temperature is so high that both -amylase and -amylase will be inactivated by the time the starch is gelatinized. Therefore, the cooking and mashing should be carried out separately.

Mashing

Mashing aims at converting starch of the malt and adjunct into sugars so as to give a

fermentable wort of the desired composition. This first starts with cooling down the

cooked adjunct to about 60C after 2-h cooking in order to prevent the enzymes in the malt from denaturing, extra sorghum malt is added and the mixture is held for 2

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h at 60C so as to convert the starch molecules of the cooked adjunct into glucose, maltose and maltooligosaccharides by - and -amylases and other related enzymes. Both fermentable and unfermentable sugars are produced at the same time. - and -amylases are the principal enzymes responsible for starch conversion. When they work together, they are capable of converting only 60 to 80% of the available starch

to fermentable sugars (Dougherty 1977). By the end of mashing, the pH of the wort

becomes 3.9 with 0.16% lactic acid and a high total solids content (Briggs 1998d).

Control of the mash pH is very important since it affects viscosity, sugar

concentration and yield of alcohol (Rooney and Serna-Saldivar 1991). Although the

mash should be thinned, complete starch hydrolysis is not the ultimate objective.

This can be prevented by inhibition of -amylase with low pH at which the mash never becomes too fluid, part of the gelatinized adjunct starch should remain at the

end of the mashing (Novellie 1966) so as to give the beer its opaque character and

high viscosity.

Straining

Straining process refers to the removal of coarse particles such as husks, pericarp

and plumules with a diameter larger than 250-m, so as to obtain a smoother texture for the opaque beer. This can be done by solid bowl centrifuges (decanters)

industrially (Novellie and De Schaepdrijver 1986) or passing through a bag of

woven grass or metal screen with appropriate mesh size domestically (Daiber and

Taylor 1995). The spent grains (also known as strainings) are usually used as animal

feed. They are rich in dense, ungelatinized starch and insoluble protein, contain 46%

starch and 25% protein on dry basis (Van Heerden 1987).

Fermentation

Fermentation is done either with the inoculation of wild yeasts from the malt or

cultivated strains of yeast. The predominant natural yeast is Saccharomyces

cereviseae, and strains of its top fermenting yeast are used to start the fermentation

after cooling down the wort to 28C. It is then kept for 48 h at 28 to 30C.

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During fermentation, yeasts multiply and fermentation develops, they convert sugar

to ethanol and carbon dioxide. Glucose, maltose and maltotriose are the only starch

hydrolysis products that can be utilized by yeast to form alcohol in this process

(Panchal and Stewart, 1979) and they can be obtained by degradation of grain

components, especially starch and soluble sugars, by both intrinsic grain enzymes

and enzymes of the fermenting media (Chavan and Kadam 1989). At the same time

more B vitamins and new protein are also produced and the bacteria continue to

develop lactic acid (Doggett 1970). Final alcoholic content of the beer varies

between 1-4% (Daiber and Taylor, 1995).

Fermentation not only involves ethanol production but also improves the nutritional

quality of sorghum by causing significant changes in chemical composition and

eliminating antinutritional factors (Chavan and Kadam 1989). Chemical changes like

amylolytic hydrolysis not only take place during mashing but simultaneously with

alcoholic fermentation (Steinkraus 1996) and combination of cooking and

fermentation, as in the case of opaque beer production, was also reported to enhance

the nutrient quality and drastically reduce the antinutritional factors to safe levels

(Obizoba and Atii 1991). Moreover, fermentation was also found to contribute to

protein digestibility and availability of amino acids (Mbugua and Njenga 1992,

Steinkraus 1996). Amino acids and peptides stimulate the growth and fermentative

activity and tolerance of yeasts as well as proteolysis and lactate production by LAB,

and these factors affect both the sensory and nutritional quality of fermented foods

(Martinez-Anaya 1996) as well.

Shelf-life of opaque beer

The shelf life of opaque beer only ranges from 1 to 4 days depending on the hygiene

of the production conditions. Therefore, the beer should be consumed promptly

before deterioration, while fermentation is still active and before the alcohol content

rises above the legal limit (Briggs 1998a). Stability of the product is the main

"hindrance" to achieve a better profit level. The opaque beer should be sold as soon

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as possible after bottling. Final distributors / sellers to consumers cannot store the

bottle or packing for long due to the active fermentation of the beer. Fermentation is

not completed when the product is packaged and continues the more the bottle or

packing is shaken; followed by a deterioration of the product into a mixture of

alcohol and rancid fine particles. The readiness to spoilage is due to the activities of

species of Acetobacter, a genus of acetic acid bacteria. Acetic acid bacteria are

particularly known in breweries for their ability to produce acetic acid which gives

vinegary off-flavors, turbidity, and ropiness. It is a kind of gram-negative, rod-

shaped bacteria which can cause the ethanol, the main product from fermentation, to

combine with oxygen to form acetaldehyde which eventually converts to acetic acid.

Low pH does not restrict growth of acetic acid bacteria (Hough et al 1982). It

develops best in wort and beer when exposed to air during early fermentation and

aeration of the beer by rousing or splashing which provides the bacteria with

sufficient oxygen for respiration. Acetic acid bacteria are therefore unable to grow

after the yeast culture has utilized the dissolved oxygen of the wort (Goldammer

2000b).

1.2.4 Amylose and Amylopectin

Starch is a polymer with anhydroglucose units as its monomers. It consists of two

types of molecules, amylose, a mainly linear structure and amylopectin, a branched

structure. Amylose has a relatively lower molecular weight (typically 20-800 g/mol),

consisting of single mostly-linear chains with 500-20,000 -(14)-D-glucose units depending on the source. It forms a helical complex with iodine giving a

characteristic blue color. Amylopectin has a very high molecular weight (typically

10,000-30,000 g/mol) with up to two million glucose units. In addition to -(14) linkages which are present in amylose and the linear segments of amylopectin, the

amylopectin molecule also has -(16) linkages which occur every 20-30 anhydroglucose units. Amylopectin molecules are grouped together in concentric

rings representing alternating semi-crystalline and amorphous lamellae while the

amylose is partly involved in double helices with amylopectin chains in the

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crystalline regions, randomly dispersed in the amorphous regions or bound in

amylose-lipid complexes (Blanshard 1987).

The relative proportions of amylose to amylopectin depend on the source of the

starch. Regular endosperm sorghum types contain 23 to 30% amylose (Horan and

Heider 1946, Ring et al 1982) while waxy varieties contain up to 5% amylose.

Normal maize starch is composed of approximately 75% amylopectin and 25%

amylose. Waxy maize starch is composed of 98% amylopectin and 2% amylose.

High amylose (greater than 70%) containing starches are also known. This

difference in ratio of amylose to amylopectin influences both gelatinization

temperature (Knutson 1990) and starch susceptibility to enzyme attack (Ring et al

1988, Holm and Bjork 1988).

1.2.5 Biochemical Changes of Starch during Opaque Beer

Production

Gelatinization

Starch gelatinization plays an important role during processing of starch products

and it takes place in the cooking stage of opaque beer production. In this process, a

series of irreversible changes such as breakage of hydrogen bonds, water uptake,

swelling, melting of crystallites or double helices, loss of birefringence and

solubilization occur, they are generally accompanied by increasing viscosity due to

water being absorbed away from the liquid phase into the starch granules.

Due to the presence of semi-crystalline arrangement of the starch molecules, the

granules are insoluble in cold water but require heat and moisture to gelatinize.

