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CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
1.1 INTRODUCTION Sorghum [Sorghum bicolor (L.) Moench] is an
important food crop in arid and semi-
arid regions of the world (Sulma et al 1991, Mukuru 1992, Owuama
1997). It is a
major food crop ranking fifth in terms of world grain
production. Fermented foods
and beverages constitute a major portion of diets for people in
Africa (Sanni 1993,
Oyewole 1997). Cereal grains like sorghum, maize and millet are
common
substrates for lactic acid-fermented gruels and beverages
(Odunfa and Adeyele
1985). Fermented sorghum or millet-based foods, alcoholic and
non-alcoholic drinks
or beverages are prepared in many African countries for human
consumption
(Ekundayo 1969, Ahmed et al 1988, Chavan and Kadam 1989,
Steinkraus 1996,
Odunfa et al 1996).
Sorghum has been used for centuries to brew opaque beer in South
Africa. Being
drought-tolerant and because of its low price, sorghum is
suitable for further
development of additional value-added uses. The potential of
sorghum as an
alternative substrate for lager beer brewing has been described
by Owuama (1997).
A better understanding of grain changes during brewing, for
example in starch
degradation, can help in further innovative development in the
brewing industry.
Although sorghum is an important staple and commercial crop of
many people in
arid and semi-arid regions of the world, there is very little
documentation about the
change in starch either quantitatively or qualitatively at each
step of the traditional
opaque beer production and the on-going of post-fermentation
process. This study
was undertaken to analyze the changes in starch degradation and
other related
components at each step of opaque beer production as well as
post-fermentation.
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The specific objectives of this study are:
1. To examine the effect of malting to starch degradation in
seven varieties of
sorghum grain.
2. To analyze the changes in starch and other components
quantitatively at each
step of sorghum opaque beer production.
3. To compare the effect of amylose content at each step of
opaque beer
production by substituting the sorghum adjunct with Hi-maize
(high amylose)
starch, normal maize starch and waxy maize starch
respectively.
4. To investigate the qualitative modification of sorghum starch
granules after
malting and at each step of opaque beer production.
5. To trace the changes in starch and other components during
the post-
fermentation of opaque beer by using sorghum and maize starch
with
different amylose contents as adjunct.
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1.2 REVIEW OF LITERATURE
1.2.1 Grain Sorghum Classification
Sorghum belongs to the grass family Poaceae (or Gramineae),
genus Sorghum
Moench. Cultivated sorghums were domesticated from wild Sorghum
bicolor around
3000 years ago in the north-east quadrant of Africa (Snowden
1936, Evelyn 1951,
Doggett 1965, 1988). Cultivated sorghums are classified into
five basic groups or
races: bicolor, guinea, caudatum, kafir, and durra, and ten
hybrid races which
combine the characteristics of any two or more basic races
(Harlan and de Wet 1972).
Sorghum bicolor (L.) Moench, is known under various names, e.g.
great millet and
guinea corn in West Africa, kafir corn in South Africa, dura in
Sudan, mtama in
eastern Africa, jowar in India and kaoliang in China.
The sorghum kernel varies in color from white through shades of
red and brown to
pale yellow to deep purple-brown. The most common colors are
white, bronze and
brown. Kernels are generally spherical but vary in size and
shape. The caryopsis can
be rounded and bluntly pointed, 4 to 8 mm in diameter
(Purseglove 1972). The grain
is partially covered with glumes. Sorghum grain that has a testa
contains tannin in
varying proportions depending on the variety.
Origin and distribution
Sorghum is an important food crop in arid and semi-arid regions
of the world (Sulma
et al 1991, Mukuru 1992, Owuama 1997). Sorghum is among the most
drought-
tolerant of cereals, becoming dormant under drought and heat
stress and then
resuming growth when conditions improve. It also withstands
flooding and is not
influenced much by acid soil.
According to the records of International Crops Research
Institute for the Semi-Arid
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Tropics (ICRISAT), sorghum originated in the northeastern
quadrant of Africa. From
Ethiopia, it was distributed along trade and shipping routes
throughout Africa, and
through the Middle East to India. It reached China via the silk
route. Sorghum was
first taken to the America through the slave trade from West
Africa. It was
reintroduced in late 19th century for commercial cultivation and
has subsequently
been introduced into South America and Australia. Nowadays,
sorghum is widely
found in the drier areas of Africa, Asia, the Americas and
Australia because of its
adaptation to adverse environments.
Production
Sorghum is the staple food for millions of people in the
semi-arid tropics of Africa,
South Asia and Central America. Sorghum is the fifth most
important cereal in the
world in terms of production, after wheat, rice, maize and
barley. In 2001, total
sorghum production was more than 58 million metric tons from
about 42.7 million
hectares and the major producers are Africa with annual
production of 19.0 million t
of grain from 22 million ha and the United States with annual
production of 13
million t of grain from 3.5 million ha (FAOSTAT 2002).
Utilization
Sorghum is mainly used for human food and animal feed. Sorghum
is commonly
used to make porridges, pancakes, breads, snacks and alcoholic
and nonalcoholic
beverages. Sorghum utilized for human consumption in Southern
Africa is usually
processed to malt for the production of opaque beer (Beta and
Dzama 1997) and up
to 200 000 tonnes of sorghum are processed to malt per annum
(Dewar and Taylor
1993).
Carbohydrate
As observed in other cereals, carbohydrates constitute the
largest percentage of
sorghum grain, with values ranging from 60 to 80 % of normal
kernels (Horan and
Heider 1946, McNeill et al 1975).
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The average starch content of sorghum is 69.5%, ranging from 56
to 73%
(Jambunathan and Subramanian, 1988). Approximately 70 to 80 % of
the sorghum
starch is amylopectin and the remaining 20 to 30% is amylose
(Deatherage et al
1955, Beta and Corke 2001). Both environmental (Ring et al 1982)
and genetic
factors (Beta and Corke 2001) were reported to affect the
amylose content of
sorghum. Waxy sorghum varieties are very low in amylose and
their starch is
practically 100% amylopectin (Deatherage et al 1955, Ring et al
1982).
