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ISOLATION AND IDENTIFICATION OF BACTERIA AND FUNGI IN PAP

INTRODUCTION

Fermented maize starch is also known as “Pap”. It is also known as “Ogi” in

the western part of Nigeria by the Yorubas or Akamu in the eastern or “Akassa” in
the north by Ibos and Hausas respectively (Parveen and Hafiz, 2003). It is
fermented maize, millet or sorghum product obtained as smooth gel or mixed with
boiling water to form a porridge, which has a sour taste. These fermented products
are largely produced from Zea mays, Sorghum valgare, Oryza sativa and Triticum
aestivum. Similar maize preparations are referred to as “Akana” and “Kenkey” in
Ghana. It is a popular staple and most popular a traditional weaning food in West
African counties (Adams and Moss, 1995; Amakoromo, 2011). It is used as
weaning food by low income earners who cannot afford the more expensive
imported weaning foods. (Ozoh and Kuyanabana, 1995; Amakoromo, 2011).

Ogi is mostly prepared using traditional fermenting and malting

technologies which are simple but do not guarantee quality and lack of
contaminations as well as lack the appropriate nutritive value (Marero et al., 1989).
It is prepared by soaking (steeping) in water for two to five days, grinding it (wet
milling) and sieved to remove the husk. The main reason for fermenting these
grains is to convert starch contents in the cereals such that it does not require
dilution. The fermenting process also removes the pathogens. Ogi provides about
20-26 Kcal/ kg per day to an infant who has an average density of 0.26 Kcal/ kg
(Brown et al., 1998).

In most parts of Africa especially in Nigeria, children are fed with mashed
adult foods. These foods are bulky and can cause malnutrition. The development of
nutritionally balanced calorie as dense, weaning foods lead to the fermentation of
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maize to produce ‘ogi’. The food must also be of the right quantity to satisfy the
infant at one feeding. It is also a choice meal for patients in need of soft and easily
digestible foods (Jay, 2005). They are important energy food rich in carbohydrates
and with traces of vitamins, proteins and minerals (Achterberg et al., 1994; FAO,
2009) and are natural antioxidants (Eaton and Nelson, 1991).

Its reputation as the most popular traditional weaning food and its
consumption by convalescents in the West African regions calls for a safe product,
free of pathogens and any potentially hazardous microorganisms. The traditional
fermentation processes of pap are usually spontaneous and uncontrolled (Odunfa,
1985) and have led to the loss of nutrients. The nutritive quality of maize porridge
is very low resulting from low quality maize proteins and substantial loss of
nutrients at the different stages of production (Nkama et al., 2000). Consequently, a
number of leguminous seeds including soya beans and okra seeds are used to
fortify and improve their protein, iron, calcium and fibre contents (Osungbaro,
2009; Anigo et al., 2009; Olunkoya et al.,1994) to eliminate the incidences of
anaemia and stunted growth often associated with malnutrition (Muhimbula and
Issa- Zacharia, 2010).

The microbiology of pap and related products has been studied (Odunfa and
Adeyele, 1985; Adegoke and Bablola, 1988; Hountunigha, 1994). New attention is
presently on the use of starter cultures, which is solving numerous problems
associated with the product capable of prevention and treatment of many water
borne diseases using bacteriogenic Lactic Acid Bacteria (LAB) (Olukoya et al.,
1994). Olasupo et al., (1997) increased the shelf-life of ‘pap’ using a bacteriocin-
producing Lactobacillus isolate.
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Ogi is fairly acidic (pH 4.8), which tends to inhibit the growth of some
bacteria. Despite the delicate health position of ‘pap’ to some consumers, the role
of spoilage microorganisms has not been investigated, nor has their potential to
produce harmful metabolites. Its spoilage is however, enhanced by some extrinsic
factors amongst which is storage. There are so many problems which arise from
fermentation of Ogi (i.e. spoilt ogi corn starch) and this may include: deriving a
complete sour taste which may result in over fermentation due to the conception
of people, varietal problems among many others and bad odour may also arise.
Also the length of fermentation can also affect the final product.

Ogi as a fermented food contains bacteria and fungi as a result of the

fermentation which takes place in the cereal starch. Evaluation of the microbial
quality of pap is to identify the contaminants associated with the improper storage
for a relatively length of time. Improper storage is likely to develop other
contaminants which can become harmful to consumers especially children; it could
even lead to food poisoning or/ and intoxication. Therefore the study was carried
out on the maize, the freshly prepared raw ogi that was kept for five days without
changing the water; to identify the organisms present and to estimate the possible
stage preventing the contamination by harmful microorganisms.

LITERATURE REWIEW

Ogi is a fermented cereal porridge from West Africa, typically made from
maize, sorghum, or millet (United Nations FAO, 2006). Traditionally, the grains
are soaked in water for up to three days, before wet milling and sieving to remove
husks. The filtered cereal is then allowed to ferment for up to three days until sour.
It is then boiled into a pap, or cooked to make a stiff porridge. The fermentation of
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ogi is performed by various lactic acid bacteria including Lactobacillus spp, and
various yeasts including Saccharomyces and Candida spp.

Maize (Zea mays L., Poaceae) is the most important cereal in the world after
wheat and rice, with regard to cultivation areas and total production (Purseglove,
1992; Osagie and Eka, 1998). The name maize is derived from the South American
Indian Arawak-Carib word mahiz. It is also known as Indian corn or corn in America
(Kochhar, 1986; Purseglove, 1992). It was introduced into Nigeria probably in the
16th century by the Portuguese (Osagie and Eka, 1998). In Nigeria, maize is known
and called by different vernacular names depending on locality like ‘agbado’,
‘igbado’ or ‘yangan’ (Yoruba); ‘masara’ or ‘dawar masara’ (Hausa); ‘ogbado’ or
‘oka’ (Ibo); ‘apaapa’ (Ibira); ‘oka’ (Bini and Isha); ‘ibokpot’ or ‘ibokpot union’
(Efik) and ‘igumapa’ (Yala).
The global production of maize is estimated to about 300 million tonnes per year.
145million (or about 50 per cent)of which are produced in USA alone (Ihelarouye
and Ngoddy, 1965; Kochhar, 1986; Purseglove, 1992). In Nigeria, its production is
quite common in all parts of the country, from the north to the south, with an annual
production of about 5.6million tonnes (Central Bank of Nigeria, 1992). The
country’s maize crop covers about 1million hectares out of nine million hectares it
occupies in Africa (Hartmans, 1985).
Maize is prepared and consumed in a multitude of ways which varies from
region to region or from one ethnic group to the other. For instance, maize grains
are prepared by boiling or roasting as paste (‘eko’), ‘abado’, and ‘elekute’ in
Nigeria and ‘kenke’ in Ghana, or as popcorn which is eaten all over West Africa.
Traditional methods of preparations and uses of maize are restricted to definite
localities or ethnic groups. This trend was also noted in the traditional preparation
and uses of cassava (Manihot esculenta Crantz, Euphorbiaceae) by Etejere and Bhat
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(1985). The current investigations on the traditional preparations and uses of maize
by various ethnic groups in Nigeria is to make available different methods of
preparations and uses to all people.

TYPES OF MAIZE

Maize, the American Indian word for corn, means literally "that which
sustains life". It is, after wheat and rice, the most important cereal grain in the
world, providing nutrients for humans and animals and serving as a basic raw
material for the production of starch, oil and protein, alcoholic beverages, food
sweeteners and, more recently, fuel. The green plant, made into silage, has been
used with much success in the dairy and beef industries. After harvest of the grain,
the dried leaves and upper part, including the flowers, are still used today to
provide relatively good forage for ruminant animals owned by many small farmers
in developing countries. The erect stalks, which in some varieties are strong, have
been used as long-lasting fences and walls.

Botanically, maize (Zea mays) belongs to the grass family (Gramineae) and
is a tall annual plant with an extensive fibrous root system. It is a cross pollinating
species, with the female (ear) and male (tassel) flowers in separate places on the
plant. The grain develops in the ears, or cobs, often one on each stalk; each ear has
about 300 to 1 000 kernels, weighing between 190 and 300 g per I 000 kernels, in a
variable number of rows (12 to 16). Weight depends on genetic, environmental and
cultural practices. Grain makes up about 42 percent of the dry weight of the plant.
The kernels are often white or yellow in colour, although black, red and mixtures
of colours are also found. There are a number of grain types, distinguished by
differences in the chemical compounds deposited or stored in the kernel.
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Special crops grown primarily for food include sweet corn and popcorn,
although dent, starchy or floury and flint maize are also widely used as food. Flint
maize is also used as feed. Immature ordinary corn on the cob either boiled or
roasted is widely consumed. Floury maize is a grain with a soft endosperm much
used as food in Mexico, Guatemala and the Andean countries. The dent type of
maize has a vitreous horny endosperm at the sides and back of the kernel, while the
central core is soft. Flint kernels have a thick, hard and vitreous endosperm
surrounding a small, granular, starchy centre.

