CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Across Africa, maize is an important cereal that is used as food and raw material for

industries. Proximate composition studies have shown that the dry maize contains moisture,

protein, fat, fibre, metabolisable energy, and minerals such as phosphorus, sodium, sulphur,

copper, magnesium calcium, iron, and potassium in varying amounts(Species, 2017). It is

therefore not surprising that in most part of the Nigeria, ogi is a delicacy consumed by both

adults and infants. It is usually the first food given to babies singly or side by side with

formulated ones. Conventionally, it is prepared from maize, however, millet or sorghum can

also be used. It preparation and microbiology is well studied(Species, 2017). Steeping and

souring are identified as the two fermentation stages involved in the longestablished process

of Ogi preparation. Its color when ready for eating depends on the cereal used in its

preparation. Where maize is used, it has a creamy or white appearance. When boiled, it turns

into a porridge called pap and can be served with protein rich foods such as beans. Typically,

the carbohydrate rich pap is usually served as weaning food for infants, as breakfast for

children and convenient meal for its convalescence.

Furthermore, nutrient loss seems inevitable during the preparation of ogi. It is

therefore not surprising that several attempts aimed at improving its nutritional status such as

enrichment with protein rich substrates have been made. In an earlier study, nutritional

enhancement with proteineous foods lowered their pasting viscosities and also sensory

qualities. Interesting, it has been shown that the intrinsic fermenters could be majorly

responsible for its nutritional improvement. Fermented cereal based foods consumed in West

Africa are important for a number of reasons. Studies have shown that the fermentation

microorganisms such as Lactobacillus and Bifidobacterium have the ability to produce

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healthy and safe products with better shelf lives, probiotics and probiotics potentials,

nutritional and health benefits, ability to dominate indigenous microbiota and even hydrolyze

starch. Others include alleviation of lactose intolerance, flavour enhancement, improvement

of immune system, neutralization of toxin effects, bioavailability of nutrients, phytate

degradation and fortification of folate amongst others. Despite westernization of infant food,

ogi prepared into pap still remains an important first line meal and the food of choice of most

West African and Nigerian babies. It has been shown that maize, millet and guinea corn paps

contain less protein and fibre which increases on fortification with soya bean milk by over

threefold(Inuwa et al., 2020).

In Nigeria and Ghana, a number of studies aimed at fortification with peanuts, soya

bean and cowpea have been carried out. The aim of the study was to improve on the protein

content of ogi by enrichment with L. brevis and L plantarum. Sorghum (Sorghum bicolor

(L.)Moench) is an important food crop in Africa and is the fifth most important cereal crop

grown in the world as well as the most important cereal food in the Northern states of Nigeria

that cover the Sahelien, Sudanian and Guinea Savannah ecological zones. Sorghum is locally

called guinea-corn or dawa, the most widely cultivated cereal crop and the most important

food crop in the Savanna areas of Nigeria.

Nigeria is the second largest producer of sorghum, grown on about 5.9 million ha with

current annual production estimated to be about 6.7 million tonnes. Sorghum is grown by

over 59% and 55% of farmers in Adamawa and Borno States, respectively. It is mostly grown

for domestic consumption and the excess sold to generate income. Among the constraints to

sorghum production are subsistence farmers who do not invest much in fertilizer and

improved varieties, rising labor cost, changing consumer food preferences, bird attacks and

parasitic weeds such as Striga.

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 Another major problem is the variable rainfall that leads to wild fluctuations in

production. Prices fall abruptly in good years, leaving traders reluctant to enter the market.

This increases the price risk that sorghum producers face; hence their reluctance to invest in

commercial sorghum production(Adisa& Enujiugha, 2020).

1.1.1 FERMENTATION

Fermentation is a metabolic process that converts sugar to acids, gases or alcohol. It

occurs in yeast and bacteria, and also in oxygen-starved muscle cells, as in the case of lactic

acid fermentation. Fermentation is also used more broadly to refer to the bulk growth of

microorganisms on a growth medium, often with the goal of producing a specific chemical

product like enzyme, vaccines, antibiotics, food product/additive etc. French microbiologist

Louis Pasteur is often remembered for his insights into fermentation and its microbial causes.