Starch is generally insoluble in water at room temperature. But when heat is applied

to progressively higher temperatures, very little changes are noted until a critical

temperature at which irreversible starch transformation takes place. The starch

granules start to swell and lose their polarization cross simultaneously. Since loss of

birefringence occurs at the time of initial rapid gelatinization (swelling of the

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granule), thus it is a good indicator of the initial gelatinization temperature of a

given starch. The larger starch granules, which are usually less compact, begin to

swell first. The amylose chains solubilize and a starch gel is formed. At this point,

the starch is easily digestible.

In general, the swollen granules are enriched with amylopectin, while the linear

amylose diffuses out of the swollen granules and makes up the continuous phase

outside the granules (Hermansson and Svegmark 1996). This is usually the case for

lipid free amylose when heated in water. But amylose can form complex with lipid

known as amylose-lipid complex. It is usually made up of a left-handed amylose

helix with six residues per turn, in which the aliphatic part of the part is embedded

with the polar group lying outside due to its large molecular size (Neszmelyi et al

1987). The melting temperature of these crystals is around 110C for endogenous cereal lipids (Becker et al 2001). The presence of monoacyl lipids in amylose can

therefore restrict swelling, dispersion of granules and solubilization of amylose. It is

the enclosed lipid molecules which contribute to the stability of the amylose helix

conformation (Becker et al 2001).

The gelatinization temperature of starches is directly correlated with amylose

content (Gerard et al 1999, Sievert and Wuesch 1993, Knutson 1990). The

gelatinization temperatures of high-amylose maize starches are higher than normal

and waxy maize starches (Colonna and Mercier 1985). Waxy and regular maize

gelatinize at 62 to 72C while for high-amylose starches, they begin to swell below

100C, and temperatures greater than 130C are required to fully disperse these

starches. During the gelatinization process, waxy starches usually swell to a greater

extent than their normal-amylose counterparts (Tester and Morrison 1990), and

amylose is proposed to act as a restraint to swelling (Hermansson and Svegmark

1996).

Starch hydrolysis

Starch degradation mainly occurs at the mashing step in the production of opaque

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beer, though it may also take place during fermentation. During starch hydrolysis,

the starch molecule is acted on by enzymes such as - and -amylases, resulting in the production of various sugars in the wort.

The effectiveness of enzymatic breakdown depends on degree of gelatinization, i.e.

whether the starch is truly dispersed, partly gelatinized, or suspended as intact

granules (Zobel and Stephen 1995). The chemical nature of the starch, particularly

the amylose and amylopectin content, is yet another factor that affects its breakdown.

It was reported that the susceptibility to hydrolysis in cooked starches was highest in

waxy maize starch (99100% amylopectin) and ordinary corn starch (approximately

25% amylose) followed by hybrid high-amylose corn starch (6466% amylose) and

100% corn amylose (Rendleman 2000), indicating that starch with a lower amylose

content is more prone to enzymatic hydrolysis.

- and -Amylases Starch hydrolysis in the mashing stage of opaque beer production is brought about

mainly by the joint action of the sorghum - and -amylases (Taylor 1989). The ratio of - to -amylases in sorghum malt varies from 2:1 to 3:1 (Novellie 1960, Dyer and Novellie 1966).

-Amylase constitutes 60 to 80% of the total diastatic activity (Dyer and Novellie 1966, Okon and Uwaifo 1985) and its production in sorghum originates in the

scutellum instead of the aleurone layer (Aisien 1982). It is an endo-acting enzyme

that catalyses the hydrolysis of (14)--glycosidic bonds at random on both amylose and amylopectin to produce an array of linear and branched dextrins and

this does not occur in the immediate vicinity of (16)-branch points. -Amylase is able to degrade starch to a complex mixture of sugars on its own depending on the

relative location of the bond under attack as counted from the end of the chain. For

amylose, glucose and maltose are produced while for amylopectin, -limit dextrins of variable composition are produced. The optimum pH range for -amylase is 4.5 - 5.0 (Botes et al 1967a) and below about 4.9 the enzyme becomes unstable (Briggs

17

1998b).

Dextrin, maltotriose, maltose and glucose are produced accordingly upon the

hydrolytic effect exerted by -amylase. Dextrins are shorter, broken starch segments that form as a result of the random hydrolysis of internal glucosidic bonds. A

molecule of maltotriose is formed if the third bond from the end of a starch molecule

is cleaved. A molecule of maltose is formed if the point of attack is the second bond.

A molecule of glucose results if the terminal bond is cleaved. This degradation

occurs more rapidly with the presence of -amylase. Whenever a starch chain is broken by -amylase, a new non-reducing chain end is formed that may be attacked by -amylase. Since -amylase can cleave amylopectin chains on either side of (16)-branch points, therefore it can by-pass branches and provides substrate for -amylase which can then degrade starch more rapidly and completely than they are able to do alone. -Amylase then begins to sequentially remove units of maltose from the non-reducing end of these large dextrins.

-Amylase is an exo-enzyme that catalyses the hydrolysis of the -(14) glucosidic bond from their non-reducing ends, liberating maltose. This enzyme does not attack

(16)-glucosidic links or (14)-links immediately adjacent to them. Thus, this enzyme is unable to degrade starch granules in the absence of other enzymes. When

acting alone, it can degrade amylose completely to maltose but for amylopectin, the

enzyme degrades its outer chains to maltose but the residue remains as a -limit dextrin. For -amylase, its optimum pH range is 5.2 - 5.5 (Botes et al 1967b). -Amylase activity of sorghum malt was found to be lower than that of barley malt

(EtokAkpan 1992, Taylor and Robbins 1993) and the -amylase activity of sorghum malt was less than 25% of barley malt level (Taylor and Robbins 1993).

-Amylase is also known as liquefying amylase due to its ability of drastically reducing the viscosity of gelatinized starch solution by splitting large chains into

various smaller sized segments and destroying the ability of the starch to give color

18

with iodine, and only relatively slowly increasing the reducing power of mixtures.

On the other hand, -amylase is known as saccharogenic amylase due to its ability of rapidly generating reducing sugars (maltose) while only slowly reducing the

iodine-staining capacity of soluble starch. The saccharification power of sorghum

malt is restricted due to limited amount of -amylase (Nout and Davies 1982), yet some sorghum cultivars do develop significant -amylase (Palmer 1992).

Diastatic power

Diastatic power (DP) is a measure of the joint - and -amylase activity (Novellie 1959) and is a parameter indicating the ability to convert complex starches into

fermentable sugars, expressed as Sorghum Diastatic Unit (SDU). The ability of the

malt to produce sufficient diastatic power is considered to be the most critical factor

in brewing opaque beer (Novellie 1968).

Diastatic activities were found to be zero in ungerminated sorghum seeds (Ahmed et

al 1996) and ranged from 16 to 58 SDU/g in sorghum malt. The minimum diastatic

power specification for sorghum malt by sorghum brewery is less than 28 SDU/g

(Taylor and Dewar 1992). Sorghum malts have lower diastatic power than barley

malts (EtokAkpan and Palmer 1990, Adejemilua 1995) and vary according to

cultivars and processing differences. However, if mashing is carried out at 60C, the optimum temperature for sugar production, and at a low pH, sorghum malt will have

a higher diastatic activity than barley malt (Novellie 1966).

Research has been done to investigate factors affecting diastatic activity in sorghum.