ABOUT YELLOW MAIZE

Yellow maize is known as Zea mays varrugosa. Yellow corn is just one
cultivar among thousands of corn varieties, heirloom, and hybrid or genetically
modified. Yellow corn is an evolutionary mutation of white corn. Most yellow corn
in the commercial marketplace is a hybrid variety developed in the 20th century.
The first hybrid yellow sweet corn, 'Red green' was released by the Connecticut
Agricultural Experimental program in 1924.

The occurrence of yellow or white colour of maize kernels is controlled by a

single gene. Each maize grain is actually a different individual (the seed of a new
plant) with a unique mix of genes inherited from its parents. The difference
between yellow and white maize grains is due to the colour of the nutritive tissue
that surrounds the embryo in the seed, called the endosperm.

Endosperm colour in maize is controlled by a single gene. The presence of a

single (dominant) Y allele leads to the production of carotenoid pigments in the
endosperm and results in yellow seeds. White maize seeds occur when two
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recessive Y alleles are present: carotenoids are not produced and the endosperm is
white.

The colour of the endosperm may be masked if the outer layer surrounding it
(called the aleurone) is also pigmented. The aleurone may be colourless (as in
white and yellow maize), purple or red.

Description/Taste

Yellow corn is a variety of sweet corn. Its ears are wrapped in tightly bound
lime-hued husks with silks and a tassel that extend out from the tip. The yellow
kernels are packed in tight almost uniform rows. A single ear of corn can contain
up to 400 kernels. Freshly harvested yellow corn at its peak ripeness is sweet,
offering flavors of almond and sugar, the kernels so succulent, the skin pops as you
bite into it. As the corn matures, the kernels lose their milky consistency giving
way to a starchy and doughy consistency. At this point, the corn is considered a
grain crop and is best suited for processing or feedstock.

Nutritional Value

Yellow corn is a significant resource of Vitamin A. As corn kernels mutated

from white to yellow, they acquired chemicals called cartenoids. Of these
cartenoids is beta carotene, which produces Vitamin A. Very little attention has
been paid on the yellow corn's significant beta carotene levels until the early 21st
century. Yellow corn, easy to grow in developing regions of Africa and Latin
America, where corn is heavily relied upon as a food source, could actually keep
millions of children from going blind. Yellow corn is now being bred to have at
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least 10 times higher amount of beta carotene than the average sweet corn
varieties.

Applications

The less the sweet corn is cooked, the better the flavor and texture. Yellow
corn can be roasted, grilled, blanched, steamed, or pureed. Its bright and sweet
flavors lend well to pastas and salads. It pairs well with tomatoes, basil cilantro,
lobster, pork, chilies, truffles, shelling beans, cream, nutty cheeses, peas, summer
squashes, fennel, citrus and scallops. Yellow corn is dried and ground into flour for
baked goods, tortillas, cereals and used as a crust/crisping agent for dishes both
savory and sweet. Corn is also used for oil, as a sweetener in foods and beverages
and as a base for beverage alcohol.

PRODUCTION

Method

Because it is cold-intolerant, maize is usually planted in spring in the

temperate regions of the world. Its root system is generally shallow, so the plant is
dependent on soil moisture. As a C4 plant (a plant that uses C4 carbon fixation),
maize is a considerably more water-efficient crop than C3 plants (plants that use
C3 carbon fixation) like the small grains, alfalfa and soybeans. Maize is most
sensitive to drought at the time of silk emergence, when the flowers are ready for
pollination. In the United States, a good harvest was traditionally predicted if the
maize were "knee-high by the Fourth of July", although modern hybrids generally
exceed this growth rate. Maize used for silage is harvested while the plant is green
and the fruit immature. Sweet corn is harvested in the "milk stage", after
pollination but before starch has formed, between late summer and early to mid-
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autumn. Field maize is left in the field very late in the autumn to thoroughly dry
the grain, and may, in fact, sometimes not be harvested until winter or even early
spring. The importance of sufficient soil moisture is shown in many parts of
Africa, where periodic drought regularly causes maize crop failure and consequent
famine. Although it is grown mainly in wet, hot climates, it has been said to thrive
in cold, hot, dry or wet conditions, meaning that it is an extremely versatile crop
(Fernandez-Armesto, Felipe, 2011).

Maize was planted by the Native Americans in the hills, in a complex system
known to some as the Three Sisters. Maize provided support for beans, and the
beans provided nitrogen derived from nitrogen-fixing rhizobia bacteria which live
on the roots of beans and other legumes; and squashes provided ground cover to
stop weeds and inhibit evaporation by providing shade over the soil (Mann,
Charles C., July 2011). This method was replaced by single species hill planting
where each hill 60–120 cm (2.0–3.9 ft) apart was planted with three or four seeds,
a method still used by home gardeners. A later technique was "checked maize",
where hills were placed 40 inches (1.0 metre) apart in each direction, allowing
cultivators to run through the field in two directions. In more arid lands, this was
altered and seeds were planted in the bottom of 10–12 cm (3.9–4.7 in) deep
furrows to collect water. Modern technique plants maize in rows which allows for
cultivation while the plant is young, although the hill technique is still used in the
maize fields of some Native American reservations.

In North America, fields are often planted in a two-crop rotation with a

nitrogen-fixing crop, often alfalfa in cooler climates and soybeans in regions with
longer summers. Sometimes a third crop, winter wheat, is added to the rotation.
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Many of the maize varieties grown in the United States and Canada are
hybrids. Often the varieties have been genetically modified to tolerate glyphosate
or to provide protection against natural pests. Glyphosate is an herbicide which
kills all plants except those with genetic tolerance. This genetic tolerance is very
rarely found in nature.

In Midwestern United States, low-till or no-till farming techniques are

usually used. In low-till, fields are covered once, maybe twice, with a tillage
implement either ahead of crop planting or after the previous harvest. The fields
are planted and fertilized. Weeds are controlled through the use of herbicides, and
no cultivation tillage is done during the growing season. This technique reduces
moisture evaporation from the soil, and thus provides more moisture for the crop.
The technologies mentioned in the previous paragraph enable low-till and no-till
farming. Weeds compete with the crop for moisture and nutrients, making them
undesirable

Before World War II, most maize in North America was harvested by hand.
This involves a large numbers of workers and associated social events (husking or
shucking bees). Some one- and two-row mechanical pickers were in use, but the
maize combine was not adopted until after the War. By hand or mechanical picker,
the entire ear is harvested, which then requires a separate operation of a maize
sheller to remove the kernels from the ear. Whole ears of maize were often stored
in corn cribs, and these whole ears are a sufficient form for some livestock feeding
use. Few modern farms store maize in this manner. Most harvest the grain from the
field and store it in bins. The combine with a corn head (with points and snap rolls
instead of a reel) does not cut the stalk; it simply pulls the stalk down. The stalk
continues downward and is crumpled into a mangled pile on the ground. The ear of
maize is too large to pass between slots in a plate as the snap rolls pull the stalk
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away, leaving only the ear and husk to enter the machinery. The combine separates
out the husk and the cob, keeping only the kernels.

For storing grain in bins, the moisture of the grain must be sufficiently low
to avoid spoiling. If the moisture content of the harvested grain is too high, grain
dryers are used to reduce the moisture content by blowing heated air through the
grain. This can require large amounts of energy in the form of combustible gases
(propane or natural gas) and electricity to power the blowers (Van Devender, Karl,
July 2011).

USES OF MAIZE

Human food

Maize and cornmeal (ground dried maize) constitute a staple food in many
regions of the world. Maize is central to Mexican food. Virtually every dish in
Mexican cuisine uses maize. One form of grain or cornmeal, maize is the main
ingredient of tortillas, tamales, pozole, atole and all the dishes based on them, like
tacos, quesadillas, chilaquiles, enchiladas, tostadas and many more. In Mexico
even a fungus of maize, known as huitlacoche is considered a delicacy.