According to previous findings, total sugar contents generally
lay in the range
between 0.81 to 6.01%. The total sugar contents of sorghum
grains were reported to
vary from 1.30 to 5.19% (Subramanian et al 1980), 2.34 to 6.01%
(Neucere and
Sumrell 1980) and 0.81 to 1.59% (Edwards and Curtis 1943)
respectively. Among
the sugars, stachyose, raffinose, sucrose, glucose and fructose
were identified while
no maltose was detected (Subramanian et al 1980). No consensus
has been reached
on the predominant sugar in sorghum grain. Sucrose was reported
to be the
predominant sugar ranging from 68.7 to 82.7% of soluble sugars
in the sorghum
cultivars (Subramanian et al 1980) while glucose and fructose
were also reported to
be the major components (Neucere and Sumrell 1980).
Adjunct
In the brewing of opaque beer, adjunct plays an important role
to provide extract at a
lower cost as a cheaper form of carbohydrate than is available
from malt and impart
the beer its characteristic viscous body (Novellie and de
Schaepdrijver 1986).
Adjunct alters the carbohydrate and nitrogen ratio of the wort,
as a result, it affects
the formation of by-products, such as esters and higher alcohols
(Goldammer 2000a).
If the level of adjunct used is too high, there may be risk of
producing wort with
insufficient insoluble nitrogen for yeast growth.
The requirements for an adjunct depend on availability,
competitive price, low fat
content and absence of mycotoxins (Daiber and Taylor 1995).
Sorghum or maize is
usually used as adjunct in opaque beer production.
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1.2.2 Sorghum malt Sorghum malt is defined as a composite
material of partially solubilized,
nutritionally and technologically beneficiated solids containing
a spectrum of
enzymes, flavor substances, microorganisms, buffers and other
components essential
for brewing (Daiber and Taylor 1995). Sorghum has been malted
for centuries and in
Africa, it is used for the production of opaque beers, weaning
foods, baby foods and
other traditional alcoholic and nonalcoholic beverages. The malt
used in preparation
of opaque beer, a traditional alcoholic beverage in Africa,
plays a role in
determining the rate of fermentation and is a source of
lactobacilli, yeasts and
essential nutrients (Agu 1995a, Ugboaja et al 1991). Cereal
flour, lactic acid bacteria
(LAB) (Spicher and Nierle 1988), and yeasts (Ogrydziak 1993)
contain proteinases
and peptidases. Cereal malts are used to initiate spontaneous
fermentation in a
number of African indigenous foods (Steinkraus 1996). The
proteolytic activities in
cereals increase considerably during germination (Gahlawat and
Sehgal 1994,
Anibaba et al 1997), and an improvement in the composition of
some of the amino
acids in sorghum has been observed (Mbugua and Njenga 1992).
Malting results in mobilization of hydrolytic enzymes such as
amylases and
proteases, which are essential for the solubilization of starch
and proteins in the
grains, thus making them susceptible to fermentation (Lorenz and
Kulp 1991). It
also brings about the modification of the grain composition and
structure. After
malting, a loss of 56-66% and 98-99% in tannin for low and high
tannin cultivars
(Elmaki et al 1999) were reported. For starch, a decrease with
only 60 and 54% were
remained in 5- and 7-day malted sorghum grain (Von Holdt and
Brand 1960). An
increase in hot water extract, diastatic activity and sugar
contents (Lasekan et al
1995) in which fermentable mono- and disaccharides are produced
depending on the
- and -amylase activities (Hulse et al 1980) were also
recorded.
Production process of sorghum malt
Malting involves steeping, germination, drying and milling of
grains.
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Steeping
Steeping refers to the soaking of grain in water in order to
hydrate the embryo for
active growth and germination. It has been widely considered as
a critical stage in a
malting process (Briggs et al 1981, French and McRuer 1990). Its
objective is to
initiate germination such that modification of the endosperm
structure will progress
at a rate producing malt of the desired quality.
Alkali treatment, steeping temperature and aeration during
steeping process all
contribute to final malt quality. By steeping sorghum grains in
dilute NaOH, this can
detoxify high-tannin sorghum cultivars, reduce steeping period
and enhance malt
quality in both condensed-tannin free and high-tannin sorghum
cultivars due to
increased water uptake and tannin inactivation (Dewar et al
1997a, Beta et al 2000).
Increase in steeping temperature up to 30C can improve malt
diastatic power and a steeping temperature of 25C brings both free
amino nitrogen and extract content to their maximum levels (Dewar
et al 1997b). For aeration, it was also shown to
enhance the extract and free amino nitrogen content of the final
malt (Dewar et al
1997b).
Germination
After being steeped, the sorghum grains are then germinated. The
main objective of
germination is to trigger the development of hydrolytic enzymes
which are absent in
the ungerminated grain during the germination of cereal grain in
moist air under
controlled condition. Germination was found to have a highly
significant effect on
enzyme activity (Uriyo and Eigel 1999). The presence of
hydrolytic enzymes such as
-amylase, protease, -glucanase and pentosanase enzymes in
sorghum malt was reported (Aniche and Palmer 1990).
Factors important for germination include adequate moisture
content, temperature
and aeration, which play essential roles in production of
enzymes and solubilization
of reserve materials in the endosperm (Daiber and Taylor 1995).
They also affect
extract yield, diastatic activity and other important malt
quality characteristics
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(Morrall et al 1986). It was found that higher germination
temperature of 30C when compared with 20C, increased the production
of total soluble nitrogen and - and -amylases (Agu and Palmer
1997). Chemical treatment by sprinkling potassium bromate at a
concentration of 125 mg/liter on germinating sorghum was found
to
increase the diastatic power of sorghum malt significantly after
4-d malting (Agu
1995b).
Drying
Drying ends the germination process by reduction of moisture to
12% or less, which
in turn preserves the malt quality and enzymes during storage
(Novellie 1962,
Pathirana et al 1983). Industrially, sorghum malt is dried by
forced-draft dryer, the
temperature of which should not be higher than 50C since higher
temperatures were reported to cause losses of enzymes especially
during the initial stages of drying
(Novellie 1962, Okon and Uwaifo 1985).
Milling
The objective of milling is to split the husk, so as to expose
the starchy endosperm.
The dried malt is milled to pass through a 0.5-mm sieve to at
least 95% together
with the roots and shoots which are found to be an important
source of FAN (Taylor
1983) and flavor (Daiber and Taylor 1995).
1.2.3 Opaque Beer
Description
Opaque beer is a traditional and popular beverage in several
countries in Africa. It is
also known as chibuku in Zimbabwe, impeke in Burundi, dolo in
Mali and Burkina
Faso and pito in Nigeria (Jambunathan and Subramanian 1995). The
beer is usually
brewed for important social and cultural gatherings like
weddings, celebrations of
success and traditional religious ceremonies (Madovi 1981,
Benhura and Chingombe
1989).