The science of fermentation is known as zymology.

To many people, fermentation simply means the production of alcohol: grains and

fruits are fermented to produce beer and wine. If a food soured, one might say it was 'off' or

fermented. Here are some definitions of fermentation. They range from informal, general

usage to more scientific definitions

1. Preservation methods for food via microorganisms (general use).

2. Any process that produces alcoholic beverages or acidic dairy products (general use).

3. Any large-scale microbial process occurring with or without air (common definition used

in industry).

4. Any energy-releasing metabolic process that takes place only under anaerobic conditions

(becoming more scientific).

5. Any metabolic process that releases energy from a sugar or other organic molecules, does

not require oxygen or an electron transport system, and uses an organic molecule as the

final electron acceptor (most scientific).

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Examples of Fermentation

Fermentation does not necessarily have to be carried out in an anaerobic environment. For

example, even in the presence of abundant oxygen, yeast cells greatly prefer fermentation to

aerobic respiration, as long as sugars are readily available for consumption (a phenomenon

known as the Crabtree effect). The antibiotic activity of hops also inhibits aerobic metabolism

in yeast. Fermentation react NADH with an endogenous, organic electron acceptor. Usually

this is pyruvate formed from the sugar during the glycolysis step. During fermentation,

pyruvate is metabolized to various compounds through several processes(Pasteur & Adp,

2020).

1. Ethanol fermentation, aka alcoholic fermentation, is the production of ethanol and carbon

dioxide

2. Lactic acid fermentation refers to two means of producing lactic acid

 Homolactic fermentation is the production of lactic acid exclusively

 Heterolactic fermentation is the production of lactic acid as well as other acids and

alcohols

Sugars are the most common substrate of fermentation, and typical examples of

fermentation products are ethanol, lactic acid, carbon dioxide, and hydrogen gas (H2).

However, more exotic compounds can be produced by fermentation, such as butyric acid and

acetone. Yeast carries out fermentation in the production of ethanol in beers, wines, and other

alcoholic drinks, along with the production of large quantities of carbon dioxide.

Fermentation occurs in mammalian muscle during periods of intense exercise where oxygen

supply becomes limited, resulting in the creation of lactic acid.

Types of Fermented Foods, Including:

i. cultured milk and yoghurt

ii. wine

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 iii. beer

iv. ciders

v. tempeh

vi. miso

vii. kimchi

viii. sauerkraut

ix. Fermented sausage.

1.1.2 USES OF SORGHUM

A majority of the domestic produce is used for household consumption by many rural

communities. It finds uses in the production of beverage, malt, sorghum meal, and livestock

feed, among others. Whole grain is ground into flour used to make traditional foods.

Sorghum is mainly used as flour or paste processed into tuwo (thick porridge), kamu (thin

diet porridge), and pate (soup like and light porridge mixed with vegetables, sometime

containing beans). A gradual increase in demand for pre-processed sorghum convenience

foods as well as for industrial sorghum products has been observed. Sorghum is also

processed into malt for malted drinks and foods, high quality flours, and as a raw material for

the poultry and fish feed industries. Sorghum is also processed into cake, biscuits, sweets and

other confectionaries(Inuwa et al., 2020).

1.2 AIM OF THE STUDY

The present study was undertaken with the aim to investigate lactic acid bacteria

present in ‘Corn slurry (Ogi) ’.

1.3 OBJECTIVES OF THE STUDY

1. To produce corn slurry (Ogi) from maize.

2. To isolate lactic acid bacteria from corn-slurry produced.

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3. To prepare pure culture of the isolates.