Introduced into Africa by the Portuguese in the 16th century, maize has become
Africa's most important staple food crop. Maize meal is made into a thick porridge
in many cultures: from the polenta of Italy, the angu of Brazil, the mămăligă of
Romania, to cornmeal mush in the US (and hominy grits in the South) or the food
called mealie pap in South Africa and sadza, nshima and ugali in other parts of
Africa. Maize meal is also used as a replacement for wheat flour, to make
cornbread and other baked products. Masa (cornmeal treated with limewater) is the
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main ingredient for tortillas, atole and many other dishes of Central American
food.
Popcorn consists of kernels of certain varieties that explode when heated,
forming fluffy pieces that are eaten as a snack. Roasted dried maize ears with semi-
hardened kernels, coated with a seasoning mixture of fried chopped spring onions
with salt added to the oil, is a popular snack food in Vietnam. Cancha, which are
roasted maize chulpe kernels, are a very popular snack food in Peru, and also
appears in traditional Peruvian ceviche. An unleavened bread called makki di roti is
a popular bread eaten in the Punjab region of India and Pakistan.
Chicha and chicha morada (purple chicha) are drinks typically made from
particular types of maize. The first one is fermented and alcoholic, the second is a
soft drink commonly drunk in Peru. Corn flakes are a common breakfast cereal in
North America and the United Kingdom, and found in many other countries all
over the world.
Maize can also be prepared as hominy, in which the kernels are soaked with
lye in a process called nixtamalization; or grits which are coarsely ground hominy.
These are commonly eaten in the Southeastern United States, foods handed down
from Native Americans, who called the dish sagamite.
The Brazilian dessert canjica is made by boiling maize kernels in sweetened
milk. Maize can also be harvested and consumed in the unripe state, when the
kernels are fully grown but still soft. Unripe maize must usually be cooked to
become palatable; this may be done by simply boiling or roasting the whole ears
and eating the kernels right off the cob. Sweet corn, a genetic variety that is high in
sugars and low in starch, is usually consumed in the unripe state. Such corn on the
cob is a common dish in the United States, Canada, United Kingdom, Cyprus,
some parts of South America, and the Balkans, but virtually unheard of in some
European countries. Corn on the cob was hawked on the streets of early 19th-
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century New York City by poor, barefoot "Hot Corn Girls", who were thus the
precursors of hot dog carts, churro wagons, and fruit stands seen on the streets of
big cities today (Solon Robinson, 1853). The cooked, unripe kernels may also be
shaved off the cob and served as a vegetable in side dishes, salads, garnishes, etc.
Alternatively, the raw unripe kernels may also be grated off the cobs and processed
into a variety of cooked dishes, such as maize purée, tamales, pamonhas, curau,
cakes, ice creams, etc.

Maize is a major source of starch. Cornstarch (maize flour) is a major

ingredient in home cooking and in many industrialized food products. Maize is
also a major source of cooking oil (corn oil) and of maize gluten. Maize starch can
be hydrolyzed and enzymatically treated to produce syrups, particularly high-
fructose corn syrup, a sweetener; and also fermented and distilled to produce grain
alcohol. Grain alcohol from maize is traditionally the source of Bourbon whiskey.
Maize is sometimes used as the starch source for beer. Within the United States,
the usage of maize for human consumption constitutes about 1/40th of the amount
grown in the country. In the United States and Canada, maize is mostly grown to
feed for livestock, as forage, silage (made by fermentation of chopped green
cornstalks), or grain. Maize meal is also a significant ingredient of some
commercial animal food products, such as dog food.

Maize is also used as a fish bait, called "dough balls". It is particularly popular in
Europe for coarse fishing.
Alternative medicine
Stigmas from female maize flowers, popularly called corn silk, are sold as
herbal supplements.
Chemicals
Starch from maize can also be made into plastics, fabrics, adhesives, and
many other chemical products.
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The corn steep liquor, a plentiful watery byproduct of maize wet milling process, is
widely used in the biochemical industry and research as a culture medium to grow
many kinds of microorganisms (Liggett, R. Winston; Koffler, H., December 1948).
Chrysanthemin is found in purple corn and is used as a food coloring.
Medicinal Uses
A crop which is highly edible and nutritious as maize, also has some
medicinal uses among the local people. It is used to cure many diseases,
which it had over the years proved to be very effective. These include:

1. Water filtered through charcoal obtained from maize stalk can be used as a
treatment to cure gonorrhea (AbdulRahaman, 1997).
2. An infusion obtained from stigma of maize inflorescence can be used for
treatment of diseases of the urinary tract or passage (AbdulRahaman, 1997).
3. Water (i.e. ‘omi-eko’ or ‘omikan’ or ‘omidun’) obtained during the
preparation of pap is used to soak bark or root of some plants (e.g. ‘dokita
igbo’). This is used to treat fever and malaria. Water obtained from the cold-
pap is more effective than that from the hot-pap.
4. Cold-pap or ‘eko-tutu’ is used more often in traditional medicines. It is
mixed with some preparations (usually granulated, black particles) to cure
some spiritual problems. It may be prescribed to provide protection against
enemies, bad occurrences or to foster posterities.
5. Holes are created or made in some maize grains to make rosary. This is
put on the hand (wrist) of a child to prevent him or her from becoming slim.
6. Whole dried maize fruit and dried yam with some charms are planted or
buried together. This preparation is done to unite or bind couple together
with effect that either of them cannot remarry to another person. It means
that they will remain husband and wife forever.
FORMS OF MAIZE CONSUMPTION
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Maize is consumed in many forms in different parts of the world, from

maize grits, polenta and corn bread to popcorn and products such as maize flakes
(Rooney and Serna-Saldivar, 1987). The grain is fermented to give ogi in Nigeria
(Oke, 1967) and other countries in Africa (Hesseltine, 1979) and is decorticated,
degermed and precooked to be made into arepas in Colombia and Venezuela
(Instituto de Investigaciones Tecnológicas, 1971; Rodriguez, 1972).
In Egypt, maize flat bread, aish merahra, is widely produced. Maize flour is
used to make soft dough spiced with five percent ground fenugreek seeds, which is
believed to increase the protein content, improve digestibility and extend the
storage life of the bread. The dough is fermented all night with a sourdough starter.
In the morning the dough is shaped into small, soft, round loaves, which are left for
30 minutes to "prove". Before baking, the loaves are made into wide, flat discs.
Aish merahra keeps fresh for seven to ten days if it is stored in airtight containers.
A similar product called markouk is eaten in Lebanon.
Maize is also widely used to make beer. In Benin, for example, malt is obtained by
germinating the grain for about five days. The malt is then exposed to the sun to
stop germination. The grains are lightly crushed in a mortar or on a grinding stone.
The malt is cooked and the extract is strained off, cooled and allowed to stand.
After three days of fermentation it is ready to be drunk as beer (FAO, 1989).
The lime-cooking process for maize is particular to Mexico and Central
America (Bressani, 1990), although today the technology has been exported to
other countries such as the United States. A dough prepared from lime-cooked
maize is the main ingredient for many popular dishes such as atole, a beverage
with a great variety of flavours, and tamalitos, made by wrapping the dough in
maize husks and steam-cooking it for 20 to 30 minutes to gelatinize the starch.
This form is usually prepared with young chipilín leaves (Crotalaria longirostrata),
the flowers of loroco (Fernaldia pandurata) or cooked beans mixed with the dough,
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thus improving the nutritional quality of the product and its flavour (Bressani,
1983). The dough is also used for tamales, a more complex preparation because of
the number of ingredients it contains, in most cases with chicken or pork meat
added to the gelatinized dough. It is also used to provide support for enchiladas,
tacos (folded tortillas containing meat, etc.) and pupusas, the latter made with fresh
cheese placed between two layers of dough and baked like tortillas. When the
dough is fried and flavoured, it yields foods such as chips and chilaquiles. If the
dough is allowed to ferment for two days, wrapped in banana or plantain leaves, it
provides a food named pozol from which a number of drinks can be made. It has
been claimed that this preparation is of high nutritional quality.
There are many ways to convert maize into interesting and acceptable forms
which, if presented in attractive and easily prepared products, could to some extent
counteract the trend toward greater consumption of wheat derived foods in arepa-
and tortilla-eating countries and elsewhere.

OGI AND OTHER FERMENTED MAIZE PRODUCTS

Acid porridges prepared from cereals are eaten in many parts of the world,
particularly in developing countries, where they may form part of the basic diet.
Some examples of acid porridges include pozol in Mexico and Guatemala, ogi in
Nigeria, uji in Kenya and kenkey in Ghana. These porridges are usually made from
fermented raw or heat-treated maize, although sorghum and millet are often used.

