CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

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

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 postfermentation of opaque beer by using sorghum and maize starch with
different amylose contents as adjunct.

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 droughttolerant 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
3

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).

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.

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.

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

(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).

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).

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).

10

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

11

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.
12

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

13

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, rodshaped 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
14

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
15

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

16

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 endproducts 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 cometabolism 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

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

0.065-0.115

0.084

Volatile acidity as g acetic acid / 100

ml

Nitrogen (%)
Sources: Novellie (1968).

24

Table 2. Nutrient Content of Industrially Brewed Opaque Beer a

Nutrient

Mean

RDAb

Content

% Contribution to
RDA by 1 L of
Beer

Alcohol (g)

25.4

NRc

Fat (g)

Trace

96d

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

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

<3,000

0.3

218

800

27.3e

Crude Fiber (g)

Sodium (mg)
Phosphorus (mg)
a

Adapted from Van Heerden (1989b)

RDA = Recommended dietary allowance.

NR = no recommendation.

Suggested daily intake.

Significant contribution to RDA (>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 nonstarch 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 8060 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

82.4

16.7

73.3

71.7

Mean (Total)

GA = Germination Activity

dml = dry matter loss

Malt GT = Gelatinization Temperature of Malt

Adjunct GT = Gelatinization Temperature of Adjunct

41

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

Sorghum

Decrease in

variety

Total Starch (% db)

Total Starch (% db)

Adjunct

Malt

starch %

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.

Decrease in
amylose % of

Sorghum
variety

% amylose of starch (db)

% amylose of starch (db)

Adjunct

Malt

starch

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.

Alpha-amylase

Beta-amylase

Diastatic power

Reduction in viscosity

(Ceralpha Unit/g)

(Betamyl Unit/g)

(SDU)

(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

165.4

45.3

53.3

42.4

Sorghum variety

Mean

44

Table 5. Sugar contents of seven sorghum cultivars.

Total non-starch
carbohydrate

Reducing sugar

Non-reducing sugar

Glucose

Fructose

(mg/g)

(mg maltose/g)

(mg sucrose/g)

(mg/g)

(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

9.1

3.4

7.9

4.8

3.2

Sorghum variety

Mean

45

Table 6. Sugar contents of seven malted sorghum cultivars.

Total non-starch
carbohydrate

Reducing sugar

Non-reducing sugar

Glucose

Fructose

(mg/g)

(mg maltose/g)

(mg sucrose/g)

(mg/g)

(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

237.8

8.7

12.9

14.4

35.3

Sorghum variety

Mean

46

PL
TX
DC-75n
Red
Brown
Pato
Pirira

300
280
260
240
220
200

Ceralpha Units/g

180
160
140
120
100
80
60
40
20
0
25

35

45
55
65
Incubation Temperature ( C)

75

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

47

80
75

PL
TX

70

DC-75
Red

65

Brown
Pato
Pirira

60
55
50

Betamyl Unit/g

45
40
35
30
25
20
15
10
5
0
25

30

35

40

45

50

Incubation Temperature (C)

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

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.
Acetic acid

Acetic acid was determined by Gas Chromatography (GC). Sample (0.5 ml) was
mixed with acetone (0.5 ml) by vortex and the mixture was centrifuged for 10 min at
14, 000 rpm. It was then filtered through Whatman 0.2 m polyethersulfone
membrane with polypropylene housing (Puradisc 25 AS). The supernatant was used
for gas chromatographic analysis using a HP-6890 GC (Hewlett-Packard, Palo Alto,
CA), equipped with a flame ionization detector and a HP-FFAP (Crosslinked FFAP)
capillary column (15 m x 0.25 mm x 0.25 m) (Agilent Technologies, 19091F-431).
The column temperature started from 60C and increased to 200C at a rate of 10C
per min. The detector temperature was kept at 260C. Nitrogen was used as the
carrier gas and the flow rate was kept at 2.0 ml per min. The injector was kept at

54

250C with an injection volume of 2.0 L under splitless injection mode. Acetic acid
concentration was determined according to standard curve prepared by acetic acid
standard mixtures representing 0 to 0.15% acetic acid.

3.4 RESULTS AND DISCUSSION

Souring
pH decreased significantly from 4.97 to 3.38 (Figure 1) after souring, most likely
due to the lactic acid produced by the lactobacillus. Lactic acid is responsible for
imparting the beer with its characteristic taste and serves as background for
distinguishing other flavors (Bvochora and Zvauya 2001). Acetic acid (Figure 2)
might also be another factor contributing to pH decrease. Since the optimum pH
ranges for - and -amylases are 4.5 to 5.0 (Botes et al 1967a) and 5.2 to 5.5 (Botes
et al 1967b) respectively, this decrease in pH was therefore unfavorable for their
activities and might lead to the virtually zero amylase activities at the end of the
souring process.
After souring, starch decreased from 40.0 mg/ml to 32.0 mg/ml (Figure 5),
representing a decrease of 17.5% whereas amylose decreased from 8.1 mg/ml to 7.9
mg/ml (Figure 6), representing a decrease of 3.0%. The lower decrease in amylose
than starch might be due to the presence of amylose-lipid complex which inhibited
the hydrolytic action of amylases. The decrease in starch and amylose concentration
together with the increase in total non-starch carbohydrate marked the action of
amylases.
There was a general increase in total non-starch carbohydrate, from 67.9 mg/ml to
234.7 mg/ml (Figure 7). Among the sugars, there was a net increase of glucose from
15.6 mg/ml to 34.7 mg/ml (Figure 8), but a net decrease of fructose from 3.8 mg/ml
to 1.7 mg/ml (Figure 9). The increase in glucose was brought about by starch
hydrolysis during souring by the diastatic enzymes present in the sorghum malt

55

while its decrease was due to utilization by lactic acid bacteria and various
microorganisms. Homofermentative lactic acid bacteria like Lactobacillus
delbrueckii inoculated for this souring process can ferment hexoses via glycolysis,
with 90% of the glucose being metabolized to lactic acid (Kontula et al 1998). The
net increase in glucose represented a greater rate of saccharification than utilization,
indicating that the sour provided more than enough glucose for metabolic activities
of micro-organisms. On the other hand, as fructose is not a product resulting from
starch hydrolysis, therefore only utilization was carried out by the microflora but no
production, thus it was found to decrease after 2-d souring. According to the data,
glucose and fructose were just part of the total non-starch carbohydrate, indicating
the presence of other sugars in the sour, which could possibly be sugars like dextrins,
maltotriose or maltose, depending on the relative location of the bond under attack
during the hydrolytic action.
Cooking

Using sorghum grains as adjunct

After souring, adjunct was added and cooked. This aimed at supplementing the
expensive malt with a cheaper source of starch which would undergo gelatinization
at the cooking stage, thus making it readily hydrolysable in the next step, mashing.
Gelatinized starch is also important for holding the ungelatinized starch in
suspension, giving the beer its creamy texture and characteristic viscous body
(Novellie and de Schaepdrijver 1986). Due to the addition of adjunct, starch
increased from 33.0 mg/ml to 36.0 mg/ml (Figure 5) while amylose increased from
8.1 mg/ml to 27.1 mg/ml (Figure 6). The extent of increase depends on the type and
cultivars of adjunct added. As adjunct contains a low content of sugar, this led to a
decrease in total non-starch carbohydrate concentration from 234.7 mg/ml to 45.7
mg/ml (Figure 7). Glucose concentration dropped from 34.7 mg/ml to 8.5 mg/ml
(Figure 8) while fructose concentration dropped from 1.7 mg/ml to 0.5 mg/ml
(Figure 9). The decrease in sugar concentration of the cooked sour was chiefly due
to the intrinsically low level of sorghum adjunct as recorded in the previous chapter.

56

The optimum incubation temperatures of - and -amylase were approximately

55C and 39C as recorded in the previous chapter while the optimum pH values
were recorded to be 4.5 to 5.0 (Botes et al 1967a) and 5.2 to 5.5 (Botes et al 1967b)
respectively. As the cooking temperature was far too high for their activities, thus in
spite of an increase in pH from 3.38 back to 4.68 (Figure 1), which was much closer
to their optimum pH ranges than in the sour, a decrease in amylase activities to
virtually zero after cooking was recorded, indicating denaturation by such a high
temperature. This is a reason why cooking and mashing cannot be carried out
simultaneously during opaque beer production because of the high gelatinization
temperature of sorghum adjunct.