Diastatic power was found to increase with an increase in steeping temperature of up

to 30C (Dewar et al 1997b) or an increase in malting temperature with maximum activity occurred at 24C (Taylor and Robbins 1993). Various treatments for malting were also reported to increase diastatic power. It was reported that a concentration of

125 mg/L potassium bromate caused a significant increase in diastatic power after 4

d germination (Agu 1995b). Steeping in NaOH or formaldehyde (HCHO) markedly

improved the DP of the tannin-containing varieties, and steeping in NaOH appears

19

to be safer than HCHO for treatment of high-tannin sorghums in the malting

industry and for other food uses (Beta et al 2000). Extended germination and finer

milling were also found to increase the diastatic activity (Laseken et al 1995).

1.2.6 Microorganisms involved in opaque beer production Lactic acid bacteria

Lactic acid bacteria contribute to both processing technology and quality of the end-

products in terms of flavor, keeping properties, safety and overall product image

(Salovaara 1998). Lactobacillus is the preferred genus for industrial lactic acid

fermentation (Vickroy 1985, Atkinson and Mavituna 1991) because of high

metabolic rate, high lactate production and the ability of sustaining lower pH

(Anuradha et al 1999). Homofermentative lactic acid bacteria such as Lactobacillus

delbrueckii, used for inoculation in the souring stage of opaque beer production, can

ferment hexoses via glycolysis, with 90% of the glucose being metabolized to lactic

acid (Kontula et al 1998). Factors affecting the metabolic products include the

available carbohydrates and growth conditions such as pH, aeration and cell density

(Kontula et al 1998). The optimum pH and temperature for growth rate of

Lactobacillus delbrueckii were 5.5 and 45C (Venkatesh et al 1993, Venkatesh 1997).

The lactic acid bacteria require substrates with high nitrogen content and have a

particular demand for B vitamins (Hofvendahl and Hgerdal 1997). This can be

satisfied by the addition of malt sprouts or yeast extract (Vickroy 1985, Atkinson and

Mavituna 1991).

Lactic acid fermentation is capable of lowering the pH to below 4 in food products,

including sorghum-based fermented cereal gruels used as infant foods (Kunene et al

1999). This is done by the conversion of carbohydrate substrate to lactic acid which

lowers the pH of the fermenting medium to levels that cannot support the growth

and activities of many spoilage microorganisms. This results in growth reduction of

pathogenic bacteria such as B. cereus, Campylobacter spp., enterotoxigenic E. coli,

20

Salmonella spp., Shigella spp. and S. aureus (Menash et al 1991, Nout 1991,

Simango and Rukure 1992, Kingamkono et al 1995). Most of the spoilage and

pathogenic microorganisms are inhibited by a combination of pH reduction, a

lowering of oxidation-reduction potential, competition for essential nutrients and the

production of inhibitory compounds such as organic acids, hydrogen peroxide,

antibiotics and antimicrobial substances (Mbugua and Njenga 1992, Hanciolu and

Karapinar 1997).

Lactic acid fermentation of cereals plays an important role in terms of nutrient

composition. It has been found to reduce the amount of phytic acid, polyphenols and

tannins and improved protein availability in sorghum (Chavan et al 1989). It has also

led to improved iron (Svanberg and Sandberg 1988), minerals and sugar (Khetarpaul

and Chauhan 1990).

The utilization of soluble carbohydrates by lactic acid bacteria and consequently,

their energy yield, and lactic and acetic acid production are greatly influenced by the

associated yeasts and vary according to the types of sugars (Gobbetti 1998). Just like

yeast, Lactobacillus species have also been reported to produce ethanol (Hansen and

Hansen 1996).

Yeast

Yeast for brewing not only converts fermentable sugar in the wort to ethanol and

carbon dioxide, but also produces a variety of volatile and nonvolatile constituents

that contribute to the overall flavor and acceptability of fermented cereal gruels

(Banigo et al 1974, Odunfa and Adeyele 1985). Most of the brewing yeasts will

prosper at a pH of 5.0 to 5.5. The principal fermentable carbohydrates in the wort

include glucose, fructose, sucrose, maltose and maltotriose. Sucrose was found to be

the first sugar which disappeared from the wort, indicating the disappearance of the

disaccharide through hydrolysis by the enzyme invertase. Glucose and fructose are

then utilized rapidly, followed by maltose (Reed and Nagodawithana 1991). Pentose

sugars are not metabolized by brewers yeasts. The unfermented carbohydrates then

21

pass through the fermentation and contribute to the caloric content of the final beer

(Reed and Nagodawithana 1991).

At the beginning of fermentation for beer production, there is an adequate level of

oxygen in the wort medium, and the yeast undergoes two different overlapping

metabolic phases, the aerobic respiration and anaerobic fermentation:

Aerobic Respiration

C6H12O 6 + 6 O2 CO2 + 6 H2O

Anaerobic Fermentation

C6H12O6 2 CO2 + 2 C2H5OH

With adequate level of oxygen in the wort, the initial aerobic growth phase continues

until all the dissolved oxygen in the wort is utilized and biomass is formed. For

anaerobic fermentation, ethanol, carbon dioxide and other flavor components of the

beer are formed.

The wild type Saccharomyces cerevisiae strains intrinsically lack significant amylase,

and therefore they are unable to metabolize starch (Marn et al 2001). For S.

cerevisiae to utilize starch, starch must be converted to fermentable sugars such as

glucose or maltose. In the production of opaque beer, gelatinization by cooking and

enzymatic liquefaction and saccharification by mashing are used to promote starch

hydrolysis so as to produce a fermentable wort for the yeast.

Apart from sugar, amino nitrogen is also important for fermentation and essential for

the growth of yeast. A low proportion of malt is used in the grist in the production of

opaque beer, and nutrients especially FAN, may limit yeast growth during

fermentation (Taylor et al 1985). A linear relationship was found between the sugar

content in opaque beer wort and the quantity of FAN required to rapidly ferment the

sugars to alcohol, and a minimum requirement of 100 mg/L for FAN in the wort was

22

proposed (Pickerell 1986).

Association of lactic acid bacteria and yeasts

Co-metabolism between LAB and yeasts are common in most of the African

indigenous fermented foods (Adegoke and Babaola 1988, Steinkraus 1996). This

increases the microbial adaptability to complex food ecosystems (Gobbetti et al

1994, Stolz et al 1995, Gobbetti and Corsetti 1997). It is possible that the co-

metabolism between LAB and yeasts benefits one another. The growth of lactic acid

bacteria could be promoted by growth factors such as vitamins and soluble nitrogen

compounds from the yeast. On the other hand, the proliferation of yeasts in foods is

favored by the low pH condition caused by LAB (Nout 1991, Gobbetti et al 1994,

Steinkraus 1996) and the bacterial end products could be used by the yeasts as an

energy source (Leroi and Pidoux 1993).

Co-metabolism between LAB and yeasts during fermentation can also improve the

final quality of the food as well. They impart taste and flavor to foods by their

metabolites (Akinrele 1970, Halm et al 1993, Brauman et al 1996, Hansen and

Hansen 1996). The production of acids and antimicrobial components in gruel

during fermentation can improve microbiological safety (Svanberg et al 1992,

Kingamkono et al 1994, 1995) and stability of the products (Menash et al 1991).