OGI MANUFACTURE
The traditional process of making ogi has a number of slight variations
described by several authors. Ogi is traditionally prepared in batches on a small
scale two or three times a week, depending on demand. The clean grain is steeped
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in water for one to three days to soften. Once soft, it is ground with a grinding
stone, pounded in a mortar or ground with a power mill. The bran is sieved and
washed away from the endosperm with plenty of water. Part of the germ is also
separated in this operation. The filtrate is allowed to ferment for 24 to 72 hours to
produce slurry which when boiled gives the ogi porridge. Ogi is usually marketed
as a wet cake wrapped in leaves, or it may be diluted to 8 to 10 percent solids in
water and boiled into a pap or cooked to a stiff gel.
Akinrele (1970) reported that the souring of the maize took place
spontaneously without the addition of inoculants or enzymes. He identified the
organisms involved in this unaided fermentation and investigated their effects on
the nutritive value of the food. He identified the moulds as Epholosporium,
Fusarium, Aspergillus and Penicillium species and the aerobic bacteria as
Corynebacterium and Aerobacter species, while the main lactic acid bacterium he
found was Lactobacillus plantarum. There were also yeasts: Candida mycoderma,
Saccharomyces cerevisiae and Rhodotorula sp.
Although ogi is supposed to have an improved B-vitamin content, the results
observed are quite variable, at least for thiamine, riboflavin and niacin. Banigo and
Muller (1972) identified the carboxylic acids of ogi fermentation. They found 11
acids, with lactic, acetic and butyric acids being the most important.
The ogi-making process is quite complex, and the porridge can also be
prepared from sorghum, rice, millet and maize. Therefore, laboratory procedures
have been developed to learn more about the process and introduce changes to
convert the grains to food more efficiently. These have been described by
Akingbala, Rooney and Faubion (1981) and Akingbala et al. (1987), whose studies
have been useful also in evaluating varieties of cereal grains for their efficiency in
making ogi. The authors also reported on the yields of ogi from whole maize
kernels (79.1 percent) and dry milled flour (79.8 percent).
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The commercial manufacture of ogi does not differ substantially from the
traditional method. Modifications have been introduced, such as the dry milling of
maize into a fine meal or flour and subsequent inoculation of the flour-water
mixture with a culture of lactobacilli and yeast. In view of the importance of ogi in
the Nigerian diet, large-scale production is indicated. The material could be dried
and packaged in polythene bags for a good shelf life. There is some problem in
achieving a controlled fermentation with pure cultures. Some modifications
include spray-drying the slurry or drum drying.

OTHER MAIZE PREPARATIONS

In Latin America there are many maize-based foods besides tortillas and
arepas. Some of these are drinks like colados, pinol and macho, basically
suspensions of cooked maize flour. These three products have a very low protein
quality. The production of humitas, a tamale-like food consumed in Bolivia and
Chile, was described by Camacho, Bañados and Fernandez (1989). Made from
immature common or opaque-2 maize to which is added a number of other
ingredients, humitas is produced from precooked maize flour which resembles the
lime-treated masa. Other products include mote, made from cooked maize and
cheese, pupusas, made from lime-treated maize and cheese, and patasca, which is
like a lime-treated maize kernel. From immature maize a sweet, tasty atole of high
nutritive value is made; Khan and Bressani (1987) described the process, which
consists of grinding the maize in water followed by filtration and cooking.
Immature maize, either common or opaque2, and sweet maize are also extensively
consumed. Chavez and Obregon (1986) reported on the incorporation of the
opaque-2 gene into sweet maize to provide a food of high nutritional quality.
Maize has also been used as a substrate for fermented beverages called
chicha. Cox et al. (1987) have reported on the microflora of these fermented
 19

products, which are made by basically the same process but using a variety of
additives.
NUTRITIONAL AND CHEMICAL CHANGES OF OGI AND OTHER
FERMENTED MAIZE PRODUCTS
Chemical changes
The process of fermenting maize, sorghum, millet or rice to produce ogi not
only removes parts of the maize kernel such as the seed-coat and the germ, but also
involves washing, sieving and decanting, all of which induce changes in the
chemical composition and nutritive value of the final product. Akinrele (1970)
reported on specific nutrients of a number of ogi samples produced in different
ways: unfermented and fermented with Aerobacter cloacae, Lactobacillus
plantarum and a mixture of the two bacteria. He compared the values found with
those from the traditionally fermented product. Judging from the ratio of amino
nitrogen to total nitrogen, the author reported that protein was degraded to a very
small amount by any bacterial species. When compared with the unfermented ogi,
A. cloacae appeared to synthesize more riboflavin and niacin, which did not take
place with L. plantarum. Traditionally produced ogi had more thiamine and slightly
lower values of riboflavin and niacin than that made with maize and A. cloacae.
In any case the changes were small, and smaller if compared with whole
maize, whereas in comparison with degermed maize, the ogi products contained
more riboflavin and niacin. Akinrele (1970) and Banigo and Muller (1972)
reported on the carboxylic acids in ogi and found lactic acid in greatest
concentration (0.55 percent) followed by acetic acid (0.09 percent) and smaller
amounts of butyric acid. The latter investigators suggested levels of 0.65 percent
for lactic acid and 0.11 percent for acetic acid, responsible for the sour taste, as
goals for flavour evaluations. Banigo, de Man and Duitschaever (1974) reported on
the proximate composition of ogi made from common whole maize which was
 20

uncooked and freeze-dried or cooked and freeze-dried after fermentation. Changes

were relatively small in all major nutrients, with a slight increase in fibre and a
decrease in ash content when compared with whole maize.
These authors also reported on amino acid content; they found no
differences between maize flour and ogi for all amino acids including the essential
ones. The ogi samples, however, had about twice the amount of serine and
somewhat higher values for glutamic acid. Adeniji and Potter (1978) reported that
ogi processing did not decrease the protein content of maize, but total and available
lysine were significantly reduced. On the other hand, tryptophan levels were more
stable and in two samples increased, probably because of fermentation. These
authors also found an increase in neutral detergent fibre and ash but no change in
lignin. Akingbala et al. (1987) found a decrease in protein, ether extract, ash and
crude fibre in ogi as compared with maize that was processed as a whole grain or
dry milled.
Nutritional changes
Nutritional evaluations of ogi and other maize-fermented products are not
readily available. Adeniji and Potter (1978) found a substantial decrease in protein
quality of drum-dried common maize ogi, which they ascribed to the drying
process. These same authors reported significant losses in lysine. Several authors
have more recently tested maize and sorghum and reported that fermentation
improved the nutritional quality of the product. Akinrele and Bassir (1967) found
net protein utilization, protein efficiency ratio and biological value of ogi inferior
to those values in whole maize, even though some increase in thiamine and niacin
was obtained. It has been indicated that some of the micro-organisms responsible
for ogi fermentation, such as Enterobacter cloacae and L. plantarum, use some of
the amino acids for growth. This together with the elimination of the germ from
kernels explains the very low protein quality of ogi and similarly produced maize
 21

products. However, there are some exceptions, such as kenkey and pozol, both
products in which the maize is fermented with the germ. Although protein quality
values are not available for kenkey, Cravioto et al. (1955) found higher levels of
tryptophan and available lysine which suggested higher protein quality than in raw
maize or lime-treated maize. More recently, Bressani (unpublished) found the
fermented product to be higher in protein quality than raw maize, but not different
in quality from lime-cooked dough.
Microbial Properties of Ogi
Three distinctive fermentative phases are characterized with Ogi production.
At steeping, gram negative organisms predominates especially Achromobacter and
Klebsiella spp. Following the milling and sieving, gram negative organisms and
lactic acid bacteria especially Streptococcus spp dominates. The final stage of
souring is dominated by non-homofermentative lactic acid bacteria especially
Lactobacillus plantarum and Pediococcus gunther leads to the involvement of
yeast as a minority component. Saccahromyces cerevisiae dominates the steeping
stage while Candida survives in the finished product. Fermentation temperature is
the major factor affecting the types of organisms involved in the production
process. The Lactic acid bacteria Lactobacillus sp, Corynebacterium sp and
Enterobacter sp were among the major organisms responsible for the fermentation
and nutritional improvement of Ogi (Akinrele, 1970).
At 150C, gram negative organisms survive in the products at the end of
5days; whereas at 330C and 370C, the flora is more heterogeneous and the end
product is accompanied by an odd odour and taste. It also leads to shelf life
reduction and makes it unfit for consumption. Large fermentation rods suggestive
of Bacillus spp. are target to be the casual agent of proteolysis and the putrial odour
of ogi. Also the gas evolution that is found during steeping is attributed to the
presence of Klebsiella aerogens.
 22