Comparison between using maize starches and sorghum as adjunct

When adjuncts (maize starch) of different amylose percentage were substituted for
cooking, some of the changes were significantly different from the one by using
sorghum as the adjunct. For starch, sorghum set reached a concentration of 35.9
mg/ml after cooking while for the maize starch sets, they reached a concentration
ranging from 46.2 mg/ml to 47.6 mg/ml. The increase was more significant in maize
starch sets than in the sorghum set (Figure 5). As the maize starch had a much higher
starch content than sorghum grains, therefore, starch concentration recorded in the
sorghum set was apparently lower than the maize starch sets.
The most obvious difference among the four sets was amylose concentration. Starch
concentration increased in all four sets, but to various extents depending on the
original amylose content, with Hi-maize set having the highest amylose
concentration (52.1 mg/ml), followed by normal maize (33.2 mg/ml), sorghum (27.3
mg/ml) and waxy maize starch (20.9 mg/ml) as shown in Figure 6. Amylose plays a
critical role in starch gelatinization, the main modification taking place during
cooking. It was reported that gelatinization temperature increased with increasing
amounts of amylose (Knutson 1990, Sievert and Wuesch 1993, Gerard et al 1999).
Waxy and regular maize starches gelatinized from 62 to 72C, while for highamylose starch, granules began to swell below 100C, but a temperature above

57

130C was needed to fully disperse these starches (Shi et al 1998).

Several factors were proposed for the high resistance of starch granules to
gelatinization. 1) Amount of amylose-lipid complex. The enclosed lipid molecules
made the amylose helix conformation more stable, thus preventing the granule from
swelling. Amylose double helices, which disassociate only at high temperature,
could also be another factor retarding swelling of starch granules (Shi et al 1998). 2)
Size of polymer. As more energy is required to break smaller polymers (amylose) as
compared to bigger ones (amylopectin) (Aggarwal and Dollimore 1998, 2000), Himaize starch is more resistant to gelatinization. Longer branch lengths found in highamylose starches were also proposed to be responsible for its high gelatinization
temperature (Gidley 2001). 3) Degree of crystallinity. Properties of starch like
gelatinization, swelling, pasting behavior and functionality are affected by the
degree of crystallinity (Lorenz and Kulp 1981). Therefore, during the 2-h cooking,
high-amylose starch granules were more resistant to gelatinization than the other
types used and it was expected to have the lowest amount of gelatinized starch in the
wort.
The pH value of sorghum set was 4.7, this was much higher than that measured in
the maize starch sets, which was 3.8, as shown in Figure 1. This indicated that the
sorghum grain intrinsically had a higher pH than the maize starch. No significant
difference among the four sets was observed for sugar and amylase activities
(Figures 3, 4, 7, 8 and 9) as adjunct generally has low sugar content and virtually
zero amylase activity after being denatured.
Mashing
Using sorghum grains as adjunct
During mashing, more sorghum malt was added to facilitate starch hydrolysis so as
to produce fermentable wort for yeast. Starch decreased from 35.9 mg/ml to 11.0
mg/ml, representing a decrease of 69.3% (Figure 5) while amylose decreased from
27.3 mg/ml to 8.0 mg/ml (Figure 6), representing a decrease of 70.6%. When

58

comparing the extent of starch hydrolysis after souring and mashing, it is worth
noting that decreases in starch and amylose were 17.5% and 1.5% after souring,
which were lower than that found after mashing. This could be due to the effect
brought about by starch gelatinization which is essential in the production of opaque
beer so as to make the adjunct readily hydrolysable during mashing by the malt
diastatic enzymes. It is known that native granules are degraded very slowly by
amylases because granules are very resistant to amylolytic digestion (Hyun and
Zekius 1985, Ring et al 1988, Gallant et al 1992) and that starch gelatinization
increases susceptibility of starch granules to amylases (Holm et al 1985, Asp and
Bjrck 1992, Bjrck and Asp 1994, Bjrck 1996, Yue and Waring 1998). During
gelatinization, amylose leached out from the starch granules, leading to breakdown
of crystalline order of starch, thus crystallinity makes starch more susceptible to
enzyme attack. Therefore, by gelatinizing the starch in the unmalted cereal adjunct,
this can render it more hydrolyzable during mashing by the malt diastatic enzyme.
As a result of starch hydrolysis by the amylases introduced by the sorghum malt,
total non-starch carbohydrate increased markedly from 45.7 mg/ml to 86.5 mg/ml
(Figure 7), representing an increase of 89.2%. Glucose increased from 8.5 mg/ml to
30.1 mg/ml (Figure 8), contributing 34.8% of the total non-starch carbohydrate in
the wort whereas fructose increased from 0.5 mg/ml to 4.0 mg/ml (Figure 9),
contributing 4.6% of the total non-starch carbohydrate in the wort. This indicated
that glucose represented a significant proportion of sugar in the wort, which would
serve as a source of energy for yeast in later part of the brewing process. The
increase in glucose concentration could be traced to the hydrolytic action of
amylases and the glucose originally present in the sorghum malt. The increase in
fructose concentration could be partly brought about by the fructose originally
present in the sorghum malt or by glucose conversion, but was not a product of the
hydrolytic enzyme.
Though extra sorghum malt was added, - and -amylase activities were still found
low by the end of 2-h mashing (Figures 3 and 4) when compared with the initial

59

amylase activities in the sorghum malt. Thus it appeared that the amylases were
exhausted after exerting their effects at the end of mashing.
The pH value of the mash (pH 4.82) was found to be higher than that (pH 3.9)
reported elsewhere (Briggs 1998a). This could be due to the difference in treatment
during malt preparation. In this study, sorghum grains were steeped in NaOH (0.3%,
w/v) for 8 h before germination. This was likely to be the reason for the higher pH
value.

Comparison between using maize starches and sorghum as adjunct

As observed in the sorghum set, starch concentration also decreased for all the maize
starch sets, but to varying extents depending on the amylose content of the adjunct.
Hi-maize starch was most resistant to starch degradation with a decrease of 13.7%,
followed by normal maize starch (25.3%) and waxy maize starch (52.5%). It
appeared that the higher the amylose content, the higher the resistance to starch
hydrolysis, as noted by the final starch concentration in the mash (Figure 5).
Amylose in all four sets decreased (Figure 6), indicating the hydrolysis of amylose
by enzyme from the malt. The final amylose concentration of the mash was in
accordance with the original amylose content of the adjunct, with Hi-maize starch
having the highest amylose concentration in the mash, followed by normal maize,
sorghum and waxy maize. Amylose contains small chains with no branch points and
is situated in the amorphous region, thus making it susceptible to enzyme hydrolysis,
resulting in its decrease in concentration.
All four sets had similar amylase activity as shown in Figures 3 and 4. Both - and
-amylase activities increased slightly after mashing, yet their activities were
relatively low when compared with the original sorghum malt amylase activities.
This indicated the exhaustion of amylase after 2-h mashing.
Variation was observed among the four sets in terms of total non-starch carbohydrate

60

concentration (Figure 7). All four sets experienced an increase in total non-starch
carbohydrate after mashing but to different extents according to their amylose and
starch contents. This increase could be partly contributed by the introduction of malt
and partly by the action of amylases present in the malt, resulting in a range of
products such as glucose, maltose and limited dextrin. Waxy maize had the highest
increase in total non-starch carbohydrate, followed by sorghum and normal maize
starch both of which had a very similar increase, while for Hi-maize, its increase in
total non-starch carbohydrate was the least. This agreed with the data as observed in
changes in starch after mashing in which Hi-maize was found with the lowest starch
hydrolysis among the four, followed by normal maize and waxy maize, thus
resulting in a lowest total non-starch carbohydrate concentration. But for sorghum,
as it had the lowest concentration of starch in the cooked sour, even its amylose
content was lower than normal maize starch, this would result in similar total nonstarch carbohydrate concentration as for the normal maize set instead of higher.
No significant difference was observed for glucose and fructose concentrations for
the four sets. In starch hydrolysis, no fructose was expected to form. Therefore, even
though fructose was found to increase after mashing (Figure 9), this was probably
contributed by the initial presence of fructose in the malt added for the sake of
mashing and/or conversion of glucose instead of being a product from starch
hydrolysis. For glucose, its concentration increased for all the four sets (Figure 8),
this was partly introduced by the addition of extra sorghum malt and partly from the
product of starch hydrolysis.
Concerning the difference in starch degradation, it was reported that the amount of
amylose in the granule does not seem to affect the rate of enzymic attack (Kimura
and Robty 1995). The difference in degree of degradation for starch granules with
various amylose contents could be explained in terms of 1) Granular size and surface
area. Initial step of -amylolysis involved the adsorption of -amylase on the
granule surface. After gelatinization, the starch granules became swollen and offered
a larger granular size than the intact ones and thus facilitated such adsorption. 2)