4. To characterize lactic acid bacteria isolated from corn-slurry (Ogi).

5. To examine the proximate analysis of the corn-slurry.

1.4 SIGNIFICANCE OF THE STUDY

The significance of the research is to assess Lactic acid Bacteria in ‘Corn Slurry

(Ogi)’ so as to further the research on their probiotic characteristics. More especially this

study would serve as a guideline for further research and investigation of the role of

fermented foods as physiological agents and at this point, hopefully expose the particular

peptides and amino acids responsible for particular physiological roles.

1.5 SCOPE AND LIMITATION

The research work is mainly to analyse microbiological and proximate analysis of

Corn-Slurry (Ogi) produced from maize.

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 CHAPTER TWO

LITERATURE REVIEW

2.0 SORGHUM PRODUCTION IN NIGERIA

Though sorghum is produced in almost all the states of Nigeria, Adamawa, Bauchi,

Benue, Borno, Gombe, Jigawa, Kaduna, Kano, Katsina, Kebbi, Kogi, Kwara, Nasarawa,

Niger, Plateau, Sokoto, Taraba, and Zamfara States are the major producers.

Cultivation Practices

Sorghum is a warm weather crop that requires high temperature for good germination and

growth. The best time to plant is when there is sufficient water in the soil. To exploit its

inherent yield potential, medium to good and fairly stable rainfall evenly distributed during

the growing season are required.

Soil Requirements

Sorghum can grow on different soils, but optimum grain and stover yields come from when

grown on deep fertile, well-drained loamy soil. Clay-loam or loam textured soils with good

water retention capacity are best suited for it. Though it can also grow in poor and sandy soils

not suitable for maize and rice, it responds better to increased soil fertility. It is more tolerant

of alkaline salts than other grain crops and can therefore be successfully cultivated on soils

with a pH (KCl) between 5.5 and 8.5, and can also tolerate short periods of waterlogging.

Climatic requirements

Temperature plays an important role in the growth and development of all crops, sorghum

included. The minimum temperature for germination varies from 7ºC to 10ºC. At a

temperature of 15ºC, 80% of seed germinates within 7 to 10 days after sowing. The best time

to plant is when there is sufficient moisture in the soil and the soil temperature is 15ºC and

above. A temperature of 27ºC to 32ºC is required for optimum growth and development.

Exceptionally high temperatures cause a decrease in yield due to pollen abortion.

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 Sorghum is a short-day plant, which means that the plant requires short days (long

nights) before proceeding to the reproductive stage. The optimum photoperiod, which will

induce flower formation, is between 10 and 11 hours. Photoperiods longer than 11 to 12

hours stimulate vegetative growth. Sorghum plants are most sensitive to photoperiod during

flower initiation. The early maturing varieties are photoperiod insensitive.

Sorghum is produced under variable average rainfall conditions between 300 mm and 1,200

mm, for optimum yield. A medium- to late-maturing sorghum cultivar (i.e., maturing within

110 to 145 days) requires approximately 450-800 mm of water during a growing season.

Daily requirement varies greatly depending on the growth stage (Fig. 1) and the type of

variety grown. Extra-early-maturing varieties have lesser water requirements than

mediumand late-maturing genotypes.

Figure 1. Growth stages of sorghum.

2.1 LAND PREPARATION

Sorghum requires a well-prepared seed bed for good establishment and well-drained

fertile land that has been left fallow for two or more years or preferably cropped with

legumes in the previous season. It is recommended that farm yard manure (FYM) at the rate

of 2-5 t/h be incorporated into the soil at ploughing. However, 1 t/ha annually is good enough

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to help improve soil structure, moisture retention, and nutrient content. Land preparation

depends on the system of sowing. In conventional tillage, plough/harrow and make ridges at

75 cm row spacing. Minimum tillage has been found to be suitable for good yields in drier

areas, aiding moisture conservation and reducing production cost(Inuwa et al., 2020).

Plate 1: (L) An animal-drawn ridger runs over the field after FYM application and (R) a well

prepared sorghum field.

Planting Date

Planting date is determined after the arrival of rainfall. The crop is normally planted from

end of May in southern Adamawa State to end of June or early July in northern Borno State,

when there is adequate moisture in the soil, depending on the location and variety to be used.