Biochemistry of Ogi
When the grain is fermented, there is an increase in pH. The raw ogi contains
much less protein than the parent cereal because some soluble proteins are lost in
steeping, washing with water and during mashing. Acid reacting substances are
present during mashing. Acid reacting substances are present during ogi
fermentation and increases as the fermentation progresses.
Lactic acid is the primary volatile acid of ogi fermentation, acetic acid is the
main volatile acid followed by butyric acid. Other volatile acids of fermentation
are formic acid, propionic acid, isobutyric acid, isohexonic acid, etc. The steeping
water contains a higher amount of volatile acids unlike finished products, because
the bulk of the acid produced in the later stages of the fermentation is leached out
into the water. These acids appear in form of film on the surface of the water.
MICROBIAL CONTAMINATION AT DIFFERENT STAGES OF OGI
PRODUCTION
The outbreak of infectious and communicable diseases in tropical parts of
the world is primarily as a result of food poisoning due to microbial contamination
(Jay, 2005). They are often responsible for acute gastroenteritis, abdominal
discomfort and pain and diarrhoea in infants and young adults (WHO, 2010;
Kimmons et al., 1999).
The maize grains were almost surfaced sterile prior to soaking. The isolated
Staphylococcus aureus in few maize samples could have arisen from contaminated
sacks used for storage and transportation of produce. Onovo and Ogaraku (2007)
discovered some bacteria and fungi on exposed Tigernut (Cyperus esculentus L.)
before processing. The presence of Aspergillus flavus, A. niger, Penicillium
oxalicum, Fusarium oxysporium, Rhizopus stolonifer, Saccharomyces cerevisia
Candida albicans Escherichia coli Klebsiella aerogenes and Staphylococcus
aureus in water from the reservoirs suggest s an extremely poor storage system,
 23

deplorable sanitary conditions available at the three sites and multiple sources of
contamination due to open access to the reservoirs. Similar and related organisms
were implicated in food and canned products by Gadaga et al. (2008), Taulo et al.
( 2009) and Oladipo and Omo-Adua (2011).
The exposure of water tanks to direct rays from the sun provided the
required warmth and physical condition for growth of these organisms. Ozoh and
Kuyanbana (1995) and Osho and Fagade (2000) equally affirmed water as the
source of Shigella spp. and E. coli in maize and other cereal porridge.
Contaminated water was linked as the main source of Vibrio cholera infection
(Shahcheraghi et al., 2009) in some population in Iran. Oranusi et al. (2007)
estimated 2-3 Log10 coliforms per 100 g mL -1 of cooked maize porridge and
linked contamination to the water used during washing and soaking of maize
grains. Heavy presence of E. coli, Klebsiella pneumonia and Streptococcus sp. was
reported (Yeboah-Manu et al., 2010) in some foods sold around the University of
Ghana campus. The introduction Salmonella sp., Rhizopus sp. and Staphylococcus
aureus in some food products have been linked to the presence of phytotoxin by
Okafor and Omodamiro (2006).
The attendants at the three study sites admitted collecting water from
streams and lakes whenever the public water supply failed and often time untreated
water was used during preparation. Good and hygienic water supply has been the
bane of many communities in the developing countries (Ehiri et al., 2001)
including Nigeria where the needed social and infrastructural facilities are grossly
inadequate.
The unlimited and open access to the water tanks allowed cross
contamination of the cooking utensils and bowls and subsequent re-contamination
of products at the later stages of production. The body swabs and underneath of
nails contained substantial counts of Staphylococcus aureus and Lactobacillus
 24

plantarum and also, E. coli, Klebsiella aerogenes and Saccharomyces cerevisiae.

The Muslin clothes used in sieving the shaft were stained and soiled and often
reused without thorough washing. The wrapping leaves and polythene were not
sufficiently rinsed or sterilized before use. Omemu and Adeosun (2010) observed
similar unhygienic practices among attendants and vendors at some production
sites in Abeokuta, Nigeria.
Air was laden with Aspergillus flavus, Penicillium oxalicum and Rhizopus
stolonifer and served as source of re-contamination of the finished products.
Wacher et al. (1993) linked the contamination of freshly prepared pozol, traditional
Mexican fermented maize dough to the surrounding air. The growth of bacteria
(Escherichia coli and Klebsiella aerogenes) declined significantly in fully
fermented wet paste as rightly observed by Byaruhanga et al. (1999) for Bacillus
cereus after 24 h fermentation. Also, Mensah et al. (1990, 1991) observed a
significant inhibition in the growth of some gram-negative bacteria. Chukeatirote
et al. (2010) observed an exponential increase in the population of bacteria and
fungi with increased pH and fermentation time of grain.
However, a re-contamination at latter stages of production by these enteric
bacteria as observed could be linked to water as it was used repeatedly during
preparation. Odugbemi et al. (1993) reported an increase in the level of faecal
coliforms in cooked Ogi under 9 h storage conditions and suggested a probable re-
introduction during storage. A similar conclusion was held by Sanni et al. (2002)
for the rise in the population of yeast from 1.0 cfu g -1 to 5.36 cfu g-1 after 12 h
fermentation. Alalade and Adeneye (2007) observed a significant correlation
between pH and coliform bacterial count in wara cheese during fermentation
process.
We observed few counts of Escherichia coli and Klebsiella aerogenes in
fully fermented products (wrapped wet Ogi). Poor handling by vendors was rightly
 25

suggested by Wacher et al. (1993) for the significant increase in enteric bacteria in
freshly prepared pozol. On the other hand, the growth of Lactobacillus plantarum
was unhindered at the different stages of production, even after 48 h fermentation.
Relatedly, an exponential increase in growth of some lactic acid bacteria was
earlier reported by Kunene et al. (1999) in both fermented and cooked maize
porridge. The critical contamination points during the preparation of Ogi included
the point of soaking of grains, mill and wrapping materials. Effective good
manufacturing practices (GMP) as recommended by Amoa-Awua et al. (2007)
would help eliminate contaminants for improved table quality and assure the health
of consumers.
APPROACHES TO IMPROVING THE NUTRITIVE VALUE OF MAIZE
Processing
Often the processing of foodstuffs stabilizes nutrients in the food, but losses
may take place when optimum conditions are exceeded. There are cases, however,
in which processing induces beneficial changes in the food; a classic case is the
elimination of antiphysiological factors in beans.
Lime-cooking
Lime-cooking of maize as described in Chapter 4 causes some losses in
nutrient content, but it also induces some important nutritional changes. Its effects
on calcium, amino acids and niacin content have already been described in Chapter
4.
Other processes
Besides the lime cooking process, other processes have been reported to
improve the quality of maize. One such process is natural fermentation of cooked
maize, which results in higher B-vitamin concentration and protein quality (Wang
and Fields, 1978). Pozol, a food made from lime-treated maize allowed to ferment
naturally, has been shown to be of higher quality than raw maize or tortillas.
 26

Germination of the grain has also been reported to improve the nutritional value of
maize by increasing lysine and to some extent tryptophan (Tsai, Dalby and Jones,
1975; Martinez, Gómez-Brenes and Bressani, 1980) and decreasing zein content. A
similar result was found with QPM.
Fortification
A third approach often used to improve the nutritive value of foods, mainly
cereal grains, is fortification. Because of the great nutritional limitations in maize,
a lot of efforts have been made to improve its quality, and particularly that of its
protein, through addition of amino acids or protein sources rich in the limiting
amino acids.
Supplementation with amino acids
Raw maize proteins have been shown to be of a low nutritive value because
of deficiencies in the essential amino acids lysine and tryptophan. Many studies
conducted with animals have demonstrated that the addition of both amino acids
improves the quality of the protein. Some workers have even found that besides
lysine and tryptophan, isoleucine is also deficient, possibly because of an excess of
leucine in maize proteins (Rosenberg, Rohdenburg and Eckert, 1960). Similar data
have been obtained from studies with animals when lime-treated maize was
supplemented with lysine and tryptophan (Bressani, Elías and graham, 1968).
These results have been confirmed in nitrogen balance studies conducted with
children as shown in Chapter 6. (Selected results are shown in Table 32.) The
finding that the addition of lysine and tryptophan at the lower levels of protein
intake gave a nitrogen retention significantly higher than at the higher level of
protein intake has often been overlooked, and the importance of protein quality has
been overshadowed by that of energy intake.
Supplementation with protein sources
 27

The results from animal and human studies in which limiting amino acids
have been added to lime-treated maize have served as the basis for evaluating the
ability of different types of protein supplements to improve its protein quality.
Studies on protein supplementation of lime-treated maize flour have been
published by many researchers using different food sources including milk,
sorghum, cottonseed flour, fish flour, torula yeast and casein. Table 40 summarizes
the results of adding small recommended amounts of various protein sources. The
quality increase is at least 200 percent of the protein quality value of maize. In tests
with young dogs, the nitrogen balances when maize was supplemented with 5
percent skim milk, 3 percent torula yeast and 4 percent fish flour were significantly
higher than those measured when maize was given alone. Most of the supplements
that have been tested have several characteristics in common. They all have
relatively high protein content and are good sources of lysine, with the exception
of cottonseed protein and sesame oil meal. The latter is a good source of
methionine. With the exception of casein and/or milk and fish protein concentrate,
they are of vegetable origin.
The improvement in quality of protein in tortilla flour is in most cases a
synergistic response to lysine and tryptophan enhancement and to a higher level of
protein, both provided by the supplement. Since soybean protein in different forms
is the supplement to tortilla flour most often tested by different investigators and
because it is almost the only one also tested in children, with results comparable to
those in studies with animals, its importance and effects are reviewed in this
section. Figure 3 depicts the PER for combinations of common maize and opaque-
2 maize with soybean flour in different ratios.
Supplementation with green vegetables
One form in which masa is eaten in some countries is the tamalifo. This is
made by wrapping the dough in maize husks and placing it over steam.
 28