23

Table 1. Analysis of Opaque Beer

Determination Range Average

pH 3.2-3.9 3.5

Lactic acid (mg %) 164-250 213.0

Volatile acidity as g acetic acid / 100

ml

0.012-0.019 0.016

Total solids (%) 2.6-7.2 4.9

Insoluble solids (%) 1.6-4.3 2.3

Alcohol (%) (by weight) 2.4-4.0 3.2

Nitrogen (%) 0.065-0.115 0.084

Sources: Novellie (1968).

24

Table 2. Nutrient Content of Industrially Brewed Opaque Beer a

Nutrient Mean

Content

RDAb % Contribution to

RDA by 1 L of

Beer

Alcohol (g) 25.4 NRc

Fat (g) Trace 96d

Crude Fiber (g) 1 NR

Protein (g) 5.4 56 9.6

Ash (g) 1.1 NR

Carbohydrate (g) 47.6 446d 10.7e

Starch (g) 29.7 NR

Food energy (kJ) 1,651 12,600 13.1e

Vitamin B1 (thiamin) (mg) 0.2 1.5 16.0e

Vitamin B2 (riboflavin) (mg) 0.4 1.8 21.7e

Niacin (mg) 2.9 20 14.7e

Copper (mg) 0.2 2 10.0e

Iron (mg) 2.0 18 11.1e

Zinc (mg) 1.8 15 12.0e

Manganese (mg) 1.5 3.8 39.5e

Calcium (mg) 53 800 6.6

Magnesium (mg) 140 400 35.0e

Potassium (mg) 276 4,000 6.9

Sodium (mg) 9 10%/L)

25

CHAPTER 2

EFFECT OF MALTING ON STARCH AND

OTHER COMPONENTS IN SEVEN VARIETIES

OF SORGHUM

2.1 ABSTRACT

The effect of malting on seven varieties of sorghum grain was compared and the

physicochemical and enzymatic properties of their malt were studied to predict their

suitability for brewing opaque beer. Malting was carried out and the germinated

sorghum samples were compared with their ungerminated counterparts. The seven

varieties showed a decrease in starch ranging from 23.2% to 48.5% and a decrease in

amylose ranging from 13.6% to 42.3% due to the action of amylolytic enzymes,

resulting in tremendous increase in sugar content. DC-75, a sorghum variety

commonly used for commercial brewing of opaque beer, had the lowest content of

starch as well as amylose yet the highest content of glucose, fructose and total non-

starch carbohydrate among the seven sorghum varieties in this study. It had a

diastatic power of 55.2 SDU/g, satisfying the minimum specification of 28 SDU/g.

DC-75 had the lowest -amylase activity yet the highest -amylase activity among

the seven sorghum varieties investigated. All these properties were involved in the

final quality of opaque beer. The correlation among the parameters was examined.

The gelatinization temperatures of sorghum malt and adjunct were significantly

correlated with each other (r = 0.92). This was believed to be due to the competition

for water between sugar and starch present in the system.

26

2.2 INTRODUCTION

Sorghum is a cereal grain well adapted to semi-arid and sub-tropical conditions. It

can yield crops under harsh environmental conditions such as drought and heat stress.

It can also withstand flooding and is not influenced much by acid soil. Apart from its

drought-tolerant characteristic, the available supply, its low price, its content of

starch and protein has enabled sorghum to become an important material for brewing

(Haln 1966). The potential of sorghum as an important source of industrial brewing

material has been long recognized since World War II. At that time, sorghum was

offered as a brewing material due to the scarcity of barley, the conventional brewing

material (Haln 1966). In Nigeria, there is already a total replacement of imported

barley malts with sorghum and maize produced locally. In the developing countries,

sorghum production is predicted to grow at 1.6 % per annum from 44 million tons in

1992-94 to 53 million tons in 2005, the rise primarily concentrated in Africa

(ICRISAT 2002).

The malt used in the preparation of opaque beer plays a role in determining the rate

of fermentation and is a source of lactobacilli, yeasts and essential nutrients (Agu

1995a, Odunfa and Adeyele 1995, Ugboaja et al 1991). Therefore, malt is an

important component contributing to the final quality of the beer and it is absolutely

essential to select the malt which suits the quality of the beer most. A lot of research

has been done to investigate changes taking place during malting.

In the present study, the aims were 1) to investigate the effect of malting on seven

varieties of sorghum grain, 2) to examine the correlation among starch and other

related parameters in this study and 3) to predict the suitability of sorghum malt for

the production of opaque beer.

27

2.3 MATERIALS AND METHODS

Sorghum Grains

Grains of five varieties of sorghum, DC-75, Brown Tsweta, Red Swazi, Pato and

Pirira, were supplied by Dr. C. E. Walker Department of Grain Science and Industry,

Kansas State University. The other two varieties, PL 1 and TX 2737, were supplied

from Kenya.

Malting

Steeping

Clean sorghum grains (50 g) were put into perforated nylon mesh bags and steeped

for 8 h at 25C in NaOH (0.3%, w/v). They were thoroughly rinsed with fresh tap water and were then immersed in sodium hypochlorite solution (2%, v/v) for 10 min

for prevention of microbial growth. They were thoroughly rinsed with tap water

again for the removal of any residual reagent. Excess water was then removed by

paper towel.

Germination

Germination was carried out at 25C and 100% RH for five days. The grains in nylon mesh bag were grown on moist paper towel with another layer of paper towel

covering on top. The germinating grains were spread evenly during germination so

as to reduce the resistance of the grain to aeration and assist the uniform watering of

the green malt. The grains were steeped in fresh tap water for 10 min twice a day.

Excess surface-held water was removed by paper towel. These steps were done with

great care so as to avoid any damage of the shoots and roots. Since they were not

protected at all, they were liable to injury which would lead to unproductive

regrowth of the seedling and give access to parasitic and potentially toxin-producing

fungi (Daiber and Taylor 1995).

28

Drying

After five days of germination, the grains were dried at 50C for 48 h in a forced-air oven.

Milling

The germinated dried grains were milled to pass through a 0.5-mm sieve with an

Udy Cyclone Mill (Udy Corporation, Ft. Collins, Colorado, USA).

Dry Matter Loss

Dry matter loss was measured by recording the weight of the grains before steeping

and after germination and was expressed as the percentage change in weight of

grains before and after malting.

Germination Activity

The germination activity was recorded by germinating 100 grains (in triplicate) on

petri dishes. After germination, the number of germinated grains was counted and

this represented the germination activity.

Germination Activity

= Number of grains germinated / Number of total grains x 100%

Gelatinization Temperature

A Mettler DSC-20 differential scanning calorimeter (Mettler-Toledo AG

Instruments, Naenikon-Uster, Switzerland) equipped with a ceramic sensor and a

Mettler TC II data analysis station was used for measuring the gelatinization

temperatures of sorghum adjunct and malt. Sample (3 mg, dwb), in duplicate, was

weighed in an aluminium pan and the final weight was made up to 12 mg by

addition of distilled water. It was equilibrated at room temperature for 1 h and then

heated from 30 C to 100C at a rate of 10C per min. The gelatinization temperature (C) was recorded.

29

Total Starch Content

Total starch content was determined by the amyloglucosidase / -amylase method using the Megazyme Total Starch Assay Kit (Megazyme, Bray, Ireland).

Amylose Content Assay

Amylose was determined using an iodine-binding spectrophotometric method.

Sample (50 mg) was weighed to the bottom of 50 ml volumetric flask. 95% Ethanol

(1 ml) and 1 M sodium hydroxide (4.5 ml) were added respectively and mixed well.