The choice of planting date is critical so that the period of critical moisture need does not

coincide with a drought period and maturity does not coincide with a drier period.

Choice of Variety

Variety choice aims to reduce risks by avoiding drought periods during the most critical

growing stages of the plant, such as flowering and seed set. Varieties differ in their reaction

to the environment and the climate. The yield potential of the farm or field should be known

as well as the long-term rainfall pattern to be able to make the best cultivar choice. Long-term

rainfall data is usually used as a guide to choose the variety with the appropriate length of

growing season suited to the target area. Characteristics such as disease and insect resistance,

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lodging and head placement are some important factors to be kept in mind when choosing a

variety

Planting

Sorghum can be planted manually with a hand pushed planter or an animal-drawn or

machine-drawn planter (a tractor-drawn seed drill with 4 coulters that simultaneously cover

seeds by a blade attached to the seed drill). Sorghum should be sown at an inter-row (between

rows) spacing of 75 cm and intra-row (within rows) spacing of 25 to 30 cm. A planting depth

of 5 cm is ideal with sufficient moisture. Under drier conditions, the seed should be planted

deeper, but no more than 5 cm.

Plate 2: (L) Manual planting and (R) an animal-drawn planter

Thinning

Thinning is recommended two weeks after sowing. The seedlings are thinned to two stands

per hill. Where gaps exist, there is a need to transplant when the soil is wet and preferably in

the evening. The transplants should carry as much root as possible and the foliage should be

slightly pruned to reduce evapo-transpiration and transplanting shock. The transplants are to

be planted upright. In both the sowing and the transplanting, the soil around the plant should

be firmed up.

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 Plate 3: Germinating sorghum field.

2.2 CHARACTERISATION AND PRODUCTION OF OGI

Several cereals can be employed for the production of ogi either independently or in

combination. Maize, sorghum and millet are the major cereals used as substrates for the

production of ogi; however, emergent cereals for ogi production include fonio grains

(Digitariaspp), rice (Oryza sativa) and amaranth (Amaranthusspp). The colour of ogi Fig. 1

on the type of cereal employed for its production. Of the millets, pearl millet

(Penisetumglaucum) ogipopularly referred to as ‘ogi-gero’ is the most common. Millet seeds

are rich in phytochemical and phytic acid which is believed to lower cholesterol, phytate on

the other hand is linked to cancer reduction. The traditional technique of ogi fermentation has

been extensively studied and involves spontaneous fermentation of the grains by soaking in

water for about 48 hours at 28 ± 2°C and milling into a smooth paste. The slurry obtained is

then sieved using a muslin cloth to remove the bran, germ and hull which is high in protein.

The filtrate is allowed to undergo a secondary fermentation for about 24–72 h in order to

develop its characteristic sour taste(Adisa& Enujiugha, 2020).

The length of the secondary fermentation depends on the extent to which sourness is

desired. In another context, the grains are soaked in hot/warm water for about 12-24 h prior to

fermentation to facilitate softening of the cotyledons. This method is usually practised by the

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traditional unskilled producers who lack adherence to good manufacturing practice and

sanitation especially in rural areas where portable water supply is a major concern. These

unsanitary practices expose the product to not only contamination from handling and

processing, but water-borne pathogens. The traditional method of ogi fermentation is labour

intensive, time consuming(Adisa& Enujiugha, 2020).

Fig. 1 (a-f). Commonly

consumed ogi from different grains

(a) White maize meal; (b) White maize steamed paste (c) Yellow maize meal; (d) Yellow

maize steamed paste (e) Sorghum meal; (f) Sorghum steamed paste

Improvements in the usual traditional process of ogi fermentation have been studied

include application of starter culture, dry milling of the grains prior soaking and fermentation,

dehulling and milling, sprouting before milling, fortification, boiling of grains before milling,

accelerated batch fermentation or back slopping. Ogi produced using lactic acid bacteria

(LAB) starter cultures and back-slopping methods presents a higher degree of sourness

compared to that produced using the traditional method of fermentation. The short period of

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acidification (24-48 h) must have been responsible for the sourer taste in the LAB fermented

ogi.