Tomalitos are often eaten instead of tortillas and have the advantage of
remaining soft for a longer period. There are various ways to prepare them, some
of which include the young leaves of native vegetables such as crotalaria and
amaranthus. Chemical and nutritional studies have demonstrated that about a 5
percent contribution of these leaves improves the protein quality of the dough
(Bressani, 1983). The reason is that they have relatively high levels of protein rich
in lysine and tryptophan. They also provide minerals and vitamins, particularly
provitamin A. Leaf protein concentrates have also been shown to improve the
protein quality of cereal grains (Maciejewicz-Rys and Hanczakowski, 1989).
Supplementation with other grains
Sorghum is another grain that has been processed by lime-cooking in
Mexico and Central America, particularly in areas where maize does not grow
well. Sorghum tortillas, however, are not of the same organoleptic or nutritional
quality as maize tortillas. Many successful efforts have been made to use blends of
both cereal grains, among others by Vivas, Waniska and Rooney (1987) and Serna-
Saldivar et al. (1987, 1988a, 1988b). Other approaches include the use of blends of
common maize, since germination has been reported to increase lysine Mixtures of
tortilla flour and rice and of tortilla flour and wheat flour have also been studied.
The rice/maize products have higher nutritive value than the wheat/maize tortillas,
as shown in Figure 4. These results show the superiority of rice over whole maize
flour and of the latter over wheat flour. More recently, blends of amaranth grain
with lime-cooked maize flour have been shown to have an improved protein
quality because of the much higher lysine and tryptophan content of amaranth as
compared with maize. The product has been reported to be of an acceptable
organoleptic quality. Other products added include potato, rice and pinto beans,
providing foods with acceptable sensory attributes.
 29

MATERIALS AND METHODS

 30

The apparatus and equipment that were used to successfully carryout the
investigation are all listed in the appendix.

METHODS

Sample Collection

Yellow maize was purchased from Eke market in Awka, the Anambra State
capital. A portion of the yellow maize was collected, dry-milled and made into
flour. This was sample 1 labeled slurry. The remaining portion of the yellow maize
was soaked (steeped) for 3 days (this was done to soften the maize so as to allow
fermentation to take place). After 3 days the steeping water was decanted, the
maize was washed with clean water and wet-milled using an electric motor. The
ground material was made into slurry with clean water, was sieved with a clean
mesh cloth and washed through with water. The product was left to sediment for an
hour. After sedimentation, the supernatant was decanted and then 100ml of the
sediment was collected as Sample 2 which was labeled fresh. The remaining was
re-suspended with clean water and left to stay for five days without changing the
water. After 5days, the supernatant was decanted and 100ml of the sample was
collected as Sample 3 and labeled 5days.

Sample Analyses

The investigation was carried out on the fresh yellow maize which was made
into powder (slurry), the freshly prepared raw ogi (fresh) and the prepared raw ogi
that was kept for 5days without changing the water (5 days). This was done in
order to estimate the possible stage and source of contamination of the ogi by some
harmful microorganisms.

Sterilization
 31

The medium, distilled water used for serial dilution and all the glass
equipment used in carrying out the experiment were sterilized in the autoclave for
15 minutes at 1210C at a pressure of 15Ib. The inoculating loops used were
sterilized before and after use by flaming over a lit Bunsen burner till it was red
hot, allowed to cool by shaking it close to the lit Bunsen burner and used under
aseptic condition. The sugars used were autoclaved for 10minutes at 115 0C at 15Ib.
The working surface used during experiment were swabbed before and after use
with cotton wool soaked in 70% ethanol.

Processing of the Samples

A ten-fold serial dilution was carried out using sterile distilled water as the
diluents. Ten test tubes containing ten milliliter volume per volume (10mll v/v) of
sterile distilled water were used for each of the samples. They were labeled and
arranged appropriately in a test tube rack. One gram weight per volume (1g w/v)
was serially diluted in test tubes containing 10ml of distilled water using a sterile
10ml syringe, each transfer followed by a gentle agitation in other to mix the
contents uniformly. The procedure was repeated for all the samples.

The serial dilution was performed aseptically, beside a lit Bunsen burner to
prevent contamination.

Isolation Procedure

Bacteria in the samples were isolated by inoculating the 1ml of dilution 10 -6

and 10-7 of the serially diluted samples in nutrient agar using the pour plate
technique. The samples were taken from the test tube aseptically using a sterile
syringe near a lit Bunsen burner. The plate was allowed to dry properly and then
incubated at 370C for 24- 48hours. This plate is known as the primary isolate.
 32

Fungi in the samples were isolated by inoculating 0.1ml of dilution 10 -6 and

10-7 of the serially diluted samples in an already prepared Petri dish of Sabouraud
dextrose agar, using the spread plate technique. The plates were incubated at 25 0C
for 3- 5days. This culture plate is known as the primary isolate.

Plate Count

The nutrient agar culture plates were examined after 24 hours incubation.
The number of bacteria colonies on the plate which had between 30- 300 colonies
were counted and the viable number/ colony forming units per milliliter (CFU/ml)
was calculated. The formula used to calculate it is:

Viable count/ CFU/ml = number of colonies formed on the plate

Volume of sample × Dilution factor

The frequency distribution (FD) of the bacterial isolates was calculated

using the formula:

FD = Frequency of bacterial isolate × 100

Total frequency of the bacterial isolates 1

Isolation of Pure Culture

These were done by randomly sub-culturing various colonies from the pour
and spread plate unto already prepared Nutrient agar (NA) and Sabouraud dextrose
agar (SDA) plates. Individual visible bacteria colonies formed on the primary
isolate of the nutrient agar were picked from the culture plates using a wire loop.
 33

They colonies picked and each colony was transfered to an already prepared and
sterilized NA plates by employing streak method. This was done by introducing a
colony from the original culture plate at the end (butt) of the new plate (NB. I was
careful not to touch the edge of the plate to avoid contamination). Using a sterile
wire loop, I streaked the innoculum along the agar in four different directions with
the last streak (tail) being done in a zig zag manner. The wire loop was flamed to
red hot and allowed to cool after each streak. The procedure was performed under
aseptic conditions near a lit Bunsen burner. The streaked nutrient agar plates were
incubated at 370C for 24 hours.

The sub-culturing of fungal isolates was carried out by transferring

individual visible colonies formed from the primary isolate to already prepared and
sterilized SDA plates. The molds formed were sub-cultured by picking a little
portion of the mold colony formed from the active periphery using a sterilized
straight wire and spot inoculating it on a freshly already prepared SDA plate. The
colony was picked by digging into the agar in order to take the some aerial part
which contains the spores and mycelia. Spot inoculation was done by stabbing the
picked colony dip into the fresh agar; this was done at the centre of the agar. This
procedure was performed under aseptic condition near a lit Bunsen burner and the
which loop was flamed till it was red hot and allowed to cool near the flame before
and after each use. It was incubated at 25 0C for 3- 5 days. The yeast colonies were
sub-cultured using a wire loop and streaked on a freshly prepared SDA agar. It was
also done under aseptic conditions. Then it was incubated at 37 0C for 24 – 48
hours.

Storage of the Organisms

 34

The organisms were aseptically transferred from the sub-cultured plates to

bijou bottle slants. Nutrient agar and Sabouraud dextrose agar were prepared and
nistain and chloramphenicol were added to it. The medium was boiled and
transferred into clean bijou bottles. The bijou bottles containing the media were
sterilized in the autoclave and kept in a slanting postioon to gel in order for the
slants to form. Then the organisms were aseptically transferred into each slant and
stored appropriately in the refrigerator.

IDENTIFICATION OF MICROBIAL ISOLATES

The identification of bacteria was carried out based on the classification scheme
given in Bergey’s manual of determinative bacteriology. Bergey’s manual relied on
an empirical classification system that separated all bacteria into 19 groups based
on the basis of morphology, physiology, growth requirement and biochemistry. The
discrete (pure) colonies of the bacteria isolates were identified based on the colony
morphology, gram staining and biochemical tests.

The mold colonies were identified based on their daily (day1 – day 4)
cultural colony characteristics on SDA and microscopic characteristics after
performing a slide culture. The yeasts were identified based on the colony
morphology on SDA, wet mount microscopy and biochemical tests.

MORPHOLOGICAL/ MICROSCOPIC EXAMINATION

Colony Description

The macroscopic morphology of the bacterial isolates which included the

opacity, colour, form, elevation, appearance and margin were observed and results
recorded.
 35

The macroscopic morphology of the fungal isolates on the SDA was

observed which included the pigmentation, colour of the underside, texture and age
were observed and the results recorded.