The samples were kept at 45C in the oven overnight. Distilled water (25 ml) was added and the samples were then kept at 100C in the oven for 60 min for starch gelatinization. They were then cooled down to room temperature and made up to

volume. The solution was centrifuged at 3,000 rpm for 10 min so as to remove the

coarse particles. An aliquot (1 ml) was transferred into a 25 ml volumetric flask, to

which 300 l 1 M acetic acid and 400 l I2-KI solution were added, made up to volume and mixed well. The solution was kept at room temperature for 20 min.

Absorbance value was read at 620 nm against blank. Amylose content was

determined according to a standard curve which was prepared by maize amylose /

amylopectin (Sigma) standard mixtures representing 0 to 70 % amylose.

Total non-starch carbohydrate

Starch in the sample was removed according to step 1 to 4 of the pretreatment of

samples containing glucose and maltosaccharides of Megazyme Total Starch Assay

Kit. The supernatants of the sorghum adjunct and malt were made to a total volume

of 50 ml and 500 ml respectively and they were estimated for their total non-starch

carbohydrate by the phenol-sulphuric acid method (Dubois et al 1956).

Reducing and nonreducing sugars

Reducing and nonreducing sugars were determined according to AACC Method 80-

60 with the following modification for sorghum malt samples. The sample weight

was reduced by half while all other factors remained the same.

30

Glucose and fructose

Glucose and fructose were analyzed by High Performance Liquid Chromatography

(HPLC). They were extracted by weighing 100-200 mg sample in 1.5 ml Eppendorf

tube. Distilled water (1 ml) was added, vortexed and rotated overnight with a rotator.

It was centrifuged at 10,000x g for 5 min. The supernatant was retained by decanting

carefully to avoid disturbing the sedimented pellet which was discarded. The

polymers present in the supernatant were precipitated by adding 4 ml 95% ethanol to

1 ml supernatant. It was left overnight and centrifuged at 10,000 x g for 5 min. The

supernatant obtained was then dried by means of nitrogen gas. Double distilled

water was added and stored in an Eppendorf tube until HPLC analysis.

The HPLC was performed with DX500 chromatography system (Dionex, U.S.A.).

L20 Chromatography Enclosure was operated with GP40 Gradient Pump and EC40

Electrochemical Detector. Chromatograms were recorded and peak areas were

analyzed with Dionex PeakNet System. Then column used was CarboPac PA-1

sized 4 mm x 250 mm.

Double distilled water and sodium hydroxide were used as eluent. Reagent water

and sodium hydroxide were degassed for an hour before HPLC analysis. The

method program used was Sa2%15m.met (with 2% of NaOH and 15 minutes for

running time of chromatography). Samples were laded using a 10 l sample loop.

Chromatography was started immediately after injection of sample. Chromatograms

were recorded and peak area of each carbohydrate was calculated automatically. A

standard carbohydrate solution contained 0.1 mg/ml of fructose, glucose and sucrose

was used to determine the retention times of the sugar peaks and for quantitative

determination of glucose and fructose in the samples.

Optimum temperature for -amylase and -amylase activities The optimum temperatures for sorghum - and -amylase were determined by the Ceralpha method using the Megazyme -amylase assay procedure and Betamyl

31

method using the Megazyme -amylase assay procedure respectively. The tests were run at various incubation temperatures (30C, 40C, 50C, 60C and 70C for -amylase and 30C, 40C, 50C and 60C for -amylase). The optimum temperatures for maximum - amylase and -amylase activities of different cultivars were determined.

-amylase and -amylase activities Sorghum - and -amylase activities were determined by Ceralpha method using the Megazyme -amylase assay procedure and Betamyl method using the Megazyme -amylase assay procedure respectively. The tests were run at their optimum

temperatures for each enzyme.

Diastatic power

Diastatic power of each sorghum malt was determined according to AACC Method

22-16.

Viscosity Reduction

Rapid Visco Analyzer (RVA) (Newport Scientific Pty. Ltd., Warriewood, Australia)

was employed to determine viscosity reduction of the sorghum malt. Maize starch (3

g) was made up to 27 g by distilled water. The slurry underwent 90C for 3 min and the temperature was gradually reduced to 55C within 5 min. Malt extract (1 ml) (Beta 1995) was immediately added and the viscosity was monitored for 3 min at

55C. A blank was prepared by replacing the malt extract with distilled water. The viscosity reduction was recorded by the difference between the final viscosities of

the blank and the sample.

32

2.4 RESULTS AND DISCUSSION

Germination Activity

The germination activity of the sorghum grains ranged from 69.4% to 94.0% (Table

1). Ideally, for malting all grains would germinate but this can hardly be achieved.

To germinate, grains must be adequately hydrated, have a supply of oxygen, must be

within a suitable temperature range and must not be exposed to harmful agents,

either toxic chemicals or physically damaging machinery (Briggs 1998e). If all the

above conditions are satisfied, yet the grain still fails to germinate, this may be due

to the fact that the grain is dead or dormant. As shown in Table 7, germination

activity correlated with -amylase (r=0.77) and diastatic power (r=0.52).

Dry Matter Loss

Dry matter loss ranged from 14.0% to 20.7% (Table 1). The result was very similar

to 15-20%, as reported by Palmer et al (1989). This loss was not correlated with

sorghum diastatic power and this agreed with previous findings that dry matter loss

was not in any way related to the simultaneous improvement in DP (Novellie 1962).

Instead, this loss is a characteristic feature of seedling growth and malting (Aisien

1982, Aisien et al 1983, Iwuoha 1988). The loss due to germination can be attributed

to respiratory loss (Chavan et al 1979, Hornsey 1999) and loss during steeping

(Hoseney 1999). Malting loss was found to be directly proportional to the number of

days allowed for soaking and germination of seeds (Pathirana and Jayatissa 1983)

and soaking for a long time led to a faster rate of germination (Pathirana and

Jayatissa 1983).

Total Starch

Starch was the major component of sorghum grain dry weight, and ranged from

62.3% to 78.6% (Table 2). This was similar to 70.9%2.03% as reported by Beta et al (1995). Other findings illustrated that when soaking was accompanied by

germination, starch content was reduced from 68% to 33.5% for a low-tannin

33

cultivar, whereas in the high-tannin cultivar, the starch was reduced from 75% to

44% (Hagir et al 1999). PL had the highest starch percentage, this was probably due

to its exceptionally large grain size.

Previous findings reported that starch content of endosperm cells was reduced

during germination despite the presence of undegraded cell walls (EtokAkpan 1992,

EtokAkpan and Palmer 1990, Palmer 1991). This is believed to be caused by the

formation of portals in endosperm cell walls of sorghum during malting, which

allows amylolytic enzymes to enter the endosperm and hydrolyze starch reserve

(Palmer 1991).

After malting, the total starch of sorghum decreased to 35.3% - 53.6% in the malt

(Table 2), representing a mean decrease of 35.7%. This indicated the degradation of

nutrient reserves like starch to soluble sugars to meet the seedling requirements

(Dalvi 1974). The final total starch content of the sorghum malt after malting was

correlated with amylose content found in malt (r=0.77) and total non-starch

carbohydrate (r=-0.66).

Amylose Content

The amylose content of sorghum grains ranged from 17.5% to 33.3% (Table 3),

indicating that they had normal, nonwaxy endosperm. The decrease of amylose in

the malt down to 10.1% - 25.1% after malting indicated the degradation of amylose

by hydrolytic enzymes. Amylose % in malt was negatively correlated with -amylase (r=-0.7), total non-starch carbohydrate in malt (r=-0.82) and fructose in malt

(r=-0.9) as shown in Table 7.