2.3 CHEMICAL PROPERTIES OF OGI

2.3.1 Physicochemical Properties

Studies on pH and titratable acidity (TTA) of the fermented slurry from different

grains revealed that as the TTA increases, pH value decreases. This trend was also observable

during starter culture fermentation of ogi production. The reason for this decrease in pH is

due to the presence and activities of lactic acid bacteria (LAB) which resulted in the

production of lactic acid during ogi fermentation. Antimicrobial and bacteriocins which are

of great value in bio-preservation and improved product shelf life by eliminating spoilage and

pathogenic organisms have also been linked to the low pH of fermented foods. During

fermentation of ogi, some of the fermentation organisms produce amylolytic enzymes which

are responsible for the disintegration of the starch substrate to reducing sugars, thereby

resulting to the decrease in the total sugar content of the ogi. In addition, lowering of the pH

is also affected by the ability of yeast and LAB present during fermentation to utilize the free

sugars. Another point of note is the increase in bacteria counts which increases as steeping

progresses favouring the growth of lactic acid bacteria, thereby increasing the acidity of the

steeped water and ogi at the end of fermentation.

2.3.2 Functional Properties of Ogi

Functional properties of ogi samples from different varieties of grain have been

reported to show variation, this trend was observed for water absorption capacity, swelling

power, solubility, bulk density and pasting characteristics. This variation has been linked with

the ratio of the amylose to amylopectin components of grain, the characteristics of each

fraction in terms of molecular weight, distribution, length of branching and conformation.

High bulk density is desirable, in order to reduce the paste thickness which is an important

13
factor in convalescent and child feeding. According to water and oil absorption capacities

increases with increase in steeping period. Water absorption capacity gives a good indication

of possibilities of protein incorporation with aqueous food formulations and finds application

in the development of ready to eat foods and a high water absorption capacity may also

enhance product effectiveness. Pasting is the phenomenon that follows gelatinisation in the

fractionalization or breaking up of starch. It is a property which involves rapid swelling of the

granules, exudation of molecular components, leaching out of amylose from starch granules

and total disruption of the granules. It is a property essential in ogi in order to predict the

behavioural pattern of the cooked pap as it is consumed as a cooked paste. It also represents a

degree of intactness of product, granule size, concentration and amylose/ amylopectin ratio

with changes observed in final viscosity and setback linked to the degree of reordering

leached amylose chains .

In a report, ogi from yellow maize stored at -10± 3°C and -20± °C presented a better

gelling ability throughout the 12 weeks of storage period. Furthermore, the study reported

that ogi stored at refrigerated temperatures of -10± 3°C and - 20±°C maintained its peak

viscosity and viscous load of the fresh fermented ogi while the peak viscosity of the samples

stored at ambient temperatures of 27±3°C and 5± 2°C decreased throughout the period of

storage with the viscous load decreasing as storage period increases. The authors explained

the decrease in the peak viscosity, final viscosity and setback observed at the ambient

temperature as probably due to the alteration in the chemical structure mediated by the

activities of the microorganisms present which have tendency to modify the chemical

structure of starch with time.

The setback viscosity is an index of retrogradation and entrapping water thus

promoting syneresis, however, retrogradation may have some nutritional benefit in producing

nutrient dense product that will not require the addition of water during infant feeding.

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Osungbaro also found out that maize of different varieties exhibited different pasting

viscosities on the amylograph and that the difference was due to the fact that the maize

varieties contain varying amounts of amylose with values ranging from 29- 34%. Pasting

temperature gives an indication of the minimum temperature required for cooking the ogi. It

has been shown that degree of fermentation influences swelling property of ogi. In terms of

pasting viscosities and consistencies 48 h fermentation of maize is appropriate for the

manufacture of ogi.