61

Degree of gelatinization. It has been widely known that native granules are degraded
very slowly by amylases because they are very resistant to amylolytic digestion
(Hyun and Zekius 1985, Ring et al 1988, Gallant et al 1992) but starch gelatinization
can increase susceptibility of starch granules to amylases (Holm et al 1985, Asp and
Bjrck 1992, Bjrck and Asp 1994, Bjrck 1996, Yue and Waring 1998). Therefore,
the greater the extent of starch being gelatinized, the greater the disruption of
crystallinity which in turn leading to a greater disruption of hydrogen bond of double
helices and hence, offering more active sites for enzyme attack. Moreover,
gelatinization might form cracks on the surface of starch granule, thus facilitating
the entry of enzyme to the interior part of the granule. 3) Amount of amylose-lipid
complex. The existence of complexes between amylose and the endogenous polar
lipids of cereal starch granules (Briggs 1998b) contributed stability to amylose
polymers and inhibited the amylose from being hydrolyzed by blocking the active
sites of amylose for amylase attack and/or blocking the channels leading to the
interior part of the starch granule. The amount of amylose-lipid complex depends on
genetic factors. The variation of decrease in starch and increase in total non-starch
carbohydrate concentration in the mash of maize starch sets implied that Hi-maize
starch had more amylose-lipid complex than the normal maize and waxy maize
starch.
Straining

Using sorghum grains as adjunct

Straining only involves the physical removal of coarser particles such as husks,
pericarp and plumules of diameter larger than 250-m by passing through a sieve, so
as to give a smooth mouth feel for the beer. In general, there was no significant
change after mashing with the exception of starch which increased from 11.0 mg/ml
to 18.5 mg/ml after straining (Figure 5). This was because the wort initially
contained a significant proportion of spent grains originated from sorghum malt and
sorghum adjunct. Once these were removed, starch, most of which was able to pass
through the sieve and remained in the wort, would then have a relatively higher
concentration. Though the starch had a relatively higher concentration after straining,

62

indicating that most of the starch was able to pass through the sieve, part of it might
still have been lost. An analysis of spent grains from various breweries showed that
the residual starch ranged from 15% to as high as 50% (Von Holdt and Brand 1960).
Glucose and fructose concentrations remained more or less the same (Figures 8 and
9), indicating that all glucose and fructose molecules had passed through the sieve
and formed part of the wort. Total non-starch carbohydrate concentration decreased
slightly (Figure 7), showing that part of the sugar, other than glucose and fructose,
was screened and lost with the spent grains.

Comparison between using maize starches and sorghum as adjunct

As shown in Figure 5, starch concentration showed a tendency to drop in the maize
starch sets, representing a loss in starch together with the spent grains removed,
while an increase in starch concentration was recorded in the sorghum set because
sorghum grain contained a higher proportion of spent grain than the maize starch
sets, after removal of spent grain the starch concentration proportionately increased.
No significant difference and change in amylose concentration was observed after
mashing and straining for the four sets (Figure 6), with Hi-maize having the highest
amylose concentration in the wort followed by normal maize, sorghum grain and
waxy maize, showing that majority of the amylose had passed through the sieve and
formed part of the wort which had an amylose concentration that varied according to
amylose % of adjunct.
No significant difference and change was observed for glucose and fructose
concentrations (Figures 8 and 9) as both were of molecular size small enough to pass
through the screen. Total non-starch carbohydrate concentration showed slight
decrease (Figure 7), implying that part of the sugar other than glucose and fructose
was screened and lost together with the spent grain. Amylase activities in all sets
remained low (Figures 3 and 4), with the sorghum set having slightly higher
activities than the maize starch sets.

63

Fermentation
Using sorghum grains as adjunct
During fermentation, the wort was cooled and inoculated with yeast Saccharomyces
cereviseae. It utilized the fermentable sugar in the wort, giving ethanol and carbon
dioxide in return. Yeasts can only grow on a limited range of carbohydrates, known
as fermentable sugars. Mbugua et al (1983) reported that the fermentable sugars in
maize and sorghum during fermentation included fructose, glucose, sucrose,
maltotriose and raffinose. Fructose and glucose are among the fermentable sugars
utilized by yeast. The glucose concentration decreased from 29.2 mg/ml to 22.9
mg/ml (Figure 8), contributing 28.2% of total non-starch carbohydrate while
fructose concentration decreased from 3.8 mg/ml to 2.2 mg/ml (Figure 9),
contributing 2.8% of total non-starch carbohydrate, which was approximately ten
times less than glucose. This indicated that fermentation was active. It should be
noted that glucose might be continuously produced by residual amylases. Under this
circumstance, the net decrease in glucose indicated that the rate of utilization of
glucose by microorganisms was greater than that of saccharification. Total nonstarch carbohydrate increased from 72.1 mg/ml to 81.1 mg/ml (Figure 7). Such
increase was most likely to be due to the accumulation of sugar from starch
hydrolysis, which could either be fermentable such as glucose and fructose or nonfermentable such as dextrins.
Starch decreased from 18.5 mg/ml to 14.5 mg/ml (Figure 5), representing a decrease
of 21.6%, indicating that the residual - and -amylases kept on exerting their effect
on starch during this 2-d fermentation period. By the time amylose reached the
fermentation step, its content was already quite low, only 5.0 mg/ml with no
significant further decrease (Figure 6). Acetic acid increased (Figure 2) by 19.7%
after 2-d fermentation, possibly a metabolic product from the microflora yet there
was no significant change in pH value (Figure 1).

Comparison between using maize starches and sorghum as adjunct

Starch concentration tended to decrease after 2-d fermentation (Figure 5), but to

64

different extents for the four sets. In fact, the starch concentrations for Hi-maize and
normal maize starch sets showed insignificant decrease only, with a final starch
concentration of 38.0 mg/ml in Hi-maize starch set and 30.6 mg/ml in normal maize
starch set. This was probably due to the presence of amylose-lipid complex which
prevented the amylose from enzyme hydrolysis. On the other hand, waxy maize
starch and sorghum sets showed more significant decrease, indicating the effect of
residual amylase activities resulting from the malt added for mashing. The lowest
starch concentration of sorghum set among the four sets could be a result of the low
initial starch content of the sorghum adjunct added when compared with maize
starch. For amylose, the four sets showed insignificant decrease (Figure 6). It
seemed that fermentation did not exert much effect on amylose. This could be due to
the presence of amylose-lipid complex and its V-complex formation which resisted
the action from amylases.
During fermentation, yeast took in glucose as energy source for metabolism and at
the same time, amylase kept on hydrolyzing starch and its components, forming
various fermentable sugars for yeast growth and metabolism. Glucose concentration
was found to have a net increase in the maize starch sets but a net decrease in
sorghum set (Figure 8). The increase was resulted from starch degradation due to the
residual amylase activities and the decrease was resulted from utilization by yeast
and other microorganisms. Net increase was observed for the maize starch sets,
which implied that the utilization of glucose was low, this was probably due to the
lack of amino nitrogen in maize starch adjunct, which was essential for yeast growth.
On the other hand, the net decrease in glucose found in the sorghum set implied that
the rate of utilization was greater than the rate of saccharification as sorghum adjunct
contained plenty of amino nitrogen which favored yeast growth, resulting in
consumption of more glucose. Only the sorghum set showed a significant decrease
in fructose concentration but no such phenomenon was observed in the maize starch
sets. Similar to glucose, this showed the sign of utilization by yeast and other
microorganisms since glucose and fructose both contributed part of the fermentable
sugars.

65

3.5 CONCLUSION
Souring, mashing and fermentation are the steps involving starch hydrolysis in the
production of opaque beer. They are important in providing the yeast with sufficient
fermentable sugar in the wort during fermentation as yeast is not able to utilize
starch polymers. Starch degradation during the production of opaque beer was
greatly affected by amylose content. Its effect was most obvious during mashing
where more sorghum malt was added for exerting amylolytic attack. Starch
degradation was least obvious in Hi-maize, followed by normal maize starch and
waxy maize starch. This could be attributed to the surface area available for
enzymatic action, degree of gelatinization and the presence of amylose-lipid
complex in the system.