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Opaque beer production involves both lactic acid and alcoholic
fermentation stages
and takes about 5 to 7 days to brew depending on ambient
temperature (Gadaga et al
1999). It is an opaque, alcoholic, effervescent, pinkish-brown
beverage with a sour
flavor resembling yoghurt and the consistency of a thin gruel
(Steinkraus 1996). Its
opaque appearance is due to the high content of suspended solids
and cells such as
undigested starch residues, yeasts and other microorganisms. Its
pinkish-brown color
is due to the solubilization of reddish anthocyanin pigments
(Glennie 1983) during
souring and mashing. Its sour flavor is due to the lactic acid
fermentation bringing
pH down to 3.2 to 3.9 (Table 1). This beer is distributed and
consumed while still
actively fermenting, thus it is held in vented containers so as
to allow escape of
carbon dioxide. In contrast to European brewing, opaque beer is
not pasteurized and
it has a short shelf life ranging from one to five days
depending on how hygienic the
condition is during preparation and fermentation (Daiber and
Taylor 1995).
Composition/Nutritive Value
Opaque beer is more a food than a beverage as shown by the
important role it plays
in the nutrition of Bantu tribesmen who work at the diamond and
gold mines. The
beer is rich in carbohydrate with an alcohol content ranging
from 1-4% and
consumption of 1-L opaque beer can supply 13.1% daily food
energy requirements
of an adult (Table 2). The beer contains a considerable amount
of protein and 1-L
opaque beer can satisfy 9.6% recommended daily dietary allowance
for protein
(Table 2). It also contains plenty of B vitamins, including
thiamin, riboflavin and
niacin (Table 2). It was reported that pellagra, which is
relatively common in people
subsisting on maize diets, was never found in people consuming
usual amounts of
opaque beer (Platt 1964). Opaque beer contains various kinds of
mineral such as
copper, iron, zinc, manganese, magnesium and phosphorus. It was
found that by
using sorghum as adjunct rather than maize grits, this increases
the nutritional value
of opaque beer in terms of vitamins and minerals (Van Heerden
1989a). When
compared with barley beer, opaque beer has a higher protein,
thiamin, riboflavin and
mineral content, but a lower alcohol and niacin content (Van
Heerden 1988).
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Opaque Beer Production
Souring
Souring refers to lactic acid fermentation, which involves the
growth of lactic acid
bacteria on slurry of approximately 8-10% sorghum malt in water
inoculated with a
pure culture of thermophilic, homofermentative Lactobacillus
delbrueckii
(leichmanni). The temperature during souring should be strictly
kept at 48-50C so as to prevent the growth of mesophilic organisms
and favour the growth of
thermophilic, homofermentative Lactobacillus delbrueckii
(leichmanni), which
produces solely lactic acid (Van der Walt 1956) from maltose or
glucose under
anaerobic conditions. It is kept for up to 2 days until pH is
about 3.0 3.3 and with a
lactic acid content of about 0.8-1.0% (Briggs 1998d).
Lactic acid fermentation can be classified into two types,
spontaneous or inoculated.
Spontaneous fermentation, though a simple way to sour, is rather
a hit or miss
process depending on the presence of the lactic acid bacteria
which form part of the
natural microflora of the sorghum malt (Daiber and Taylor 1995).
It involves a
complex microbial process (Daeschel et al 1987) and may result
in a product of
variable quality (Novellie and de Schaepdrijver 1986, Kingamkono
et al 1995).
Inoculated fermentation can be done either by means of 10% by
volume of the
previous sour or isolated pure strains of Lactobacillus. This is
normally adopted in
industrial brewing and in many home brewing processes (Daiber
and Taylor 1995).
Souring serves three functions. The lactic acid produced imparts
the beer with its
characteristic sour taste and lowers the pH of the beer, thus
slows down the rate of
microbial spoilage and inhibits the growth of pathogenic
organisms. This low pH
also prevents the complete hydrolysis of starch into sugars
during mashing by
retarding the rate of enzyme activity (Novellie 1966, Taylor
1989) because the
resulting residual starch in the beer is important for giving
the beer with its opaque
and viscous character (Daiber and Taylor 1995).
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Cooking
Cooking involves the gelatinization of starch in the adjunct.
When the correct level
of acidity is obtained after souring, unmalted cereal, known as
adjunct, and more
water are added and the mixture is boiled for 2 hr. The starch
in the mixture, mainly
from the adjunct then undergoes gelatinization, thus making it
readily hydrolyzable
during mashing by the diastatic enzymes of the malt because
ungelatinized (raw)
starch is very resistant to amylolytic digestion and is only
slowly attacked by the
malt -amylase and not at all by the -amylase (Hyun and Zekius
1985, Daiber and Taylor 1995). The gelatinized starch also serves
to give the beer its characteristic
creamy body and keeps the particles of grain and malt in
suspension (Novellie and
Schutte 1961). Both gelatinized and ungelatinized starches are
present in
considerable amounts in the end product (Novellie and Schutte
1961, Novellie 1966)
and both starches provide calories to the consumer.
Sorghum starch granules are tightly enclosed by endosperm
protein which creates a
barrier to starch gelatinization (Chandrashekar and Kirleis
1988). This can be solved
by the softening effect of lactic acid, which is produced in
previous souring step,
thus allowing more rapid water uptake by the starch granules and
speeds up
gelatinization (Novellie 1968).
Cooking and mashing cannot be carried out simultaneously because
the temperature
at which the starch of adjuncts (maize, sorghum or millets) is
gelatinized falls in the
range 62 to 75C (Briggs et al 1981). This gelatinization
temperature is so high that both -amylase and -amylase will be
inactivated by the time the starch is gelatinized. Therefore, the
cooking and mashing should be carried out separately.
Mashing
Mashing aims at converting starch of the malt and adjunct into
sugars so as to give a
fermentable wort of the desired composition. This first starts
with cooling down the
cooked adjunct to about 60C after 2-h cooking in order to
prevent the enzymes in the malt from denaturing, extra sorghum malt
is added and the mixture is held for 2
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h at 60C so as to convert the starch molecules of the cooked
adjunct into glucose, maltose and maltooligosaccharides by - and
-amylases and other related enzymes. Both fermentable and
unfermentable sugars are produced at the same time. - and -amylases
are the principal enzymes responsible for starch conversion. When
they work together, they are capable of converting only 60 to 80%
of the available starch
to fermentable sugars (Dougherty 1977). By the end of mashing,
the pH of the wort
becomes 3.9 with 0.16% lactic acid and a high total solids
content (Briggs 1998d).