Gram-Staining Reaction

A thin smear of each colony of the bacterial and yeast isolates (which must
be an 18 – 24 hours culture) were made on clean grease free slide by a drop of
water on the slide, collecting the colony with a sterile wire loop and spreading it on
the slide (this was done under aseptic conditions near a lit Bunsen burner). It was
allowed to dry and heat fixed by passing it over a Bunsen burner. The flamed
smear was flooded with 0.5% of aqueous solution of crystal violet and left to stand
for a minute. The stain was washed off with clean water. The smear was then
stained flooding with lugol’s iodine and allowed to stand for 60 seconds. The
iodine was washed off with clean water and the slide was the decolourized with
acetone alcohol for 10 seconds and washed off immediately with clean water. The
smear was then counterstained with 0.5% safranin solution, allowed to stand for
30seconds and washed off with clean water. The slide was then air dried and
examined microscopically under the oil immersion lens. The same procedure was
repeated for all the colomies.

Slide Culture Technique

The slide culture technique was carried out on the mold isolates. A square
centimeter agar block of Sabouraud dextrose agar supplemented with 0.5mg/ml of
chloramphenicol was formed by dropping the media on a microscope slide which
was put in a Petri dish and allowed to gel. It was done aseptically. The mold isolate
was inoculated at the middle of the media aseptically and sterile cover slip was
 36

gently placed on top of the agar block, about 5- 10mls of distilled water was
poured into the petridish and incubated for 3 days at 250C.

After incubation, the slide was gently picked, using forceps the cover slip
was removed and a drop of lactophenol blue was dropped on the slide. The slide
was covered with the cover slip, placed under the microscope and examined
microscopically with a ×40 objective lens.

Wet Mount

This method was used to examine yeast cells for the presence of ascospores,
hyphae and pseudohyphae. Sabouraud dextrose broth was prepared appropriately
and the media was aseptically dispensed into sterile test tubes and inoculated with
the test organisms (fungi isolates). The tubes were examined for growth and
pellicle formation. Wet mount was then prepared on a clean grease free slide and
examined microscopically under high and low objective clenses (×40 and×10 lenes
respectively).

Germ Tube Test

This was done by dispensing 3 drops of 0.5ml of human serum into sterile
test tubes, using a Pasteur pipette. A pure yeast colony was lightly, touched with a
wire loop and inoculated in the serum. The preparation was incubated at 35 0C-
370C for 3 hours. After incubation, a drop of the suspension was placed on a clean
glass slide and a cover slip placed on it. The preparation was examined under the
microscope using the low and high power objective lenses (×10 and ×40 lenses).
 37

BIOCHEMICAL TESTS

Motility Test

This was used to determine the presence of flagella (external appendages

used for movement) in bacteria isolates. The presence of flagella indicates motility.
The isolates were grown on nutrient broth for 24 hours, after which a drop of it was
placed on a slide. The edges slide where the medium was dropped was firmly
sealed with chewing gum and then covered with a sterile cover slip. The
preparation was viewed using ×10 and ×40 objective lenses. A positive result was
indicated by a sharp darting movement across the field of view.

Catalase Test

This test was conducted to check for the production of catalase enzyme by
the bacterial and yeast isolates. Most organisms posses this enzyme which is
capable of breaking down hydrogen peroxide. Organisms containing the enzyme
will form oxygen bubbles when exposed to hydrogen peroxide. A fairly thick
emulsion of the isolates was smeared aseptically on a clean grease free slide and
few drops of hydrogen peroxide were placed on it. Positive results were shown by
an immediate effervescence or formation of gas bubbles.

Coagulase Test

This test was conducted to differentiate Staphylococcus aureus from other

Staphylococcus species. Coagulase is an enzyme produced by Staphylococcus
aureus that converts fibrinogen to fibrin. The test was carried out by aseptically
making a thin smear of the bacterial isolates on clean grease free slides and few
drops of plasma was added. Positive results were indicated by formation of clots.

Sugar Fermentation Test

 38

The test was used to distinguish gram negative enteric based on

biochemistry and was also used in the identification of yeast. The principle is based
on the ability of microorganisms to metabolize sugars, produce gas and acid.

The test was carried out using 5 sugars (maltose, glucose, lactose, sucrose
and galactoste). 4.5gram of peptone water and 1gram of each sugar were dissolved
in 100ml of distilled water each in six different conical flasks. A drop of
bromothymol blue indicator was added in each of the flasks containing the sugars.
5 milliliter of the solution each was dispensed to different test tubes. Durham’s
tubes were inverted inside the tubes and incubated at tubes and the whole set up
was sterilized at 1150C for 10minutes, after which they were allowed to cool and
the isolates inoculated into the test 37 0C and 250C for 24 hours for bacterial and
yeast isolates respectively. A positive result was indicated by a colour change of
the broth from green to orange shown the production of acid and the presence of
gas bubbles in the Durham’s tube showing gas production.

Methyl Red Test

This was done by inoculating the bacterial and isolates into test tubes which
contain sterile glucose phosphate peptone water. It was incubated at 370C for 48-
72 hours. After 48 or 72 hours, five drops of methyl reagents were added and
mixed, and then the results were read and recorded. The production of bright red
and yellow colour indicates a positive and negative result respectively.

Indole Test

This was conducted by inoculating the bacterial and yeast isolates in test
tubes containing 3 milliliter of sterile peptone water. It was incubated for 24 hours
at 370C , after the 24 hours incubation Kovac’s reagent was added. Formation of
 39

red ring at the surface indicated a positive result while retention of brown- yellow
colour indicated a negative result.

Citrate Utilization Test

The medium used to perform the test was simmon citrate agar. The test was
used to identify the organisms that can utilize citrate as the sole source of carbon
for metabolism. It was used in the differentiation of the organisms in the
Eubacteriaceae and other genera. A preparation (24 hours growth) of bacterial and
yeast isolates were inoculated individually in test tubes containing the citrate
medium slants and incubated at370C for 24 hours. A positive result was indicated
by a colour change from green to blue.

Voges Proskaeur Test

Bacterial isolates were inoculated in test tubes containing glucose phosphate

peptone water and was incubated at 37 0C for 48-72 hours. After either 48 or 72
hours, five drops of Bamitis A (Alpha naphthol) and Bamitis B (potassium
hydroxide) reagents were added, mixed and the results were read. A positive result
was indicated by a pink burgundy colour.

Oxidase Test

This test was conducted to detect the bacterial isolates with the ability to
produce the enzyme oxidase. It was done by mixing two drops of 1% aqueous
solution of tetramethyl-para-phenylene diamine hydrochloride with the test
organism on a filter paper. A positive result was indicated by the development of a
colour change from maroon to pink within 10-30 seconds.
 40

RESULTS

A total of Ten (10) organisms were isolated and identified from the samples
and they include bacteria and fungi isolates. The bacteria and fungi isolates
identified as stated in table 3 and table 4 include: Aspergillus niger var tieghem
(A), Aspergillus fumigates Fresenius (B), Rhizopus oryzae went (C),
Saccharomyces cervisae (D), Candida albican (E), Escherichia coli .(F),
Pseudomonas aeuroginosa (G), Lactobacillus plantarum (H), Lactobacillus casei
(I) and Staphylococcus aureus (J).

The occurrence of these organisms in the samples were shown in table six
(7) and seven (8). It showed that Saccharomyces cerevisae and Candida albicans
are the dominant fungi present in ogi. It also showed that Lactobacillus plantarum
and Lactobacillus casei are present in all the samples, which means that they are
the dominant bacteria in ogi.

Colonial Characteristics

Table three (4) showed the colony morphology of the fungal (mold) isolates
identified on Sabouraud dextrose agar. The table shows the different colonial
appearances of the molds on SDA agar. The colonial appearance of the bacterial
isolates identified was shown in table four (5). Table five (6) shows the colonial
appearance of the yeast isolates on SDA. They tables described the appearance,
colour and texture of the bacteria and fungi isolates individually. Table three (4)
showed that Rhizopus oryzae went formed white colonies which became light
 41

yellow to grey and blackish grey with age. It also showed that the colour of the
underside was light yellow and that it had a fluffy texture on SDA.

Morphological Characteristics

The morphological characteristics of the mold isolates identified were shown

and described in table three (3). The morphological characteristics of the bacterial
isolates of were described in table four (4) and the morphological characteristics of
the yeast isolates were shown in table five (5). These tables described the
microscopic appearance of the bacteria and fungi isolates identified. Table 3
showed that Aspergillus niger var tieghem has conidal heads which are dark brown
to black, they are radiate and biseriate with metulae twice as long as the phialides.
Their conidia are globose, brown and rough walled.