The author established that fermentation of ogi extending beyond 96 h resulted in the

paste exhibiting poor quality, gelling tendency and consistency. In a similar trend,

Apotiolaobserved the pasting characteristics of sorghum ogi powder decreased with increased

soaking period. Values of peak viscosity observed in his study were lower compared with

values obtained by Fasasiet al. Likewise, a related reduction in viscosities on fermentation of

samples of sorghum flour. This trend of variation of viscosities of ogi samples among

different varieties of grain have been reported by other studies for sorghum, maize, pearl

millet(Species, 2017).

2.4 NUTRITIONAL QUALITY OF OGI

Quite a lot of nutrient loss is experienced during the processing of cereal for the

production of ogi. These losses occur as a result of steeping, discarding of steeping water,

sifting to remove the bran and germ which contains much of the protein and also discarding

of the ogi supernatant thereby resulting in loss of minerals, fibre, protein, iron, phosphorus,

calcium, vitamins such as riboflavin, thiamine, niacin, folic, pantothenic acid and other

nutrients. This therefore results in reduction of net protein utilization, protein energy ratio and

biological values. In developing countries where consumption of this resultant low

nutritional product is high, deficiency of protein and energy usually results in negative

clinical manifestations such as kwashiorkor and marasmus in children, with severe cases

15
leading to death. In order to improve the nutritional quality of ogi several fortification with

protein rich substrates have been studied which include, functional and pasting characteristics

of fermented maize with nile tilapia (Oreochromisnicoliticus); protein enriched soy-ogi;

enrichment of sorghum ogi flour with cocoa; instant ogi from blends of fermented maize,

conophor nuts and melon seeds; co-fermentation of maize/cowpea and sorghum/cowpea ogi

as instant complimentary food; nutritive value of sorghum ogi fortified with groundnut seed

(Arachishypogaea L.); dietary fortification of sorghum-ogi using crayfish

(Paranephropsplanifrons) as supplements in infancy ; fortification of maize ogi with okra

seed meal; fortification of ogi with okra seed flour. Oyarekua and Eleyinmiin their study on

the nutritional quality of corn, sorghum and millet ogi reported the amino acid (AA)

composition of ogi prepared from these grains with leucine and proline as the most abundant

amino acids in corn and millet while for sorghum ogi, phenylalanine, glycine, arginine and

valine were the most abundant.

As a result, in the choice of weaning foods and for children, sorghum ogi would be

preferable because of its high value of arginine 91.5 compared to 33.2 and 43.1 of millet and

corn ogi which is an essential amino acid for children. Considering the total essential amino

acid of ogi flours from the three varieties of cereals as reported by millet ogi (275.2 mg g-1

crude protein), corn ogi (373.2 mg g-1 CP), and sorghum (721.9 mg g-1 CP), comparatively,

only the ogi from sorghum had values that can be compared with the egg reference protein

(566 mg g-1 CP). Therefore, since sorghum had values that have been shown to satisfy the

amino acid requirement of all age groups it can be regarded as a high quality protein.

Although, generally lysine is reported to be low for all the cereals, however, sorghum ogi has

a higher content than the other cereals. In order to improve their nutritional quality as

weaning foods, ogi from these cereals could be supplemented with milk or legume high in

lysine.

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2.5 MICROBIOLOGICAL PROPERTIES OF OGI

The microbiology of ogi is quite complex. The traditional fermentation is initiated as

a result of chanced inoculation by uncontrolled microorganisms from the environment

involving a build-up of bacteria and fungi. Some of these microorganisms may participate in

parallel, while others act in succession with a changing dominant biota during the course of

fermentation. Different studies have been able to isolate and enumerate possible

microorganisms associated with the fermentation of ogi. The following genera predominates

the bacterial fermentation, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus,

Streptococcus, Micrococcus and Bacillus The fungal genera are include representatives of

Saccharomyces, Candida, Aspergillus, Fusarium, Cladosporium and Penicillium amongst

others Lactic acid bacteria (LAB) are one the most common microorganisms responsible for

cereal fermentations, they are notable for the beneficial role of preservation, enhanced

nutritional value, detoxification, lactic acid, flavour and aroma production with Lactobacillus

plantarum reported as the most dominant specie(Adisa& Enujiugha, 2020).