Control of the mash pH is very important since it affects
viscosity, sugar
concentration and yield of alcohol (Rooney and Serna-Saldivar
1991). Although the
mash should be thinned, complete starch hydrolysis is not the
ultimate objective.
This can be prevented by inhibition of -amylase with low pH at
which the mash never becomes too fluid, part of the gelatinized
adjunct starch should remain at the
end of the mashing (Novellie 1966) so as to give the beer its
opaque character and
high viscosity.
Straining
Straining process refers to the removal of coarse particles such
as husks, pericarp
and plumules with a diameter larger than 250-m, so as to obtain
a smoother texture for the opaque beer. This can be done by solid
bowl centrifuges (decanters)
industrially (Novellie and De Schaepdrijver 1986) or passing
through a bag of
woven grass or metal screen with appropriate mesh size
domestically (Daiber and
Taylor 1995). The spent grains (also known as strainings) are
usually used as animal
feed. They are rich in dense, ungelatinized starch and insoluble
protein, contain 46%
starch and 25% protein on dry basis (Van Heerden 1987).
Fermentation
Fermentation is done either with the inoculation of wild yeasts
from the malt or
cultivated strains of yeast. The predominant natural yeast is
Saccharomyces
cereviseae, and strains of its top fermenting yeast are used to
start the fermentation
after cooling down the wort to 28C. It is then kept for 48 h at
28 to 30C.
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During fermentation, yeasts multiply and fermentation develops,
they convert sugar
to ethanol and carbon dioxide. Glucose, maltose and maltotriose
are the only starch
hydrolysis products that can be utilized by yeast to form
alcohol in this process
(Panchal and Stewart, 1979) and they can be obtained by
degradation of grain
components, especially starch and soluble sugars, by both
intrinsic grain enzymes
and enzymes of the fermenting media (Chavan and Kadam 1989). At
the same time
more B vitamins and new protein are also produced and the
bacteria continue to
develop lactic acid (Doggett 1970). Final alcoholic content of
the beer varies
between 1-4% (Daiber and Taylor, 1995).
Fermentation not only involves ethanol production but also
improves the nutritional
quality of sorghum by causing significant changes in chemical
composition and
eliminating antinutritional factors (Chavan and Kadam 1989).
Chemical changes like
amylolytic hydrolysis not only take place during mashing but
simultaneously with
alcoholic fermentation (Steinkraus 1996) and combination of
cooking and
fermentation, as in the case of opaque beer production, was also
reported to enhance
the nutrient quality and drastically reduce the antinutritional
factors to safe levels
(Obizoba and Atii 1991). Moreover, fermentation was also found
to contribute to
protein digestibility and availability of amino acids (Mbugua
and Njenga 1992,
Steinkraus 1996). Amino acids and peptides stimulate the growth
and fermentative
activity and tolerance of yeasts as well as proteolysis and
lactate production by LAB,
and these factors affect both the sensory and nutritional
quality of fermented foods
(Martinez-Anaya 1996) as well.
Shelf-life of opaque beer
The shelf life of opaque beer only ranges from 1 to 4 days
depending on the hygiene
of the production conditions. Therefore, the beer should be
consumed promptly
before deterioration, while fermentation is still active and
before the alcohol content
rises above the legal limit (Briggs 1998a). Stability of the
product is the main
"hindrance" to achieve a better profit level. The opaque beer
should be sold as soon
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as possible after bottling. Final distributors / sellers to
consumers cannot store the
bottle or packing for long due to the active fermentation of the
beer. Fermentation is
not completed when the product is packaged and continues the
more the bottle or
packing is shaken; followed by a deterioration of the product
into a mixture of
alcohol and rancid fine particles. The readiness to spoilage is
due to the activities of
species of Acetobacter, a genus of acetic acid bacteria. Acetic
acid bacteria are
particularly known in breweries for their ability to produce
acetic acid which gives
vinegary off-flavors, turbidity, and ropiness. It is a kind of
gram-negative, rod-
shaped bacteria which can cause the ethanol, the main product
from fermentation, to
combine with oxygen to form acetaldehyde which eventually
converts to acetic acid.
Low pH does not restrict growth of acetic acid bacteria (Hough
et al 1982). It
develops best in wort and beer when exposed to air during early
fermentation and
aeration of the beer by rousing or splashing which provides the
bacteria with
sufficient oxygen for respiration. Acetic acid bacteria are
therefore unable to grow
after the yeast culture has utilized the dissolved oxygen of the
wort (Goldammer
2000b).
1.2.4 Amylose and Amylopectin
Starch is a polymer with anhydroglucose units as its monomers.
It consists of two
types of molecules, amylose, a mainly linear structure and
amylopectin, a branched
structure. Amylose has a relatively lower molecular weight
(typically 20-800 g/mol),
consisting of single mostly-linear chains with 500-20,000
-(14)-D-glucose units depending on the source. It forms a helical
complex with iodine giving a
characteristic blue color. Amylopectin has a very high molecular
weight (typically
10,000-30,000 g/mol) with up to two million glucose units. In
addition to -(14) linkages which are present in amylose and the
linear segments of amylopectin, the
amylopectin molecule also has -(16) linkages which occur every
20-30 anhydroglucose units. Amylopectin molecules are grouped
together in concentric
rings representing alternating semi-crystalline and amorphous
lamellae while the
amylose is partly involved in double helices with amylopectin
chains in the
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crystalline regions, randomly dispersed in the amorphous regions
or bound in
amylose-lipid complexes (Blanshard 1987).
The relative proportions of amylose to amylopectin depend on the
source of the
starch. Regular endosperm sorghum types contain 23 to 30%
amylose (Horan and
Heider 1946, Ring et al 1982) while waxy varieties contain up to
5% amylose.
Normal maize starch is composed of approximately 75% amylopectin
and 25%
amylose. Waxy maize starch is composed of 98% amylopectin and 2%
amylose.
High amylose (greater than 70%) containing starches are also
known. This
difference in ratio of amylose to amylopectin influences both
gelatinization
temperature (Knutson 1990) and starch susceptibility to enzyme
attack (Ring et al
1988, Holm and Bjork 1988).