DC-75 had the lowest amylose % and showed the highest % decrease in both starch

and amylose after malting among the seven sorghum varieties. This might indicate

the presence of smaller quantity of amylose-lipid complex in DC-75, resulting in a

higher decrease in starch and amylose.

34

It was reported that iodine-binding method for amylose determination can

overestimate amylose content if there are branched molecules with long side chains

which bind iodine, or underestimate if there are low-molecular weight linear

molecules which bind less iodine than normal amylose (Shi et al 1998). The

presence of amylose-lipid complex may also underestimate amylose content.

Total Non-starch Carbohydrate

The primary sugars present in sorghum grain are sucrose, maltose, raffinose,

fructose and glucose (Anglani 1998). Among the sugars investigated, sucrose was

the predominant sugar in the grain with a concentration ranging from 6.0 to 11.4

mg/g in the seven sorghum cultivars (Table 5). This agreed with previous findings

(Subramanian et al 1980) which reported that sucrose was the predominant sugar

ranging from 68.7% to 82.7% of the total non-starch carbohydrate.

As shown in Table 7, reducing sugar in malt correlated with non-reducing sugar in

malt (r=0.94) and in grain (r=0.80) as well. The total non-starch carbohydrate

concentration increased greatly from 4.6 15.2 mg/g (Table 5) to 157.8 354.3

mg/g (Table 6) after malting, representing an increase of 252 to 515%. During

malting, the enzymes were released from the grain to break down the food reserves

into their respective components so as to provide energy and support the growth of

the germinating grain. In this case, starch was broken down by amylases to sugar

thus resulting in a large increase in total non-starch carbohydrate after malting. This

indicated that the rate of sugar production by hydrolytic enzymes was greater than

rate of utilization for various metabolic activities of the micro-organisms. It was

reported that sugar concentration decreased in the first day of germination due to

consumption for various metabolic processes after recovering from dormancy, but

then increased rapidly in subsequent days until after the fourth day where no further

substantial increase in sugar concentration was found (Lasekan 1995).

As a result of starch hydrolysis, glucose increased from 0.1 11.2 mg/g (Table 5) to

91.1 270.0 mg/g (Table 6), representing an increase of 71.3% to 99.7%. Among

35

the sugars investigated, glucose showed the greatest increase after malting. Fructose

concentration also increased, this could possibly originate from the conversion of

glucose to fructose.

DC-75 showed the highest content of total non-starch carbohydrate, glucose and

fructose among the seven varieties of sorghum malt. Sugar in the malt is very

important for supporting the metabolic activity of yeast and other microorganisms

through its utilization in the fermentation stage.

Diastatic Power

Diastatic power is a measure of joint -amylase and -amylase activities. The test was done by peptone extraction instead of water because peptone competes with

amylases for reaction with tannins present in the sorghum grain and reacts

preferentially, thus preventing the amylases from reacting with tannins (Beta et al

2000). Diastatic power ranged from 32.7 to 70.9 SDU/g (Table 4) among the seven

varieties of sorghum malt. All varieties assayed satisfied the minimum specification

of 28 SDU/g for sorghum malt by a sorghum brewery (Taylor and Dewar 1992).

Others reported diastatic power of 16 to 58 SDU/g in 16 sorghum cultivars (Beta et

al 1995). DC-75, a sorghum variety commonly used for opaque beer production in

commercial brewing (Beta and Corke 2001), had a DP of 55.2 SDU/g (Table 4)

which was similar to previous findings of 46.9 SDU/g (Beta et al 2000) in a 5-day

germination.

- and -Amylase activities Both - and -amylases are required for hydrolysis of starch and production of fermentable sugars (Beta et al 1995). Ratio of -amylase to -amylase in these seven sorghum varieties ranged from 0.07 to 0.54. Similar findings were also reported

(Islas-Rubio 1993, Beta et al 1995). The production of amylases during sorghum

germination is affected by cultivar and environmental factors such as temperature,

moisture and humidity (Ratnavathi and Ravi 1991). -Amylase was reported to increase with germination time (Uriyo and Eigel 1999).

36

The optimum temperature for -amylase activity of the seven sorghum malt varieties was approximately 55C (Figure 1). This temperature was therefore chosen for the analysis of -amylase activity of the seven varieties of sorghum malts. The -amylase activity of the seven sorghum varieties ranged from 104.3 to 279.7 CU/g (Table 4) while other findings reported a range of 25 to 187 CU/g (Beta et al 1995).

The deviation might be due to the difference in incubation temperature. The malt

produced from Brown Tsweta had an activity as high as 279.7 CU/g while

commercial barley had an -amylase activity of 189 CU/g (Beta et al 1995).

The optimum temperature for -amylase activity of the seven sorghum varieties was approximately 38 to 39C (Figure 2), indicating that it had a lower optimum incubation temperature than -amylase and thus was more vulnerable to heat. Not only its optimum incubation temperature, but also its activity was lower than -amylase, ranging from 12.7 to 56.3 BU/g (Table 4). This was similar to previous

findings (Beta et al 1995). Only low levels of -amylase have been reported in sorghum and ungerminated cereals (Uriyo and Eigel 1991), such inability to detect

activity may be due to the interaction of -amylase with polyphenols during aqueous extraction to form insoluble polyphenol-enzyme complexes (Ratnavathi and Ravi

1991).

DC-75 showed the highest -amylase activity but lowest -amylase activity among the seven sorghum varieties. This implied that malt produced from DC-75 had the

greatest saccharification power but the lowest liquefying power in later stages of

beer production. This contributed to the final quality of the opaque beer by providing

more reducing sugars in the wort for yeast metabolism due to the greater

saccharification power and also giving a creamy body to the beer due to the lower

liquefying power when compared with other sorghum varieties.

37

Reduction in Viscosity

Reduction in viscosity correlated with malting loss (r=0.91), reducing (r=0.50) and

non-reducing sugar (r=0.67) in the sorghum malt (Table 7). It has been reported

(Beta et al 1995) that reduction in viscosity is correlated with -amylase and DP, therefore measuring the reduction in viscosity could serve as an estimation for both

parameters. No such correlation was observed in this study, so -amylase activity of the samples is recommended to be analyzed directly using the Megazyme Ceralpha

Assay Kit, instead of estimation by RVA.

Gelatinization Temperature

Peak gelatinization temperature of sorghum malt ranged from 69.8 to 77.1C (Table 1) among the seven sorghum varieties, and that of the ungerminated samples ranged

from 69.3 to 75.1C (Table 1). The range of gelatinization temperature determined by differential scanning calorimetry for sorghum starch was 71 to 80C (Sweat et al

1984). From Table 7, the gelatinization temperature of sorghum grain and its malt

were significantly correlated (r = 0.92) with each other, with the gelatinization

temperature of sorghum malt always higher than that of sorghum grain. This was a

result of the higher sugar content present in sorghum malt. It has been shown that

various sugars, including sucrose, fructose and glucose, can raise the temperature of

starch gelatinization and delay the increase in viscosity and the effect on the

gelatinization phenomenon increases with increasing sugar concentration

(Chungcharoen and Lund 1987, Paredez-Lopez and Hemandez-Lopez 1991,

Eliasson 1992). By means of differential scanning calorimetry (DSC) technique, the

swelling of starch granules was shown to decrease in the presence of sugars (Maaurf

et al 2001). This was proposed to be due to the ability of sugar to compete with

starch for water, thus reducing the water activity of the system (DAppolonia 1972,

Derby et al 1975). Therefore, for sorghum malt gelatinization, with a high sugar

content, there is more sugar to compete with starch polymers for interaction with

water molecules, so starch polymers in the malt would have to interact among

themselves as less water was available. For sorghum adjunct gelatinization, with less

38

sugar than in sorghum malt, competition for interaction with water by sugar would

be less, and thus starch polymers would interact with the water molecules more

easily. Since the interaction among starch polymers is stronger than that between

starch polymer and water, more energy would be needed to break the interactions

among starch polymers in the sorghum malt. Hence, a higher gelatinization

temperature was always found sorghum malt than for sorghum adjunct.