66

5.5

5.0

pH

4.5

4.0

Sorghum
Hi-maize
Normal
Waxy

3.5

3.0
Before sour After sour

Cook

Mash

Strain

Ferment

Fig. 1. Changes in pH in opaque beer production

0.014
0.012

Acetic acid %

0.010
0.008
0.006
0.004

Sorghum
Himaize
Normal
Waxy

0.002
0.000
Before sour After sour

Cook

Mash

Strain

Ferment

Fig. 2. Changes in acetic acid during opaque beer production

67

Sorghum
Hi-maize
Normal
Waxy

Alpha-amylase activity (CU/ml)

0
Before sour

After sour
Cook
Mash
Strain
Ferment
Fig. 3. Changes in alpha-amylase activity in opaque beer production.

0.14
Sorghum
Hi-maize
Normal
Waxy

Beta-amylase (BU/ml)

0.12

0.10

0.08

0.06

0.04

0.02

0.00
Before sour

After sour
Cook
Mash
Strain
Ferment
Fig. 4. Changes in beta-amylase activity in opaque beer production.

68

Sorghum
Hi-maize
Normal
Waxy

55
50
45
40

Starch (mg/ml)

35
30
25
20
15
10
5
0
Before sour
After sour
Cook
Mash
Strain
Ferment
Fig. 5. Changes in starch concentration during opaque beer production.
60
55

Sorghum
Hi-maize
Normal
Waxy

50
45

Amylose (mg/ml)

40
35
30
25
20
15
10
5
0
Before sour

After sour

Cook

Mash

Strain

Ferment

Fig. 6. Changes in amylose concentration in opaque beer production.

69

Sorghum
Hi-maize
Normal
Waxy

250

Total sugar (mg/ml)

200
150
100
50
0
Before sour
40

After sour
Cook
Mash
Strain
Fig. 7. Changes in total sugar in opaque beer production.

Ferment

35

Glucose (mg/ml)

30
25
20
15
10
5
0
Before sour
After sour
Cook
Mash
Strain
Ferment
Fig. 8. Changes in glucose concentration in opaque beer production.
4.0

Fructose (mg/ml)

3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Before sour
After sour
Cook
Mash
Strain
Ferment
Fig. 9. Changes in fructose concentration in opaque beer production.

70

CHAPTER 4
QUALITATIVE ANALYSIS OF STARCH
DEGRADATION IN THE PRODUCTION OF
OPAQUE BEER BY SCANNING ELECTRON
MICROSPCOPE
4.1 ABSTRACT
Qualitative changes at each step of production were analyzed. Starch granules
became gelatinized and swelled after cooking, leading to an increase in granule size.
This provided a larger surface area for enzyme to adsorb on the starch surface for
attack, thus facilitating the next step, mashing, where starch hydrolysis was involved.
The effect of amylose on the on-going process of production was investigated.
Starch granules became pitted, revealing layers in the internal region. Unlike
sorghum starch granules, Hi-maize was not much affected by cooking. After cooking,
waxy maize starch granules showed a diameter as large as 34.5 m, followed by
normal maize (13 - 18 m) and Hi-maize starch (5 - 9 m). Several factors were
proposed to be the cause of this, including the amount of amylose-lipid complex in
the system, size of polymer and disruption of crystallinity. This difference in degree
of gelatinization would then affect the subsequent steps of the brewing process, thus
contributing to the final quality of the beer. The order of extent of starch degradation
after mashing followed that of the degree of gelatinization. This was possibly due to
the surface area available for enzyme attack, disruption of crystallinity and the
amount of amylose-lipid complex present. No further significant modification in
starch granule was observed in straining and fermentation.

71

4.2 INTRODUCTION
Starch is an important component in determining the final quality of opaque beer as
it affects factors like degree of gelatinization, the amount of fermentable sugar
available for yeast and thus the ethanol content, the creaminess as well as the
sweetness of the beer. Much research has been done with scanning electron
microscopy (SEM) to investigate gelatinization of starch (e.g. Liu and Zhao 1990)
and starch hydrolysis (e.g. Aggarwal and Dollimore 1998, 2000) respectively, but
their combined effect was seldom analyzed. In this study, the on-going changes in
sorghum starch granules and the effect of amylose on starch degradation during the
production of opaque beer were examined.

4.3 MATERIALS AND METHODS

Sorghum Grain
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 provided by
Starch Australasia Limited.

Lactobacillus
Culture of Lactobacillus delbrueckii subsp. lactis (ATCC Number 4797), type strain
of Lactobacillus leichmannii, was purchased from the American Type Culture
Collection (ATCC).
Starch extraction
Starch was extracted following the methods used by Watson et al (1955), Zhao and
Whistler (1994), Wu et al (1995) and Beta and Corke (2001).

72

Sorghum grain (100 g) was steeped at 5C for 24 h in NaOH (0.25%, w/v). The
steeped grains were washed and ground with water (1:1, w/v) in a Waring blender.
The slurry was filtered through a 200-mesh sieve (75 m screen). The material
remaining on the sieve was then rinsed with water and subjected to repeated
grinding and filtering processes before it was discarded. The filtrate was centrifuged
at 760 x g for 10 min, and the gray-colored protein layer that formed on top was
removed. Excess water was added to resuspend the sample and centrifugation was
carried for 3 min. The process was repeated until the top starch layer was white. The
starch was dried for 24 h at 40C.
Preparation of Sorghum Malt
Same preparation procedures as in chapter 2.
Preparation of Sorghum Adjunct
Same preparation procedures as in chapter 3.
Brewing
Same preparation procedures as in chapter 3.
Scanning Electron Microscope (SEM)
Samples collected were freeze-dried and attached to metal stubs and sputter coated
with gold-palladium with a thickness of 10 to 20 nm. Stubs were examined in a BAL
TEC SCD 005 scanning electron microscope.

73

4.4 RESULTS AND DISCUSSION

Extracted Sorghum Starch Granules
Before extraction, the starch granules from adjunct (Figure 1) were covered with a
thin layer of protein which was removed by centrifugation followed by scraping
after starch extraction and thus was shown to be absent from the extracted starch
granules. The granule size of the sorghum starch was about 13 to 14 m. They were
of various shapes, generally spherical, angular and/or dimpled (Figure 2). Similar
findings were previously observed (Hoseney et al 1974, Craig and Stark 1984,
Fannon et al 1992). It was reported that sorghum starch granules from the soft or
opaque endosperm are round while those from the hard or translucent endosperm are
polygonal (Hoseney et al 1974).
Small visible pores were randomly distributed on the surface of some of the starch
granules. These pores were also found in starch granules of maize and millet, both of
which are closely related species in the same family with sorghum (Liu and Zhao
1990, Fannon et al 1992). The indentations on the surface of the starch granules
were caused by matrix protein pressing into the interfacial edges of the starch
granules (Hoseney et al 1974). These were reported to be a characteristic of some
particular species of starch rather than artifacts produced by the drying, isolation,
specimen preparation or observation techniques used (Leach and Schoch 1961,
Fannon et al 1992).
It was suggested that these pores on the granules are large enough for enzymes to
gain access to the interior of the granules, facilitating attacks of the enzyme
molecules on starch granules during degradation (Kimura and Robty 1995, Sarikaya
et al 2000). It was assumed that the number of pores per granule was correlated with
the rate of digestion by amylases (Hall and Sayre 1970) and the variation in the
number of pores per granule might also be an important factor in providing a
controlled and steady breakdown of starch during seed germination (Fannon et al

74

1992).
Sorghum Malt
After 5-day germination, the starch granules generally retained their spherical shape.
Figure 3 showed starch granules pitted with holes on the surface revealing the
interior layers of the starch granules. This formation of porous starch granules was a
result of the amylolytic enzymes which attacked the surface of the starch granules
either by entering through the pores initially present in the starch granules or boring
holes first. This way of being attacked was similar to that in large starch granules of
barley, both types were attacked by pitting instead of by surface erosion, which is
characteristic as observed in small starch granules of barley (MacGregor 1980). The
action of amylolytic enzymes was further demonstrated by the decrease in starch
after malting as shown in Chapter 2.
Starch degradation did not occur uniformly, the starch granules were pitted to
different extents (Figure 4). The difference in degree of degradation between
individual starch granules was affected by the structure of starch granule which
made some granule surfaces more susceptible to amylase attack than others
(Aggarwal and Dollimore 2000). The presence of pores on the starch granule surface
would facilitate the attack of by enzyme molecules during degradation (Kimura and
Robty 1995, Sarikaya et al 2000). Moreover, the presence of channels on granule
surface could also provide a pathway for the enzyme to enter the granules directly,
making enzyme hydrolysis more efficient. The number and size of starch granules
were reported to affect enzyme attack (Hollinger and Marchessault 1975, Gallant et
al 1992, Kimura and Robty 1995, Baldwin et al 1997) because smaller granules
would represent a larger surface area exposed for starch hydrolysis.
Examination of starch granules after malting showed that the protein layer originally
adhered to the granule surface in sorghum adjunct (Figure 1) had disappeared by 5day of germination. It was reported that the protein matrix began to disappear during
steeping (Glennie 1983). Activation of protease during germination could be a