1.2.5 Biochemical Changes of Starch during Opaque Beer
Production
Gelatinization
Starch gelatinization plays an important role during processing
of starch products
and it takes place in the cooking stage of opaque beer
production. In this process, a
series of irreversible changes such as breakage of hydrogen
bonds, water uptake,
swelling, melting of crystallites or double helices, loss of
birefringence and
solubilization occur, they are generally accompanied by
increasing viscosity due to
water being absorbed away from the liquid phase into the starch
granules.
Due to the presence of semi-crystalline arrangement of the
starch molecules, the
granules are insoluble in cold water but require heat and
moisture to gelatinize.
Starch is generally insoluble in water at room temperature. But
when heat is applied
to progressively higher temperatures, very little changes are
noted until a critical
temperature at which irreversible starch transformation takes
place. The starch
granules start to swell and lose their polarization cross
simultaneously. Since loss of
birefringence occurs at the time of initial rapid gelatinization
(swelling of the
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granule), thus it is a good indicator of the initial
gelatinization temperature of a
given starch. The larger starch granules, which are usually less
compact, begin to
swell first. The amylose chains solubilize and a starch gel is
formed. At this point,
the starch is easily digestible.
In general, the swollen granules are enriched with amylopectin,
while the linear
amylose diffuses out of the swollen granules and makes up the
continuous phase
outside the granules (Hermansson and Svegmark 1996). This is
usually the case for
lipid free amylose when heated in water. But amylose can form
complex with lipid
known as amylose-lipid complex. It is usually made up of a
left-handed amylose
helix with six residues per turn, in which the aliphatic part of
the part is embedded
with the polar group lying outside due to its large molecular
size (Neszmelyi et al
1987). The melting temperature of these crystals is around 110C
for endogenous cereal lipids (Becker et al 2001). The presence of
monoacyl lipids in amylose can
therefore restrict swelling, dispersion of granules and
solubilization of amylose. It is
the enclosed lipid molecules which contribute to the stability
of the amylose helix
conformation (Becker et al 2001).
The gelatinization temperature of starches is directly
correlated with amylose
content (Gerard et al 1999, Sievert and Wuesch 1993, Knutson
1990). The
gelatinization temperatures of high-amylose maize starches are
higher than normal
and waxy maize starches (Colonna and Mercier 1985). Waxy and
regular maize
gelatinize at 62 to 72C while for high-amylose starches, they
begin to swell below
100C, and temperatures greater than 130C are required to fully
disperse these
starches. During the gelatinization process, waxy starches
usually swell to a greater
extent than their normal-amylose counterparts (Tester and
Morrison 1990), and
amylose is proposed to act as a restraint to swelling
(Hermansson and Svegmark
1996).
Starch hydrolysis
Starch degradation mainly occurs at the mashing step in the
production of opaque
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 end-
products in terms of flavor, keeping properties, safety and
overall product image
(Salovaara 1998). Lactobacillus is the preferred genus for
industrial lactic acid
fermentation (Vickroy 1985, Atkinson and Mavituna 1991) because
of high
metabolic rate, high lactate production and the ability of
sustaining lower pH
(Anuradha et al 1999). Homofermentative lactic acid bacteria
such as Lactobacillus
delbrueckii, used for inoculation in the souring stage of opaque
beer production, can
ferment hexoses via glycolysis, with 90% of the glucose being
metabolized to lactic
acid (Kontula et al 1998). Factors affecting the metabolic
products include the
available carbohydrates and growth conditions such as pH,
aeration and cell density
(Kontula et al 1998). The optimum pH and temperature for growth
rate of
Lactobacillus delbrueckii were 5.5 and 45C (Venkatesh et al
1993, Venkatesh 1997).
The lactic acid bacteria require substrates with high nitrogen
content and have a
particular demand for B vitamins (Hofvendahl and Hgerdal 1997).
This can be
satisfied by the addition of malt sprouts or yeast extract
(Vickroy 1985, Atkinson and
Mavituna 1991).
Lactic acid fermentation is capable of lowering the pH to below
4 in food products,
including sorghum-based fermented cereal gruels used as infant
foods (Kunene et al
1999). This is done by the conversion of carbohydrate substrate
to lactic acid which
lowers the pH of the fermenting medium to levels that cannot
support the growth
and activities of many spoilage microorganisms. This results in
growth reduction of
pathogenic bacteria such as B. cereus, Campylobacter spp.,
enterotoxigenic E. coli,
20
-
Salmonella spp., Shigella spp. and S. aureus (Menash et al 1991,
Nout 1991,
Simango and Rukure 1992, Kingamkono et al 1995). Most of the
spoilage and
pathogenic microorganisms are inhibited by a combination of pH
reduction, a
lowering of oxidation-reduction potential, competition for
essential nutrients and the
production of inhibitory compounds such as organic acids,
hydrogen peroxide,
antibiotics and antimicrobial substances (Mbugua and Njenga
1992, Hanciolu and
Karapinar 1997).
Lactic acid fermentation of cereals plays an important role in
terms of nutrient
composition. It has been found to reduce the amount of phytic
acid, polyphenols and
tannins and improved protein availability in sorghum (Chavan et
al 1989). It has also
led to improved iron (Svanberg and Sandberg 1988), minerals and
sugar (Khetarpaul
and Chauhan 1990).
The utilization of soluble carbohydrates by lactic acid bacteria
and consequently,
their energy yield, and lactic and acetic acid production are
greatly influenced by the
associated yeasts and vary according to the types of sugars
(Gobbetti 1998). Just like
yeast, Lactobacillus species have also been reported to produce
ethanol (Hansen and
Hansen 1996).
Yeast
Yeast for brewing not only converts fermentable sugar in the
wort to ethanol and
carbon dioxide, but also produces a variety of volatile and
nonvolatile constituents
that contribute to the overall flavor and acceptability of
fermented cereal gruels
(Banigo et al 1974, Odunfa and Adeyele 1985). Most of the
brewing yeasts will
prosper at a pH of 5.0 to 5.5. The principal fermentable
carbohydrates in the wort
include glucose, fructose, sucrose, maltose and maltotriose.
Sucrose was found to be
the first sugar which disappeared from the wort, indicating the
disappearance of the
disaccharide through hydrolysis by the enzyme invertase. Glucose
and fructose are
then utilized rapidly, followed by maltose (Reed and
Nagodawithana 1991). Pentose
sugars are not metabolized by brewers yeasts. The unfermented
carbohydrates then
21
-
pass through the fermentation and contribute to the caloric
content of the final beer
(Reed and Nagodawithana 1991).