Biochemical Characteristics

Table 4 and 5 show the results of all the biochemical tests of the bacteria and
yeast isolates. Table 4 showed the different reactions of bacterial isolates to
catalase test, coagulase test, indole test, voges proskaeur test, oxidase test, methyl
red test, sugar fermentation test, citrate utilization test and motility test. Table 5
showed the different reactions of the yeast isolates to urease, germ tube, gram
staining, sucrose, glucose, galactose, maltose and lactose.
 42

Table 1: Bacterial Counts of the Samples

Samples Bacterial viable count

(CFU/ml)

1 6.3 × 107
2 2.0 × 109
3 1.82 × 107

Samples
 43

1 – Slurry

2 – Fresh

3 – 5 days

Table 2: Frequency distribution of Bacterial Isolates

Isolates Frequency Percentage

occurrence (%)

Escherichia coli 1 9.1

 44

Pseudomonas aueroginosa 2 18.2

Lactobacillus plantarum 3 27.3

Lactobacillus brevis 3 27.3

Staphylococcus auerus 2 18.2

Total 11 100.1

Table 3: Frequency distribution of Fungi Isolates

 45

Isolates Frequency Percentage

occurrence (%)

Aspergillus niger 1 8.3

Aspergillus fumigatus 1 8.3

Rhizopus oryzae 1 8.3

Saccharomyces cerevise 3 25

Candida cervisae 3 25

Total 12 74.9
 46

Table 4: Colonial and Morphological Features of the Fungi (Molds) isolates in

Ogi

Isolates Colonial Features on Morphological Features Organisms

Sabouraud Dextrose
Agar
Dark brown colonies Conidial heads are dark brown Aspergillus
A with light yellow and to black, radiate and biseriate niger var
white periphery. with metulae twice as long as tieghem
Colour of the the phialides. Condia are
underside is black with globose, brown and rough
light yellow edges. It walled.
has a globose texture.
The dark brown splits
into loose colonies
with age.
B The pigmentation was Conidial heads are typically Aspergillus
brown with milky columnar and uniserate with the fumigates
centre. Colour of the phialides limited to the upper fresenies
underside was two thirds of the vesicle and
yellowish. The texture curving to be roughly parallel to
was globose to sub- each other.
globose (powdery).
The brown colour
expands and intensifies
 47

with age as the milky

centre disappear.

C White colonies were Non- septate mycelia, Rhizopus

formed which became sporangiospores are ovoid in oryzae went
light yellow to grey shape and are directly opposite
and to blackish grey to branched rhizoids. Sporangia
with age. Light yellow and the columella are
under side was subgloose, sporangiophoresare
observed with fluffy smooth walled.
texture.
 48

Table 5: Morphological and Biochemical Characteristics of Bacteria Isolates in

Ogi

Isolates F G H I J
Gram - - + + +
Staining
Shape Short rods Rods Rod Rod Irregular
in clusters cocci in
clusters
Motility + + + _ _
Catalase + + + _ +
Coagulas - - +
e
Indole + - _ _ _
Citrate - + + _ _
utilizatio
n
Methyl + - +
red
VP - - +
Oxidase - + _
 49

Lactose +/ AG + + + +/ A
Glucose +/AG - _ + +/A
Sucrose + - + + +/AG
Maltose + + + + +
Galactos + _ + + +
e
Probable Escherichi Pseudomon Lactobacill Lactobacill Staphylococc
Organis a coli as us us casei us auerus
m aueroginosa plantarum

Table 7: Occurrence of the Fungal Isolates in Ogi

Samples A B C D E

1 + + + + +

2 - - - + +

3 - - - + +

+ = Detected and - = Not detected

Samples

1 – Slurry
 50

2 – Fresh

3 – 5 days

Isolates

A – Aspergillus niger var tieghem

B – Aspergillus fumigates fresenius

C – Rhizopus oryzae went

D- Saccharomyces cerevisae

E – Candida albicans

Table 8: Occurrence of the Bacterial Isolates in Ogi

Samples F G H I J

1 - - + + -

2 + + + + +

3 - + + + +

+ = Detected and - = Not detected

 51

Samples

1 – Slurry

2 – Fresh

3 – 5 days

Isolates

F - Escherichia coli

G - Pseudomonas aueroginosa

H – Lactobacillus plantarum

I – Lactobacillus casei

J – Staphylococcus auerus

DISCUSSION

The bacterial counts gotten from the samples were found to be highest in
the sample 2 which is the fresh. The higher microbial load in sample 2 was as a
result of the organisms that are dominant in the maize grain which are responsible
for the fermentation of the ogi and also as a result of the organisms that
 52

contaminate the freshly prepared raw ogi. The high viable count of organisms in
the fresh sample is also as a result of the abundant availability of nutrients in the
ogi available for the growth of microorganisms. The contamination was as a result
of the improper handling of the grain or was as a result of inappropriate
manufacturing practices. The contamination could also have been from the water
used in the production of the ogi (that is the steeping and processing water).
Microorganisms that were isolated ogi included: Escherichia coli, Pseudomonas
aueroginosa, Lactobacillus plantarum, Lactobacillus casei, Staphylococcus
aureus, Candida albicans, Saccharomyces cerevisae, Aspergillus niger var
tieghum, Aspergillus fumigatus Fresenius and Rhizopus oryzae went.

The predominant organisms in the ogi are the Lactobacillus species. These
organisms were responsible for the fermentation of the maize grain to form ogi
(Odunfa, 1985; Amusa et al., 2005; Ozoh and Kuyanbana, 1995). Other organisms
dominant in the ogi are the yeasts which include Saccharomyces cerevisae and
Candida albicans, which are also responsible for the fermentation of the maize
grain. The presence of the Lactobacillus species and the yeasts lead to the
nutritional improvement of the ogi. This statement agrees with the work of
Akinrele, (1970) as stated above in the literature review.

The organisms isolated from the maize grain included: Lactobacillus

plantarum, L. casei, Aspergillus niger, A. fumigatus, Rhizopus oryzae,
Saccharomyces cerevisae and Candida albicans. As a result of the fermentation
which took place in the fresh sample by lactic acid chbacteria and yeast, their
metabolic action led to the production of organic acids, gas, diacetyl, bacteriocins,
etc which lead to the inhibition of the growth of some microorganisms. The
microorganisms that were inhibited include Aspergillus niger, A. fumigatus and R.
oryzae. The presence of Escherichia coli, Staphylococcus aureus and
 53

Pseudomonas aureoginosa in the fresh sample is as a result of contamination

through handling and processing (Amusa et al., 2005).in the

As a result of increase in the length of the fermentation, it led to the

reduction in the nutrient availability of the microorganisms in the ogi, leading to a
reduction in the viable count of organisms in the 5days; this is as a result of
decrease in the nutrient availability, competition for space and nutrients by
dominant microorganisms and the production of metabolites (e.g. organic acids,
diacetyl, etc) by the organisms responsible for the fermentation. These leads to the
inhibition of some gram negative organisms (Escherichia coli) and some molds.
This statement agrees with the report of Mensah et al., (1990, 1991) as stated in the
literature review. There increase in the viable count of the lactic acid bacteria in the
fresh sample as a result of the fermentation of the maize porridge. This agrees with
the report by Kunene et al., (1999) as stated above in the literature review. The
presence of Staphylococcus aureus and Pseudomonas aueroginosa in the 5days
sample was as a result of contamination which are of public health importance
(Prescott et al., 2008), resulting from handling and processing environment as
stated by Amusa et al., (2005).
 54

CONCLUSION/ RECOMMENDATION

The presence of lactic acid bacteria and yeast in ogi leads to the
fermentation and nutritional improvement of the ogi. The lactic acid bacteria and
the yeasts that were isolated include: Lactobacillus plantarum, Lactobacillus casei,
Saccharomyces cerevisae and Candida albicans. The production of metabolites
(organic acids, diacetyl, gas bacteriocins, etc) during fermentation by these
organisms leads to the inhibition of other organisms which may be harmful to
humans when consumed. The production of these metabolites inhibited Aspergillus
niger, A. fumigatus and Rhizopus oryaze in the study. Escherichia coli,
Staphylococcus aureus and Pseudomonas aueroginosa are contaminants which
may pose serious public health importance. Therefore effective good
manufacturing practices (GMP) are recommended which may help eliminate the
contaminants for improved table quality and assure the health of consumers.

The study showed that improper storage of ogi (keeping ogi without
changing the water daily or refrigerating) and storing ogi for long periods (5days)
at room temperature will increase the flora of microorganisms; thereby leading to
the production of odd odours and taste, shelf-life reduction, unfit for consumption.
 55

Therefore ogi should be properly stored, it shoud not be kept for long periods and
the water should be changed on a daily basis.

REFERENCES
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