75

reason for the disappearance of protein matrix initially found adhered on the starch
granule surface.
Changes in sorghum starch granules during opaque beer production
Souring
Souring was started by using sorghum malt, water and inoculation of lactic acid
bacteria as the raw materials. Before souring, the starch granules in the ingredient
mixture were already degraded to different extents (Figures 5 and 6) as a result of
malting. Figure 5 shows a slightly pitted starch granule while Figure 6 shows starch
granules with more degradation. In Figure 6, the starch granule at the top was
digested to such an extent that the hollow interior with clearly defined interior
concentric sphere was exposed. After souring, such phenomenon could still be found.
After 18-h of souring, the starch granules were generally degraded to an even greater
extent (Figure 7) due to the action of amylases. This could be further supported by
the decrease in starch and increase in sugar content after souring as demonstrated in
the previous chapter. Though the activities of - and -amylases were reduced by
the acidic pH of the souring medium, the initial high amylase activity of the sour
explained the further attack of amylases on the starch granules during the 18-h
souring. Some of the degradation was so advanced that only the skeleton of the
granule was left (Figure 8).
It was reported that small starch granules are more rapidly hydrolyzed than bigger
ones due to a relatively larger available surface area (Franco et al 1988). So it could
be deduced that as the starch granules in the sorghum malt were already pitted
before souring, this resulted in a larger surface area for the enzymes to act upon.
Therefore, pitting might be a way leading to increase in the rate of starch hydrolysis.

Cooking
In this step, adjunct and more water were added before cooking. Before the onset of
cooking, the polysaccharide in the starch granules from the adjunct were deposited

76

in an orderly, semicrystalline state as shown by the dark Maltese cross which was
apparent when granules were viewed in a polarizing microscope with the layered
structure arranged around a dark spot, the hilum (Briggs 1998b). At this stage, the
starch granules in the sorghum adjunct were insoluble in water, and swelled only
slightly due to diffusion and absorption of water into the amorphous regions. This
swelling was reversible on drying. But when cooking started and the sour was heated
to progressively higher temperatures, at which swelling became irreversible and
structural order disappeared (Aggarwal and Dollimore 1998), together with much
water uptake, the starch granules would then undergo gelatinization. The orderly
structure of starch granules broke up. At first they swelled, some components
leached out and then they gelatinized. The granules became ruptured and the
contents dispersed (Briggs 1998b).
After 2 h cooking, the sorghum starch granules had a granular size of 27 to 43 m
which was larger than that found in sorghum adjunct due to the swelling effect
brought about by gelatinization. The sorghum starch granules were melted with their
original granular appearance completely lost, forming sponge-like structure of
irregular shapes. They were eventually connected with each other as shown in Figure
9 (broad view) and Figure 10 (close view). This was the degradation of starch
granules by heat.

Mashing
Pits were found on the surface of both gelatinized and ungelatinized starch granules
(Figure 11) after mashing, this was due to the hydrolytic action of diastatic enzymes
present in the sorghum malt added for the purpose of mashing. Non-melted starch
granules probably originated from residual starch granules of the sorghum malt
added while the gelatinized starch granules were obviously a result of gelatinization
from the previous cooking step. It appeared that amylases worked quite effectively.
Some of the sorghum starch granules disappeared completely, leaving a semispherical hole in which the starch granule was supposed to be originally embedded
(Figure 12).

77

Gelatinization is essential in the production of opaque beer. It aims at making the

adjunct able to be readily hydrolyzed during mashing by the malt diastatic enzymes.
It has been widely known that native granules are degraded very slowly by amylases
because granules are very resistant to amylolytic digestion (Hyun and Zekius 1985,
Ring et al 1988, Gallant et al 1992) and that starch gelatinization can increase
susceptibility of starch granules to amylases (Holm et al 1985, Asp and Bjrck 1992,
Bjrck and Asp 1994, Bjrck 1996, Yue and Waring 1998). During gelatinization,
the starch granules would lose their semi-crystalline structure and start to disperse
into solution (Briggs 1998c). Therefore, by gelatinizing the starch in the unmalted
cereal adjunct, this could render it more hydrolyzable during mashing by the malt
diastatic enzyme.

Straining
Figure 13 showed a broad view of the sorghum starch after straining. The starch
granules were rather similar to those found in mashing because this step only
involved the physical removal of coarse particles such as pericarp, husks and rootlets
by passing through a 250-m sieve. There was no alteration in the structure of the
starch granules in this process. The picture showed starch granules which were
hydrolyzed to different levels. This further supported previous finding that
hydrolysis does not occur uniformly but with some areas more susceptible to attack
than others (Aggarwal and Dollimore 1998).

Fermentation
The sorghum starch granules present in the end product of the opaque beer were
hydrolyzed to various extents showing different characteristics, some were only very
slightly pitted (Figure 14), some were seriously pitted (Figure 15) while some were
completely degraded, just leaving the skeleton (Figure 16). Starch granules showing
the effect of both gelatinization and hydrolytic action of amylases were also found
(Figure 17).

78

During fermentation, the starch granules were further hydrolyzed due to the residual
amylase activity from the sorghum malt added for mashing as shown by the decrease
in starch and increase in sugar after fermentation in the previous chapter. As there
was no further treatment before consumption, so the residual amylase activity would
keep on exerting a hydrolytic effect on the starch granules during the postfermentation period while the opaque beer was still actively fermenting.
Comparison of changes in starch degradation among sorghum set and maize starch
sets of different amylose content during production of opaque beer
Cooking
The maize starch sets of different amylose content swelled to various degrees.
Unlike the sorghum set which swelled, melted and connected with each other by the
end of cooking, most of the Hi-maize starch granules generally retained their shape
without being melted (Figure 18) after 2-h cooking. For the normal maize starch
granules, they swelled and appeared to start deformation (Figure 19). This showed
signs of gelatinization in which the swollen granules are believed to be rich in
amylopectin while the linear amylose diffuses out of the swollen granules and forms
part of the continuous phase outside the granules (Hermansson and Svegmark 1996).
For the waxy maize starch granules, they were completely gelatinized and melted
forming a whole piece, just leaving behind the skeleton in which the starch granules
were believed to be originally embedded (Figure 20).
After cooking, the size of the Hi-maize starch granules was 5 - 9 m. For normal
maize starch, it was 13-18 m while the waxy maize starch had a granular size as
large as 34.5 m. This agreed with the general belief that waxy starches usually
swell to a greater extent than their normal-amylose counterparts (Tester and
Morrison 1990). The limited swelling of cereal starch granules during cooking when
compared with their waxy counterpart was believed to be due to the presence of
amylose-lipid complexes which only melted at temperature higher than the
gelatinization temperature of starch, thus contributed to the stability of amylose.
It was generally concluded that melting temperature of starch is directly correlated to

79

the amylose content (Sievert and Wuesch 1993, Gerard et al 1999). High-amylose
starches were reported to have a higher gelatinization temperature than normal and
waxy maize starches (Colonna and Mercier 1985). Waxy and regular maize starches
gelatinize at 62 to 72C. But for high-amylose starch, its granules begin to swell
below 100C, but a temperature of higher than 130C is needed to fully disperse
these starches (Shi et al 1998). This indicated that Hi-maize starch was most
resistant to gelatinization which led to starch deformation, therefore, Hi-maize starch
granules remained intact after 2 h cooking.
Several factors were proposed for the high resistance of starch granules to
gelatinization. 1) Amount of amylose lipid-complex. The enclosed lipid molecules
made the amylose helix conformation more stable. The association among
themselves would form an insoluble film on the surface of starch granule, thus
delaying water uptake and thus preventing the granule from swelling. Amylose
double helices which only disassociate at high temperature could also be another
factor retarding swelling of starch granules (Shi et al 1998). 2) Size of polymer. As
more energy is required to break smaller sized polymers (amylose) as compared to
bigger ones (amylopectin) (Aggarwal and Dollimore 1998; Aggarwal and Dollimore
2000). Since short chain polymers could move freely and interact easily with each
other through hydrogen bonds, therefore, more energy was needed to break down
these bonds, making Hi-maize starch more resistant to gelatinization. Longer branch
lengths found in high-amylose starches were also proposed to be responsible for its
high gelatinization temperature (Gidley 2001). 3) Degree of crystallinity. Properties
of starch like gelatinization, swelling, pasting behavior and functionality are affected
by the degree of crystallinity (Lorenz and Kulp 1981). A higher degree of
crystallization would make gelatinization difficult.