At the beginning of fermentation for beer production, there is
an adequate level of
oxygen in the wort medium, and the yeast undergoes two different
overlapping
metabolic phases, the aerobic respiration and anaerobic
fermentation:
Aerobic Respiration
C6H12O 6 + 6 O2 CO2 + 6 H2O
Anaerobic Fermentation
C6H12O6 2 CO2 + 2 C2H5OH
With adequate level of oxygen in the wort, the initial aerobic
growth phase continues
until all the dissolved oxygen in the wort is utilized and
biomass is formed. For
anaerobic fermentation, ethanol, carbon dioxide and other flavor
components of the
beer are formed.
The wild type Saccharomyces cerevisiae strains intrinsically
lack significant amylase,
and therefore they are unable to metabolize starch (Marn et al
2001). For S.
cerevisiae to utilize starch, starch must be converted to
fermentable sugars such as
glucose or maltose. In the production of opaque beer,
gelatinization by cooking and
enzymatic liquefaction and saccharification by mashing are used
to promote starch
hydrolysis so as to produce a fermentable wort for the
yeast.
Apart from sugar, amino nitrogen is also important for
fermentation and essential for
the growth of yeast. A low proportion of malt is used in the
grist in the production of
opaque beer, and nutrients especially FAN, may limit yeast
growth during
fermentation (Taylor et al 1985). A linear relationship was
found between the sugar
content in opaque beer wort and the quantity of FAN required to
rapidly ferment the
sugars to alcohol, and a minimum requirement of 100 mg/L for FAN
in the wort was
22
-
proposed (Pickerell 1986).
Association of lactic acid bacteria and yeasts
Co-metabolism between LAB and yeasts are common in most of the
African
indigenous fermented foods (Adegoke and Babaola 1988, Steinkraus
1996). This
increases the microbial adaptability to complex food ecosystems
(Gobbetti et al
1994, Stolz et al 1995, Gobbetti and Corsetti 1997). It is
possible that the co-
metabolism between LAB and yeasts benefits one another. The
growth of lactic acid
bacteria could be promoted by growth factors such as vitamins
and soluble nitrogen
compounds from the yeast. On the other hand, the proliferation
of yeasts in foods is
favored by the low pH condition caused by LAB (Nout 1991,
Gobbetti et al 1994,
Steinkraus 1996) and the bacterial end products could be used by
the yeasts as an
energy source (Leroi and Pidoux 1993).
Co-metabolism between LAB and yeasts during fermentation can
also improve the
final quality of the food as well. They impart taste and flavor
to foods by their
metabolites (Akinrele 1970, Halm et al 1993, Brauman et al 1996,
Hansen and
Hansen 1996). The production of acids and antimicrobial
components in gruel
during fermentation can improve microbiological safety (Svanberg
et al 1992,
Kingamkono et al 1994, 1995) and stability of the products
(Menash et al 1991).
23
-
Table 1. Analysis of Opaque Beer
Determination Range Average
pH 3.2-3.9 3.5
Lactic acid (mg %) 164-250 213.0
Volatile acidity as g acetic acid / 100
ml
0.012-0.019 0.016
Total solids (%) 2.6-7.2 4.9
Insoluble solids (%) 1.6-4.3 2.3
Alcohol (%) (by weight) 2.4-4.0 3.2
Nitrogen (%) 0.065-0.115 0.084
Sources: Novellie (1968).
24
-
Table 2. Nutrient Content of Industrially Brewed Opaque Beer
a
Nutrient Mean
Content
RDAb % Contribution to
RDA by 1 L of
Beer
Alcohol (g) 25.4 NRc
Fat (g) Trace 96d
Crude Fiber (g) 1 NR
Protein (g) 5.4 56 9.6
Ash (g) 1.1 NR
Carbohydrate (g) 47.6 446d 10.7e
Starch (g) 29.7 NR
Food energy (kJ) 1,651 12,600 13.1e
Vitamin B1 (thiamin) (mg) 0.2 1.5 16.0e
Vitamin B2 (riboflavin) (mg) 0.4 1.8 21.7e
Niacin (mg) 2.9 20 14.7e
Copper (mg) 0.2 2 10.0e
Iron (mg) 2.0 18 11.1e
Zinc (mg) 1.8 15 12.0e
Manganese (mg) 1.5 3.8 39.5e
Calcium (mg) 53 800 6.6
Magnesium (mg) 140 400 35.0e
Potassium (mg) 276 4,000 6.9
Sodium (mg) 9 10%/L)
25
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CHAPTER 2
EFFECT OF MALTING ON STARCH AND
OTHER COMPONENTS IN SEVEN VARIETIES
OF SORGHUM
2.1 ABSTRACT
The effect of malting on seven varieties of sorghum grain was
compared and the
physicochemical and enzymatic properties of their malt were
studied to predict their
suitability for brewing opaque beer. Malting was carried out and
the germinated
sorghum samples were compared with their ungerminated
counterparts. The seven
varieties showed a decrease in starch ranging from 23.2% to
48.5% and a decrease in
amylose ranging from 13.6% to 42.3% due to the action of
amylolytic enzymes,
resulting in tremendous increase in sugar content. DC-75, a
sorghum variety
commonly used for commercial brewing of opaque beer, had the
lowest content of
starch as well as amylose yet the highest content of glucose,
fructose and total non-
starch carbohydrate among the seven sorghum varieties in this
study. It had a
diastatic power of 55.2 SDU/g, satisfying the minimum
specification of 28 SDU/g.
DC-75 had the lowest -amylase activity yet the highest -amylase
activity among
the seven sorghum varieties investigated. All these properties
were involved in the
final quality of opaque beer. The correlation among the
parameters was examined.
The gelatinization temperatures of sorghum malt and adjunct were
significantly
correlated with each other (r = 0.92). This was believed to be
due to the competition
for water between sugar and starch present in the system.
26
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2.2 INTRODUCTION
Sorghum is a cereal grain well adapted to semi-arid and
sub-tropical conditions. It
can yield crops under harsh environmental conditions such as
drought and heat stress.