Knowing the gelatinization temperature range of sorghum is important because

gelatinization of sorghum starch would be involved in the later stages of opaque beer

production. This temperature allows us to estimate the energy requirements during

the industrial production of opaque beer.

39

2.5 CONCLUSION After 5-d malting, the seven sorghum varieties showed a decrease in starch and

amylose but an increase in total non-starch carbohydrate, glucose and fructose

respectively. Diastatic power of the sorghum malt ranged from 32.7 to 70.9 SDU/g,

satisfying the minimum specification of 28 SDU/g. The optimum incubation

temperature for - and -amylase of the seven sorghum malt varieties was approximately 55C and 39C respectively with -amylase contributing a higher proportion of the amylolytic activity of the sorghum malt. Gelatinization of sorghum

adjunct and malt ranged from 69.3 to 75.1C and 69.8 to 77.1C respectively and they were significantly correlated with each other. DC-75, the sorghum variety

commonly used for commercial brewing of opaque beer, had the lowest content of

starch and amylose but the highest content of glucose, fructose and total non-starch

carbohydrate among the seven sorghum varieties in this study. These might have

been the characteristics contributing to its role it played as being the commonly used

variety in commercial brewing of opaque beer.

40

Table 1. Germination activity, dry matter loss and gelatinization temperature of seven sorghum varieties.

Sorghum variety GAa (%) dmlb (%) Malt GTc(C) Adjunct GTd(C)

DC-75 94.00.8 16.30.5 73.20.1 70.10.0

Brown Tsweta 92.20.8 17.40.9 69.80.3 69.30.2

Red Swazi 88.90.1 16.80.1 70.00.2 69.30.5

Pato 69.41.0 14.60.0 73.30.2 72.00.2

Pirira 74.00.1 14.01.7 72.80.0 71.10.2

PL 1 71.70.4 17.10.21 77.10.4 74.90.1

TX 2737 87.00.0 20.70.91 77.10.3 75.10.2

Mean (Total) 82.4 16.7 73.3 71.7

aGA = Germination Activity

bdml = dry matter loss

cMalt GT = Gelatinization Temperature of Malt

dAdjunct GT = Gelatinization Temperature of Adjunct

41

Table 2. Changes in total starch % of sorghum during malting.

Sorghum

variety Total Starch (% db) Total Starch (% db)

Decrease in

starch %

Adjunct Malt

Mean Min Max Mean Min Max

DC-75 68.6 64.8 72.0 35.3 32.6 37.7 48.5

Brown Tsweta 69.9 65.5 75.1 50.2 45.4 59.6 28.2

Red Swazi 67.4 64.1 70.8 41.3 39.9 43.7 38.7

Pato 69.5 64.5 74.1 46.5 44.5 49.5 33.1

Pirira 69.8 65.1 75.7 53.6 50.2 59.1 23.2

PL 1 78.6 72.0 87.0 41.8 39.4 44.8 46.8

TX 2737 62.3 60.0 64.5 43.9 40.6 47.2 29.5

Mean 69.4 44.7 35.7

42

Table 3. Changes in amylose % of sorghum during malting.

Sorghum

variety % amylose of starch (db) % amylose of starch (db)

Decrease in

amylose % of

starch

Adjunct Malt

Mean Min Max Mean Min Max

DC-75 17.5 16.9 18.7 10.1 9.4 10.8 42.3

Brown Tsweta 27.3 26.1 28.2 23.6 22.0 24.9 13.6

Red Swazi 25.2 24.8 25.7 17.9 17.1 19.3 29.0

Pato 28.9 27.2 29.9 23.2 22.0 24.3 19.7

Pirira 30.2 29.0 31.0 25.1 24.1 26.3 16.9

PL 1 30.8 30.4 31.1 21.5 20.7 22.2 30.2

TX 2737 33.3 32.6 33.9 22.6 21.7 23.6 32.1

Mean 27.6 20.6 25.5

43

Table 4. Alpha-amylase activity at 55 C, beta-amylase activity at 40 C, diastatic power and reduction in viscosity in seven

varieties of sorghum malt.

Sorghum variety

Alpha-amylase

(Ceralpha Unit/g)

Beta-amylase

(Betamyl Unit/g)

Diastatic power

(SDU)

Reduction in viscosity

(RVU)

DC-75

104.30.8 56.31.9 55.22.4 40.52.5

Brown Tsweta 279.73.3 34.82.6 70.92.15 45.01.3

Red Swazi 129.90.5 42.31.7 65.52.2 45.72.4

Pato 177.42.0 12.70.1 45.82.1 35.21.0

Pirira 133.43.6 13.70.4 32.72.1 24.00.7

PL 1 175.70.7 26.40.0 50.60.9 47.41.3

TX 2737 157.30.7 36.30.1 52.50.2 59.01.9

Mean 165.4 45.3 53.3 42.4

44

Table 5. Sugar contents of seven sorghum cultivars.

Sorghum variety

Total non-starch

carbohydrate

(mg/g)

Reducing sugar

(mg maltose/g)

Non-reducing sugar

(mg sucrose/g)

Glucose

(mg/g)

Fructose

(mg/g)

DC-75 10.51.2 4.50.4 6.00.0 5.7 3.9

Brown Tsweta 6.50.8 4.90.0 6.70.0 4.7 1.4

Red Swazi 4.60.2 3.60.0 6.90.2 0.1 1.4

Pato 15.20.8 3.10.0 8.20.1 11.2 3.2

Pirira 8.90.4 2.80.3 6.50.3 5.7 1.4

PL 1 11.40.5 2.50.0 11.40.0 1.2 4.5

TX 2737 6.51.6 2.50.0 9.50.0 3.9 7.1

Mean 9.1 3.4 7.9 4.8 3.2

45

Table 6. Sugar contents of seven malted sorghum cultivars.

Sorghum variety

Total non-starch

carbohydrate

(mg/g)

Reducing sugar

(mg maltose/g)

Non-reducing sugar

(mg sucrose/g)

Glucose

(mg/g)

Fructose

(mg/g)

DC-75 354.313.9 6.70.2 11.50.1 270.0 73.3

Brown Tsweta 211.024.1 5.90.3 10.40.0 150.9 33.7

Red Swazi 277.20.2 6.40.1 10.10.3 155.8 49.8

Pato 260.88.5 6.80.3 7.90.75 195.5 15.1

Pirira 157.811.6 5.60.0 6.20.0 170.8 18.5

PL 1 209.13.7 14.50.0 21.60.0 114.1 28.5

TX 2737 194.44.9 15.00.1 22.30.3 91.9 28.3

Mean 237.8 8.7 12.9 14.4 35.3

46

020

40

60

80

100

120

140

160

180

200

220

240

260

280

300

25 35 45 55 65 75Incubation Temperature (C)

Cer

alph

a U

nits

/g

PLTXDC-75nRed BrownPato Pirira

Figure 1. Effect of temperature on sorghum -amylase activity.