Mashing
Pitting is one of the indicators showing the degree of mashing. After mashing stage,
the Hi-maize starch granules were only very slightly pitted (Figure 21) when
compared with the sorghum set. Merely being gelatinized, Hi-maize starch granules

80

were degraded relatively slowly by amylases. On the other hand, being gelatinized,
normal maize starch granules were more vulnerable to enzyme attack and therefore
more pitting was found in this set (Figure 22) than the high-amylose maize starch.
For waxy maize starch set, the waxy starch granules should have been completely
deformed as shown in the previous step because they were highly susceptible to
enzyme hydrolysis. The starch granules shown in Figure 23 might be originated
from sorghum malt added for mashing instead of the adjunct waxy maize starch.
It has been widely known that native granules are degraded very slowly by amylases
because they are very resistant to amylolytic digestion (Hyun and Zekius 1985, Ring
et al 1988, Gallant et al 1992) but starch gelatinization can increase susceptibility of
starch granules to amylases (Holm et al 1985, Asp and Bjrck 1992, Bjrck and Asp
1994, Bjrck 1996, Yue and Waring 1998). This explained the difference in degree
of pitting for maize starch sets of various amylose contents.
The difference in degree of degradation for starch granules with various amylose
contents could be explained by 1) Granular size and surface area. Initial step of amylolysis involved the adsorption of -amylase on the granule surface. Being
swollen, the starch granules after gelatinization possessed a larger granular size than
the intact ones and thus facilitated such adsorption. According to previous section,
the maize starch sets experienced different extents of gelatinization. The Hi-maize
starch granules were swollen the least, followed by normal maize and waxy maize
starch granules, thus offering different size of granule surface for -amylase
adsorption. This indicated that Hi-maize starch granules were most resistant to
enzymatic hydrolysis, followed by normal maize and waxy maize starch granules.
Therefore, different extents of pitting were found in the maize starch sets. Moreover,
pitting also served to offer a larger surface area for -amylase adsorption, thus
facilitating starch hydrolysis. 2) Disruption of crystallinity. As mentioned, starch
hydrolysis involved the adsorption of -amylase on the granule surface. Thus
crystalline disruption near the granule surface upon gelatinization of starches could
facilitate the rapid entry of -amylase into the granule interior. 3) Amount of
81

amylose-lipid complex. The existence of complexes between amylose and the

endogenous polar lipids of cereal starch granules (Briggs 1998b) contributed
stability to amylose polymers and inhibited the amylose from being hydrolyzed. Its
amount is depended on genetic factor. As shown by the difference in starch
degradation of the maize starch sets, it appeared that Hi-maize starch was composed
of a greater amount of amylose-lipid complex than the normal maize and waxy
maize starch.

Straining
This process did not involve any chemical change in starch granule structure, but
only the physical removal of coarse particles like pericarp, husks and rootlets.
Therefore, the characteristics of starch granules in this step were rather similar to
those obtained after mashing.

Fermentation
According to Figure 24, a large proportion of the Hi-maize starch granules still
remained intact after 2-d fermentation in the final beer product. This indicated that
Hi-maize starch granules were so resistant that they had escaped both the
gelatinization under normal cooking condition and the action of hydrolytic enzymes
exerted, thus making most of the starch granules remained not much modified even
after the whole brewing process.
Unlike Hi-maize starch granules, the normal maize starch granules showed
characteristics of deformed (melted), pitted or both (Figure 25), whereas waxy maize
starch granules were melted forming a sheet by the end of fermentation, just leaving
behind the holes in which the granules were originally embedded (Figure 26).

4.5 CONCLUSION
82

Sorghum starch granules became swollen after cooking and pitted after mashing
during the production of opaque beer, resulting in starch granules possessing
characteristics of both gelatinization and starch hydrolysis in the end-product. The
effect of amylose on this brewing process was significant. The higher the amylose
content of the adjunct, the more resistant the starch granules to both gelatinization
and starch hydrolysis. For Hi-maize starch, it was so resistant to starch degradation
that its granules generally remained intact even after the whole brewing process.

83

Figure 1. Scanning electron photomicrograph of sorghum starch granules in sorghum

adjunct (Magnification: x 2.75K).

Figure 2. Scanning electron photomicrograph of sorghum starch granules in sorghum

adjunct after extraction (Magnification: x 3.09K).
84

Figure 3. Scanning electron photomicrograph of sorghum starch granules in sorghum

malt (Magnification: x 3.47K).

Figure 4. Scanning electron photomicrograph of sorghum starch granules in sorghum

malt (Magnification: x 4.17K).
85

Figure 5. Scanning electron photomicrograph of sorghum starch granules before

souring (Magnification: x 4.64K).

Figure 6. Scanning electron photomicrograph of sorghum starch granules before

souring (Magnification: x 3.57K).
86

Figure 7. Scanning electron photomicrograph of sorghum starch granules after

souring (Magnification: x 3.86K).

Figure 8. Scanning electron photomicrograph of sorghum starch granules after

souring (Magnification: x 3.04K).
87

Figure 9. Scanning electron photomicrograph (broad view) of sorghum starch

granules after souring (Magnification: x 1.53K).

Figure 10. Scanning electron photomicrograph (close view) of sorghum starch

granules after souring (Magnification: x 2.93K).
88

Figure 11. Scanning electron photomicrograph of sorghum starch granules after

mashing (Magnification: x 1.26K).

Figure 12. Scanning electron photomicrograph of a skeleton left behind after

mashing (Magnification: x 2.75K).
89

Figure 13. Scanning electron photomicrograph of sorghum starch granules after

straining (Magnification: x 1.10K).

Figure 14. Scanning electron photomicrograph of sorghum starch granules after

fermentation (Magnification: x 3.29K).
90

Figure 15. Scanning electron photomicrograph of sorghum starch granules after

fermentation (Magnification: x 6.56K).

Figure 16. Scanning electron photomicrograph of sorghum starch granules after

fermentation (Magnification: x 732K).
91

Figure 17. Scanning electron photomicrograph of sorghum starch granules after

fermentation (Magnification: x 3.36K).

Figure 18. Scanning electron photomicrograph of Hi-maize starch granules after

cooking (Magnification: x 2.98K).
92

Figure 19. Scanning electron photomicrograph of normal maize starch granules after
cooking (Magnification: x 1.74K).

Figure 20. Scanning electron photomicrograph of waxy maize starch granules after
cooking (Magnification: x 717K).
93

Figure 21. Scanning electron photomicrograph of Hi-maize starch granules after

mashing (Magnification: x 2.97K).

Figure 22. Scanning electron photomicrograph of normal maize starch granules after
mashing (Magnification: x 2.06K).
94

Figure 23. Scanning electron photomicrograph of waxy maize starch granules after
mashing (Magnification: x 2.44K).

Figure 24. Scanning electron photomicrograph of Hi-maize starch granules after

fermentation (Magnification: x 2.45K).
95

Figure 25. Scanning electron photomicrograph of normal maize starch granules after
fermentation (Magnification: x 2.01K).

Figure 26. Scanning electron photomicrograph of waxy maize starch granules after
fermentation (Magnification x 1.08K).
96

CHAPTER 5
CHANGES IN STARCH AND OTHER
COMPONENTS IN THE POST-FERMENTATION
OF OPAQUE BEER
5.1 ABSTRACT
The quantitative changes in starch and other related components were analyzed
during the 9-d post-fermentation period of opaque beer brewed with sorghum grain
(DC-75), Hi-maize starch, normal maize starch and waxy maize starch as adjunct
respectively. The Hi-maize starch samples showed a relatively stable starch
concentration during post-fermentation while the other three sets showed a general
decrease. All the four sets generally showed a decrease in amylose concentration
with Hi-maize still being the one with the highest amylose concentration. All of
them showed very similar glucose concentrations, with an increase from 21.5 mg/ml
- 24.5 mg/ml on the first day to 27.3 mg/ml - 31.0 mg/ml on the ninth day of postfermentation. This increase indicated a greater rate of saccharification than
utilization. On the other hand, sorghum samples showed a gradual decrease in
fructose concentration from 1.9 mg/ml to 1.4 mg/ml, this indicated sugar utilization
by various microorganisms. Sorghum set yielded the highest concentration of
ethanol, ranging from 0.22 to 0.29 g/L while the maize starch sets only yielded an
ethanol concentration of 0.04 to 0.09 g/L. The low ethanol yield in maize starch sets
was due to the shortage of amino nitrogen by using maize starch as adjunct. The
sorghum sets showed an accumulation of acetic acid from 0.0086% to 0.0118% and
both the - and -amylase activities remained low during the post-fermentation
period.