It can also withstand flooding and is not influenced much by
acid soil. Apart from its
drought-tolerant characteristic, the available supply, its low
price, its content of
starch and protein has enabled sorghum to become an important
material for brewing
(Haln 1966). The potential of sorghum as an important source of
industrial brewing
material has been long recognized since World War II. At that
time, sorghum was
offered as a brewing material due to the scarcity of barley, the
conventional brewing
material (Haln 1966). In Nigeria, there is already a total
replacement of imported
barley malts with sorghum and maize produced locally. In the
developing countries,
sorghum production is predicted to grow at 1.6 % per annum from
44 million tons in
1992-94 to 53 million tons in 2005, the rise primarily
concentrated in Africa
(ICRISAT 2002).
The malt used in the preparation of opaque beer plays a role in
determining the rate
of fermentation and is a source of lactobacilli, yeasts and
essential nutrients (Agu
1995a, Odunfa and Adeyele 1995, Ugboaja et al 1991). Therefore,
malt is an
important component contributing to the final quality of the
beer and it is absolutely
essential to select the malt which suits the quality of the beer
most. A lot of research
has been done to investigate changes taking place during
malting.
In the present study, the aims were 1) to investigate the effect
of malting on seven
varieties of sorghum grain, 2) to examine the correlation among
starch and other
related parameters in this study and 3) to predict the
suitability of sorghum malt for
the production of opaque beer.
27
-
2.3 MATERIALS AND METHODS
Sorghum Grains
Grains of five varieties of sorghum, DC-75, Brown Tsweta, Red
Swazi, Pato and
Pirira, were supplied by Dr. C. E. Walker Department of Grain
Science and Industry,
Kansas State University. The other two varieties, PL 1 and TX
2737, were supplied
from Kenya.
Malting
Steeping
Clean sorghum grains (50 g) were put into perforated nylon mesh
bags and steeped
for 8 h at 25C in NaOH (0.3%, w/v). They were thoroughly rinsed
with fresh tap water and were then immersed in sodium hypochlorite
solution (2%, v/v) for 10 min
for prevention of microbial growth. They were thoroughly rinsed
with tap water
again for the removal of any residual reagent. Excess water was
then removed by
paper towel.
Germination
Germination was carried out at 25C and 100% RH for five days.
The grains in nylon mesh bag were grown on moist paper towel with
another layer of paper towel
covering on top. The germinating grains were spread evenly
during germination so
as to reduce the resistance of the grain to aeration and assist
the uniform watering of
the green malt. The grains were steeped in fresh tap water for
10 min twice a day.
Excess surface-held water was removed by paper towel. These
steps were done with
great care so as to avoid any damage of the shoots and roots.
Since they were not
protected at all, they were liable to injury which would lead to
unproductive
regrowth of the seedling and give access to parasitic and
potentially toxin-producing
fungi (Daiber and Taylor 1995).
28
-
Drying
After five days of germination, the grains were dried at 50C for
48 h in a forced-air oven.
Milling
The germinated dried grains were milled to pass through a 0.5-mm
sieve with an
Udy Cyclone Mill (Udy Corporation, Ft. Collins, Colorado,
USA).
Dry Matter Loss
Dry matter loss was measured by recording the weight of the
grains before steeping
and after germination and was expressed as the percentage change
in weight of
grains before and after malting.
Germination Activity
The germination activity was recorded by germinating 100 grains
(in triplicate) on
petri dishes. After germination, the number of germinated grains
was counted and
this represented the germination activity.
Germination Activity
= Number of grains germinated / Number of total grains x
100%
Gelatinization Temperature
A Mettler DSC-20 differential scanning calorimeter
(Mettler-Toledo AG
Instruments, Naenikon-Uster, Switzerland) equipped with a
ceramic sensor and a
Mettler TC II data analysis station was used for measuring the
gelatinization
temperatures of sorghum adjunct and malt. Sample (3 mg, dwb), in
duplicate, was
weighed in an aluminium pan and the final weight was made up to
12 mg by
addition of distilled water. It was equilibrated at room
temperature for 1 h and then
heated from 30 C to 100C at a rate of 10C per min. The
gelatinization temperature (C) was recorded.
29
-
Total Starch Content
Total starch content was determined by the amyloglucosidase /
-amylase method using the Megazyme Total Starch Assay Kit
(Megazyme, Bray, Ireland).
Amylose Content Assay
Amylose was determined using an iodine-binding
spectrophotometric method.
Sample (50 mg) was weighed to the bottom of 50 ml volumetric
flask. 95% Ethanol
(1 ml) and 1 M sodium hydroxide (4.5 ml) were added respectively
and mixed well.
The samples were kept at 45C in the oven overnight. Distilled
water (25 ml) was added and the samples were then kept at 100C in
the oven for 60 min for starch gelatinization. They were then
cooled down to room temperature and made up to
volume. The solution was centrifuged at 3,000 rpm for 10 min so
as to remove the
coarse particles. An aliquot (1 ml) was transferred into a 25 ml
volumetric flask, to
which 300 l 1 M acetic acid and 400 l I2-KI solution were added,
made up to volume and mixed well. The solution was kept at room
temperature for 20 min.
Absorbance value was read at 620 nm against blank. Amylose
content was
determined according to a standard curve which was prepared by
maize amylose /
amylopectin (Sigma) standard mixtures representing 0 to 70 %
amylose.
Total non-starch carbohydrate
Starch in the sample was removed according to step 1 to 4 of the
pretreatment of
samples containing glucose and maltosaccharides of Megazyme
Total Starch Assay
Kit. The supernatants of the sorghum adjunct and malt were made
to a total volume
of 50 ml and 500 ml respectively and they were estimated for
their total non-starch
carbohydrate by the phenol-sulphuric acid method (Dubois et al
1956).
Reducing and nonreducing sugars
Reducing and nonreducing sugars were determined according to
AACC Method 80-
60 with the following modification for sorghum malt samples. The
sample weight
was reduced by half while all other factors remained the
same.
30
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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
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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
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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
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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
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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
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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
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2.5 CONCLUSION After 5-d malting, the seven sorghum varieties
showed a decrease in starch and
amylose but an increase in total non-starch carbohydrate,
glucose and fructose
respectively. Diastatic power of the sorghum malt ranged from
32.7 to 70.9 SDU/g,
satisfying the minimum specification of 28 SDU/g. The optimum
incubation
temperature for - and -amylase of the seven sorghum malt
varieties was approximately 55C and 39C respectively with -amylase
contributing a higher proportion of the amylolytic activity of the
sorghum malt. Gelatinization of sorghum
adjunct and malt ranged from 69.3 to 75.1C and 69.8 to 77.1C
respectively and they were significantly correlated with each
other. DC-75, the sorghum variety
commonly used for commercial brewing of opaque beer, had the
lowest content of
starch and amylose but the highest content of glucose, fructose
and total non-starch
carbohydrate among the seven sorghum varieties in this study.