47

05

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

25 30 35 40 45 50

Incubation Temperature (C)

Bet

amyl

Uni

t/g

PLTXDC-75RedBrownPatoPirira

Figure 2. Effect of temperature on sorghum -amylase activity.

48

CHAPTER 3

QUANTITATIVE ANALYSIS OF STARCH AND

OTHER COMPONENTS IN THE PRODUCTION

OF OPAQUE BEER

3.1 ABSTRACT

The changes in starch and other related components at each step of opaque beer

production were monitored in this study and the effect of amylose in this brewing

process was also investigated. For the sorghum set, starch concentration decreased

from 38.0 mg/ml to 33.0 mg/ml after souring. With the addition of sorghum grain as

adjunct, starch concentration increased to 35.9 mg/ml after cooking, but then

decreased sharply to 11.0 mg/ml after mashing due to the amylolytic action from the

sorghum malt. As straining only involved the physical removal of coarser particles,

the relative starch concentration increased to 18.5 mg/ml. Starch concentration

decreased further to 14.5 mg/ml after fermentation in the end product. The decrease

in starch concentration during souring, mashing and fermentation was attributed to

starch hydrolysis by amylolytic action. Total non-starch carbohydrate increased

accordingly for each case, but for glucose, the concentration decreased after

fermentation. This was due to glucose utilization by the yeast inoculum. Sorghum

adjunct was substituted with maize starch with different amylose contents and its

effect in the brewing process was investigated. Hi-maize starch was comparatively

resistant to amylolytic attack, it showed the lowest decrease in starch after mashing,

followed by normal maize starch and waxy maize starch.

49

3.2 INTRODUCTION

The opaque sorghum beer industry in southern Africa is of major significance as an

industrial user of sorghum. Tribal brewing evolved into commercial production of

opaque beer. In South Africa, the brewing of Bantu beer has become industrialized,

being the first and only industry based on an African tribal art (Novellie 1968).

Opaque beer production involves souring, cooking, mashing, straining and

fermentation. Sorghum grain was the major raw material for the brewing of sorghum

opaque beer. It is usually used directly as adjunct or germinated to produce malt for

the brewing. Sorghum grain contains 60-70% starch and its malt contains 45-60%

(Daiber and Taylor 1995). Gelatinization and starch degradation were the two major

modifications taking place during the brewing process. Starch is involved in both

processes and it plays an important role in the quality of the beer. Similar research

(Bvochora and Zvauya 2001) has been done to investigate the changes in lactic acid,

ethanol, free amino nitrogen and organic acids along the production flow, but not

starch. Therefore, a better understanding of starch degradation during opaque beer

production can help the brewer to improve the quality of the beer.

In the present study, the aims were 1) to investigate the changes in starch and other

related components along the production flow of opaque beer processing and 2) to

examine the effect of amylose on beer production by substituting sorghum adjunct

with Hi-maize starch, normal maize starch and waxy maize starch respectively.

50

3.3 MATERIALS AND METHODS

Sorghum Grains

Sorghum (DC-75) was supplied by Dr. C. E. Walker Department of Grain Science

and Industry, Kansas State University.

Maize Starches

Hi-maize starch, normal maize starch and waxy maize starch were supplied by

Starch Australasia Limited (Australia).

Lactobacillus Culture of Lactobacillus delbrueckii subsp. lactis (ATCC Number 4797), type strain

of Lactobacillus leichmannii, was purchased from the American Type Culture

Collection (ATCC).

Preparation of Sorghum Malt

Same preparation procedures as in chapter 2.

Preparation of Sorghum Adjunct

Milling

Sorghum adjunct was prepared by grinding the sorghum grains in Udy Cyclone

sample mill (Udy Corporation, Ft. Collins, Colorado, USA) fitted with a 0.5 mm

sieve.

Brewing

Souring

Pure culture of Lactobacillus delbrueckii was inoculated to 2.8 g sorghum malt and

25 ml distilled water in a test tube, mixed and incubated at 48C for 18 h.

51

Cooking

20 g adjunct (sorghum adjunct, Hi-maize starch, normal maize starch and waxy

maize starch respectively) and 168 ml water were added to the sour in an

Erlenmeyer flask, mixed and cooked with continuous stirring for 120 min.

Mashing

The mixture was then cooled. Sorghum malt (6.2 g) and distilled water (13 ml) were

added to the flask and incubated at 60C for 120 min.

Straining

The mash was passed through a 250-m metal sieve to remove the spent grain.

Fermentation

The wort was cooled to 28C and active dried yeast (0.055 g) was added and fermented for 48 h at 28C.

pH

pH was determined using a pH meter with a glass electrode after calibrating with

commercial buffer (Merck) (pH 4 and 7).

Total Starch Content

Total starch content was determined by the amyloglucosidase / -amylase method using the Megazyme Total Starch Assay Kit (Megazyme, Bray, Ireland).

Amylose Content Assay

Amylose was determined using an iodine-binding spectrophotometric method.

Sample (50 mg) was weighed to the bottom of 50 ml volumetric flask. 95% Ethanol

(1 ml) and 1 M sodium hydroxide (4.5 ml) were added respectively and mixed well.

The samples were kept at 45C in the oven overnight. Distilled water (25 ml) was added and the samples were then kept at 100C in the oven for 60 minutes for starch gelatinization. They were then cooled down to room temperature and made up to

volume. The solution was centrifuged at 3,000 rpm for 10 min so as to remove the

52

coarse particles. An aliquot (1 ml) was transferred into a 25 ml volumetric flask, to

which 300 l 1 M acetic acid and 400 l I2-KI solution were added, made up to volume and mixed well. The solution was kept at room temperature for 20 min.

Absorbance value was read at 620 nm against blank. Amylose content was

determined according to a standard curve which was prepared by maize amylose /

amylopectin (Sigma) standard mixtures representing 0 to 70 % amylose.

Total non-starch carbohydrate

Sample was prepared according to step 1 to 4 of the pretreatment of samples

containing glucose and maltosaccharides of Megazyme Total Starch Assay Kit. The

supernatants of the sorghum adjunct and malt were made to a total volume of 50 ml

and 500 ml respectively and they were estimated for their total non-starch

carbohydrate by the phenol-sulphuric acid method (Dubois et al 1956).

-amylase activity Sorghum -amylase activity was determined by Ceralpha method using the Megazyme -amylase assay procedure. The tests were run at 55C.

-amylase activity Sorghum -amylase activity was determined by Betamyl method using the Megazyme -amylase assay procedure. The tests were run at 39C.

Glucose and fructose

Glucose and fructose were analyzed by High Performance Liquid Chromatography

(HPLC). They were extracted by weighing 0.5 ml sample in 1.5 ml Eppendorf tube.

Distilled water (0.5 ml) was added, vortexed and rotated overnight with a rotator. It

was centrifuged at 10,000x g for 5 min. The supernatant was retained by decanting

carefully to avoid disturbing the sedimented pellet which was discarded. The

polymers present in the supernatant were precipitated by adding 4 ml 95% ethanol to

1 ml supernatant. It was left overnight and centrifuged at 10,000 x g for 5 min. The

53