97

5.2 INTRODUCTION
Opaque beer is a traditional and popular beverage in Africa. It is usually brewed for
important social and cultural gatherings like weddings, celebrations of success and
traditional religious ceremonies (Madovi 1981, Benhura and Chingombe 1989).
However, unlike lager beer, the shelf life of opaque beer only ranges from 1 to 4
days depending on how hygienic the production conditions were. After bottling, the
opaque beer should be sold within 24 hours. Final distributors / sellers to consumers
cannot store the bottle or packing for more than 24 hours as the fermentation of
sorghum beer remains active. Deterioration will start after 48 hours, resulting in a
mixture of alcohol and rancid fine particles. The susceptibility 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 vinegary offflavors, turbidity and ropiness. It causes 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 which has been exposed to air during early
fermentation, and the more the bottle is shaken the more the bacteria were provided
with oxygen for respiration. The short shelf life of opaque beer is a main hindrance
for the breweries to achieve better profit. Little has been published on the on-going
changes in the post-fermentation of opaque beer. A better understanding of changes
during post-fermentation, for example in starch degradation, can help in further
innovative development in the brewing industry and prolonging the shelf life of the
beer.
This present study aimed 1) to examine the change in starch properties during the 9d post-fermentation period and 2) to investigate the effect of amylose during postfermentation by using adjunct of different amylose contents.

98

5.3 MATERIALS AND METHODS

Sorghum Grain
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 donated by Starch
Australasia Limited.

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
Refer to chapter 2.
Preparation of Sorghum Adjunct
Refer to chapter 2.
Brewing
Refer to chapter 3.
Post-fermentation
Samples prepared were kept at 28C in the incubator and samples were collected for
quantitative analysis every single day for nine consecutive days altogether starting
from the end of the fermentation.
pH
pH was determined using a pH meter with a glass electrode after calibrating with
commercial buffer (Merck) (pH 4 and 7).

99

Total Starch Content

Refer to chapter 2.
Amylose Content Assay
Amylose was determined using an iodine-binding spectrophotometric method.
Sample (1 ml) 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
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 the total non-starch carbohydrate concentration was
estimated 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.

100

-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. 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 Eppendorf tube before 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.

101

Acetic acid
Acetic acid was determined by Gas Chromatography (GC). 0.5 ml sample was
mixed with 0.5 ml acetone by vortex and the mixture was centrifuged for 10 min at
14, 000 rpm. It was then filtered through Whatman 0.2 m polyethersulfone
membrane with polypropylene housing (Puradisc 25 AS). The supernatant was used
for gas chromatographic analysis using a HP-6890 GC (Hewlett-Packard, Palo Alto,
CA), equipped with a flame ionization detector and a HP-FFAP (Crosslinked FFAP)
capillary column (15 m x 0.25 mm x 0.25 m) (Agilent Technologies, 19091F-431).
The column temperature started at 60C and increased to 200C at a rate of 10C per
min. The detector temperature was kept at 260C. Nitrogen was used as the carrier
gas and the flow rate was kept at 2.0 ml per min. The injector was kept at 250C
with an injection volume of 2.0 L under splitless injection mode. Acetic acid
concentration was determined according to standard curve prepared by acetic acid
standard mixtures representing from 0 to 0.15% acetic acid.
Ethanol
Ethanol concentration was determined according to UV-method using Enzymatic Kit
on Ethanol of Bio-pharm (Cat No E0176290).

5.4 RESULTS AND DISCUSSION

Starch
During the 9-d post-fermentation, sorghum generally decreased from 4.7 mg/ml to
1.4 mg/ml (Figure 1). This marked the presence of residual amylase activity of the
beer which was still actively fermenting. When comparing with the other maize
starch sets, starch concentration in the sorghum set was exceptionally low because of
the lower starch content of the sorghum grain than maize starch added as adjunct
before cooking.
Among the maize starch sets (Figure 1), Hi-maize showed the highest starch

102

concentration throughout the post-fermentation period, ranging from 18.2 mg/ml to

24.2 mg/ml, followed by normal maize starch, which decreased from 11.3 mg/ml on
the first day to 5.6 mg/ml on the ninth day of post-fermentation, and waxy maize
starch, which decreased from 7.5 mg/ml on the first day to 4.2 mg/ml on the ninth
day of post-fermentation. The highest starch concentration of Hi-maize starch set
had been noted since mashing. The starch concentration for the maize starch sets
over post-fermentation generally tended to decrease just like the sorghum set, with
the exception of Hi-maize which showed no tendency to decrease. This was most
likely due to 1) Granular size and surface area of the starch granule. Hi-maize
experienced nearly no gelatinization and thus not much swelling even after being
cooked, and this would provide less surface area for amylolytic action. 2) Degree of
gelatinization. Without gelatinization, most of the Hi-maize starch granules
remained native without any disruption of crystallinity and order brought about by
heat, thus making them more resistant to enzyme degradation. 3) Amount of
amylose-lipid complex. It was possible that the existence of complexes between
amylose and the endogenous polar lipids of cereal starch granules (Briggs 1998b)
contributed stability to Hi-maize starch granules and inhibited them from being
hydrolyzed.
Amylose
Amylose concentration (Figure 2) in the sorghum set decreased from 4.7 mg/ml to
1.4 mg/ml during the 9-d post-fermentation. Amylose molecules are small chains
with no branch points located in the amorphous region of the starch granule, and
therefore, are highly susceptible to enzymatic hydrolysis. As a result, with the
presence of residual enzyme activity from the sorghum malt, amylose was
continuously hydrolyzed during the 9-d post-fermentation period. A similar
phenomenon was also observed among the maize starch sets, the amylose
concentration tended to decrease during the 9-d post-fermentation period. Hi-maize
set was the one with the highest amylose concentration, followed by normal and
waxy maize starch sets according to their respective amylose contents.

103

- and -amylase activities

The residual -amylase activities for all the four sets were less than 1 CU/ml (Figure
3). These were very low when compared with the -amylase activity of the DC-75
sorghum malt which was found to be 104.3 CU/g as in Chapter 2. This indicated the
exhaustion of -amylase since mashing. There was no significant change in amylase activity for the four sets during the 9-d post-fermentation. Sorghum showed
the highest -amylase activity among the four sets, followed by waxy, normal and
Hi-maize starch.
-Amylase (Figure 4) contributes a smaller proportion to the amylolytic activity
when compared with -amylase. The -amylase activity of sorghum set was very
low, ranging from only 0.023 mg/ml to 0.028 mg/ml over the 9-d post-fermentation
period. As in the case of -amylase, the four sets showed no significant change
during the 9-d post-fermentation, with sorghum set having the highest -amylase
activity while the three maize starch sets showed very similar ones.
Sugars
There was no definite increase or decrease over the 9-d post-fermentation period for
the four sets in terms of total non-starch carbohydrate (Figure 5), with waxy maize
starch being the one with the highest total non-starch carbohydrate concentration
ranging from 89.0 mg/ml to 119.8 mg/ml, immediately followed by sorghum,
normal maize starch and Hi-maize starch respectively. This probably depended on
the amount of amylose-lipid complex present in the beer. The higher the amylose
content, the more the amylose-lipid complex would be expected to present. As
amylose-lipid complex contributed to the stability of amylose against amylolytic
action, thus a lower amount of sugars would be formed.
All the four sets showed very similar concentrations of glucose (Figure 6) which
gradually increased from 21.5 mg/ml - 24.5 mg/ml to 27.3 mg/ml - 31.0 mg/ml. This
indicated that rate of saccharification was greater than that of glucose utilization.
The increase in glucose concentration of sorghum set was opposed to the decrease
104