These might have
been the characteristics contributing to its role it played as
being the commonly used
variety in commercial brewing of opaque beer.
40
-
Table 1. Germination activity, dry matter loss and
gelatinization temperature of seven sorghum varieties.
Sorghum variety GAa (%) dmlb (%) Malt GTc(C) Adjunct GTd(C)
DC-75 94.00.8 16.30.5 73.20.1 70.10.0
Brown Tsweta 92.20.8 17.40.9 69.80.3 69.30.2
Red Swazi 88.90.1 16.80.1 70.00.2 69.30.5
Pato 69.41.0 14.60.0 73.30.2 72.00.2
Pirira 74.00.1 14.01.7 72.80.0 71.10.2
PL 1 71.70.4 17.10.21 77.10.4 74.90.1
TX 2737 87.00.0 20.70.91 77.10.3 75.10.2
Mean (Total) 82.4 16.7 73.3 71.7
aGA = Germination Activity
bdml = dry matter loss
cMalt GT = Gelatinization Temperature of Malt
dAdjunct GT = Gelatinization Temperature of Adjunct
41
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Table 2. Changes in total starch % of sorghum during
malting.
Sorghum
variety Total Starch (% db) Total Starch (% db)
Decrease in
starch %
Adjunct Malt
Mean Min Max Mean Min Max
DC-75 68.6 64.8 72.0 35.3 32.6 37.7 48.5
Brown Tsweta 69.9 65.5 75.1 50.2 45.4 59.6 28.2
Red Swazi 67.4 64.1 70.8 41.3 39.9 43.7 38.7
Pato 69.5 64.5 74.1 46.5 44.5 49.5 33.1
Pirira 69.8 65.1 75.7 53.6 50.2 59.1 23.2
PL 1 78.6 72.0 87.0 41.8 39.4 44.8 46.8
TX 2737 62.3 60.0 64.5 43.9 40.6 47.2 29.5
Mean 69.4 44.7 35.7
42
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Table 3. Changes in amylose % of sorghum during malting.
Sorghum
variety % amylose of starch (db) % amylose of starch (db)
Decrease in
amylose % of
starch
Adjunct Malt
Mean Min Max Mean Min Max
DC-75 17.5 16.9 18.7 10.1 9.4 10.8 42.3
Brown Tsweta 27.3 26.1 28.2 23.6 22.0 24.9 13.6
Red Swazi 25.2 24.8 25.7 17.9 17.1 19.3 29.0
Pato 28.9 27.2 29.9 23.2 22.0 24.3 19.7
Pirira 30.2 29.0 31.0 25.1 24.1 26.3 16.9
PL 1 30.8 30.4 31.1 21.5 20.7 22.2 30.2
TX 2737 33.3 32.6 33.9 22.6 21.7 23.6 32.1
Mean 27.6 20.6 25.5
43
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Table 4. Alpha-amylase activity at 55 C, beta-amylase activity
at 40 C, diastatic power and reduction in viscosity in seven
varieties of sorghum malt.
Sorghum variety
Alpha-amylase
(Ceralpha Unit/g)
Beta-amylase
(Betamyl Unit/g)
Diastatic power
(SDU)
Reduction in viscosity
(RVU)
DC-75
104.30.8 56.31.9 55.22.4 40.52.5
Brown Tsweta 279.73.3 34.82.6 70.92.15 45.01.3
Red Swazi 129.90.5 42.31.7 65.52.2 45.72.4
Pato 177.42.0 12.70.1 45.82.1 35.21.0
Pirira 133.43.6 13.70.4 32.72.1 24.00.7
PL 1 175.70.7 26.40.0 50.60.9 47.41.3
TX 2737 157.30.7 36.30.1 52.50.2 59.01.9
Mean 165.4 45.3 53.3 42.4
44
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Table 5. Sugar contents of seven sorghum cultivars.
Sorghum variety
Total non-starch
carbohydrate
(mg/g)
Reducing sugar
(mg maltose/g)
Non-reducing sugar
(mg sucrose/g)
Glucose
(mg/g)
Fructose
(mg/g)
DC-75 10.51.2 4.50.4 6.00.0 5.7 3.9
Brown Tsweta 6.50.8 4.90.0 6.70.0 4.7 1.4
Red Swazi 4.60.2 3.60.0 6.90.2 0.1 1.4
Pato 15.20.8 3.10.0 8.20.1 11.2 3.2
Pirira 8.90.4 2.80.3 6.50.3 5.7 1.4
PL 1 11.40.5 2.50.0 11.40.0 1.2 4.5
TX 2737 6.51.6 2.50.0 9.50.0 3.9 7.1
Mean 9.1 3.4 7.9 4.8 3.2
45
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Table 6. Sugar contents of seven malted sorghum cultivars.
Sorghum variety
Total non-starch
carbohydrate
(mg/g)
Reducing sugar
(mg maltose/g)
Non-reducing sugar
(mg sucrose/g)
Glucose
(mg/g)
Fructose
(mg/g)
DC-75 354.313.9 6.70.2 11.50.1 270.0 73.3
Brown Tsweta 211.024.1 5.90.3 10.40.0 150.9 33.7
Red Swazi 277.20.2 6.40.1 10.10.3 155.8 49.8
Pato 260.88.5 6.80.3 7.90.75 195.5 15.1
Pirira 157.811.6 5.60.0 6.20.0 170.8 18.5
PL 1 209.13.7 14.50.0 21.60.0 114.1 28.5
TX 2737 194.44.9 15.00.1 22.30.3 91.9 28.3
Mean 237.8 8.7 12.9 14.4 35.3
46
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020
40
60
80
100
120
140
160
180
200
220
240
260
280
300
25 35 45 55 65 75Incubation Temperature (C)
Cer
alph
a U
nits
/g
PLTXDC-75nRed BrownPato Pirira
Figure 1. Effect of temperature on sorghum -amylase
activity.
47
-
05
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
25 30 35 40 45 50
Incubation Temperature (C)
Bet
amyl
Uni
t/g
PLTXDC-75RedBrownPatoPirira
Figure 2. Effect of temperature on sorghum -amylase
activity.
48
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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
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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
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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
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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
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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