found in the 2-d fermentation. This could be due to the lag phase of the yeast during
post-fermentation which was less active than during fermentation, thus leading to the
fading out of glucose utilization. On the other hand, together with the action of
residual amylase, it was possible that glucose was accumulated due to starch
hydrolysis during post-fermentation.
Sorghum set showed a general decrease in fructose concentration (Figure 7) which
was not shown in the maize starch sets. Fructose, being fermentable, was utilized by
yeasts and possibly other various microorganisms, resulting in a gradual decrease.
This was not observed in the maize starch sets because they lacked the amino
nitrogen essential for yeast growth, thus resulting in less sugar utilization. As a result,
there was no significant change in fructose concentration.
pH
The pH value (Figure 8) for the sorghum beer in the post-fermentation period ranged
from 4.9 to 5.03, this was relatively higher than the commercial opaque beer which
was found to be in the range of 3.2 to 3.9. This could be due to the difference in malt
preparation. In this study, sorghum malt was prepared by first steeping in NaOH
(0.3%, w/v) for 8 hr before germination. This was most likely the reason for such
high pH value in the sorghum beer.
The pH range, 4.9 to 5.03, recorded in sorghum set was higher than that recorded in
the maize starch sets, 4.31 to 4.68 (Figure 8). As it was the same source of sorghum
malt being added into each of the four sets, so it appeared that the sorghum grain
adjunct intrinsically had a higher pH than the maize starch.
Ethanol
Sorghum showed the highest ethanol yield during the post-fermentation period
ranging from 0.22 g/L to 0.29 g/L while the Hi-maize, normal maize and the waxy
maize starch sets had a similar ethanol yields of just 0.04 g/L to 0.09 g/L (Figure 10).
Lacking amino nitrogen essential for yeast growth was an unfavorable condition that

105

hindered the utilization of fermentable sugar to form ethanol in the maize starch sets,
thus leading to the low ethanol yield obtained.
When compared with the average ethanol concentration of commercial opaque beer,
25.4 g/L (Novellie 1968), the ethanol yield of sorghum beer produced in this study
was relatively low. A number of factors could affect this. A higher content of
fermentable sugar was believed to result in a higher ethanol yield. Amount of
oxygen available could affect the pathway of yeast metabolism. Sugar utilized
through aerobic respiration would result in carbon dioxide plus water, no ethanol
would be formed in this pathway. On the other hand, if sugar was utilized through
anaerobic fermentation, carbon dioxide and ethanol would be formed, thus a higher
ethanol yield would be resulted under such circumstance. As the amount of amino
nitrogen available in the wort was very critical for yeast growth, hence, it might also
affect the final ethanol yield in the end product.
Acetic Acid
Acetic acid % generally increased in the sorghum set from 0.0086% to 0.0118%
(Figure 9), representing the accumulation of acetic acid during the 9-d postfermentation period. By the end of the 9-d post-fermentation, sorghum set showed
the highest acetic acid % immediately followed by Hi-maize, waxy and normal
maize starch sets. This probably indicated the relatively higher activity of spoilage
microorganisms like Acetobactor, and thus resulted in more metabolic products such
as acetic acid due to the higher amount of amino nitrogen available in the sorghum
set than in the maize starch sets. According to Figure 9, amylose did not exert
significant effect on the production of acetic acid.

5.5 CONCLUSION
By using sorghum grain as adjunct, both the starch and amylose concentrations
decreased giving rise to an increase in glucose during the 9-d post fermentation
period. This indicated a greater rate of saccharification than utilization. The ethanol
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concentration of the beer ranged from 0.22 g/L to 0.29 g/L and it showed an
accumulation of acetic acid throughout the 9-d post-fermentation. Amylose content
affected starch degradation, maize starch with higher amylose content showed
greater stability against amylolytic attack. Despite its resistance to starch
degradation, amylose seems not to affect the production of acetic acid.

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Sorghum
Hi-maize
Normal
Waxy

30

25

Starch (mg/ml)

20

15

10

0
Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 1 Changes in starch concentration of opaque beer during post-fermentation.

25

Amylose (mg/ml)

20

15

10

0
Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 2 Changes in amylose concentration of opaque beer during post-fermentation.

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Sorghum
Hi-maize
Normal
Waxy

Alpha-amylase activity (Ceralpha Unit/ml)

1.2

1.0

0.8

0.6

0.4

0.2

0.0
Day 1
0.035

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 3 Changes in alpha-amylase activity of opaque beer during post-fermentation.

Beta-amylase activity (Betamyl Unit/ml)

0.030

0.025

0.020

0.015

0.010

0.005

0.000
Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 4 Changes in beta-amylase activity of opaque beer during post-fermentation.

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Sorghum
Himaize
Normal
Waxy

160
140
total sugar (mg/ml)

120
100
80
60
40
20
0
Day 1
40

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 5 Changes in total sugar concentration of opaque beer during post-fermentation.

Glucose (mg/ml)

35
30
25
20
15
10
Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 6 Changes in glucose concentration of opaque beer during post-fermentation.

5.0
4.5

Fructose (mg/ml)

4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 7 Changes in fructose concentration of opaque beer during post-fermentation.

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6.0

Sorghum
Hi-maize
Normal
Waxy

5.5

pH

5.0

4.5

4.0

3.5

3.0
Day 1

Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9

Fig. 8 Changes in pH of opaque beer during post-fermentation.

0.012

Acetic acid %

0.010

0.008

0.006

0.004

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Fig. 9 Changes in acetic acid % of opaque beer during post-fermentation

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0.40

sorghum
himaize
waxy
normal

0.35

0.30

Ethanol (g/L)

0.25

0.20

0.15

0.10

0.05

0.00
Day1

Day2

Day3

Day4

Day5

Day6

Day7

Day8

Day9

Figure. 10 Changes in ethanol during post-fermentation.

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CHAPTER 6
IMPLICATIONS AND RECOMMENDATIONS
By applying adjunct with higher amylose content could provide greater resistance
against starch degradation during opaque beer production. For example, by
supplementing part of the adjunct with Hi-maize starch, the starch present in the
system tended to be more resistant to cooking and mashing. As a result, more starch,
a larger proportion of ungelatinized starch and less sugar would present in the
opaque beer. The process did not terminate at this point. Instead, fermentation was
actively carried on. The resistance of Hi-maize starch against cooking resulted in a
higher proportion of ungelatinized starch. Together with the resistance to enzymatic
attack, using Hi-maize as adjunct would cause a delay in production of sugar. As
result, sugar would be released at a slower rate, thus ethanol production by means of
anaerobic respiration of yeast would also be delayed. Therefore, the time when
acetic acid bacteria utilized ethanol for producing enough acetic acid to cause
spoilage would also be postponed. Thus, shelf-life extension could be made possible
by applying adjunct with higher amylose content in the brewing of opaque beer.
By controlling percentage of amylose of adjunct not only contributes to shelfextension, but also final quality of opaque beer. As mentioned previously, amylose
offers resistance against starch degradation. It is critical to the final quality of
opaque beer by controlling degree of gelatinization and hydrolysis of starch.
Gelatinization, which takes place during cooking stage in opaque beer production
giving rise to gelatinized starch, determines the ratio of gelatinized starch to
ungelatinized starch, the amount of starch and the viscosity in the end product.
These in turn contribute to the final quality of opaque beer in terms of mouth feel
through creaminess and appearance through dispersion of suspended particles.
Starch hydrolysis, which takes place during souring, mashing, fermentation and
post-fermentation of opaque beer production giving rise to various sugars, controls
the sugar profile of the opaque beer. Sugar offers sweetness to the beer itself and is

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essential for both lactic acid fermentation, which affects sourness and yeast
fermentation, which affects the amount of ethanol produced. Therefore, starch
hydrolysis affects the sensory profile of the opaque beer in terms of sweetness,
sourness as well as alcohol content. Thus, percentage of amylose in the adjunct also
helps in attaining opaque beer of desired quality.
It does not necessarily mean that 100% adjunct should all be supplemented with Himaize, rather, a combination of adjunct with different amylose content can be
considered to get the right proportion to extend shelf-life on one hand while attain
beer of desired quality on the other. Apart from supplementing part of the adjunct
with Hi-maize starch, further research could also be done by using sorghum flour
with similar composition but different amylose contents so as to help lower the
production cost. Other possible ways such as application of modified starch and
atmosphere packaging could also be considered for the sake of shelf-life extension.
It was foreseen that opaque beer would continue to perform the role of being a
significant beverage in Africa in the future (Daiber and Taylor 1995). Therefore,
more research needs to be conducted to solve the greatest barrier to distribution of
the beer, being short shelf-lived, so as to enhance storage of the beer, thus reducing
loss due to spoilage.

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