These organisms have been studied to competitively eliminate other organisms

especially pathogens from the fermentation process. The synergy between LAB and yeast is

common in food and beverage fermentations with LAB creating the acidic environment for

yeast growth and yeast providing the vitamins and other growth factors necessary for the

survival of Lab.

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 CHAPTER THREE

MATERIALS AND METHODS

3.1 PRODUCTION OF CORN-SLURRY (OGI) FLOW CHART

(Screening)
Guinea corn

Steeping (24 hours)

Grinding

Sieving

Filtrate Residue

Corn-slurry (ogi) Discard

Fermentation
(96 hours)

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3.2 COLLECTION OF SAMPLE

Guinea corn was gotten from Owode market in Offa Kwara State and it was put in a clean

polythene bag and was transported to the Biological Science Laboratory for further analysis.

3.3 LIST OF APPARATUS/MATERIALS

The apparatus used for this research work include: Refrigerator, Heating drying oven,

Weighing balance, Laboratory incubator, Autoclave, Magnetic stirrer, Hotplate, pH meter,

Light microscope, Thermostat, Water bath and Colony counter.

Other materials include: Thermometer, Micropipette, Inoculating loops, McCartney

bottles and Spirit lamp,500ml conical flask, 500ml beaker, Sterile petridishes, 500ml

measuring cylinder, cover slip and slides, swap stick, cotton wool, needle and syringe sterile

test tubes, sterile distilled water.

3.4 LIST OF REAGENTS

MRS Agar (De Man Rogossa and Shapee), Crystal violet, Lugol’s iodine, Methylene

blue, 70% ethanol, Safranin, Kovac’s reagent, hydrogen peroxide and methyl red.

3.5 STERILIZATION OF MATERIALS

All the glassware or apparatus used were thoroughly washed with detergents and

rinsed very well with distilled water, then sterilized in an oven at 160 0C for 1 hour. The

media used were also sterilized in an autoclave at the temperature of 121 0c 15 minutes at is

Pascal. The workbench was swabbed with 90% alcohol. The wire loop was heated red hot

and allowed to cool down before using it for any inoculation.

3.6 PREPARATION OF MEDIA

The media use in the research work is De Man Rogossa and Shapee (MRS) agar,

which was prepared according to the manufacturer’s instruction that is 68.24 of powdered De

19
man rogossa shapee agar was to be measured and to be dissolved with 1000ml of sterile

distilled water. 34.12g of the powdered agar was weighed on the weighing balance and it was

poured into a clean conical flask and dissolved with 500ml of sterile distilled water, it was

shaked vigorously and the mouth of the flask was corked with cotton wool wrap with

aluminum foil. The conical flask was gently put in a water bath to homogenize the mixture, it

was then removed from the water bath for sterilization. The medium was then sterilized in an

autoclave at 121 degree Celsius for 15minute and 15pascal

3.7 ISOLATION OF BACTERIA

The isolation was carried out by using pour plate. Serial 10 fold decimal dilutions of

sample contaminated suspension up to 109 were made i.e 1ml of corn-slurry was diluted in

9ml of sterile distilled water. It was agitated for 2 minutes, this serve as stock. From this

dilution, 1ml of the aliquot was transferred into fresh 9ml of sterile distilled water in sterile

test tube. Subsequent dilutions were made up to 10 9. Odd number of the serial dilution was

chosen which is 10-1, 10-3, 10-5, 10-7, 10-9.

1ml of the sample was poured into each petri dish which has been labeled appropriately. The

prepared media was then dispensed into each petri dish aseptically and allowed to Gel

(Solidified).

All petri dishes containing our sample were incubated for 24 hours at 20 – 250c.

3.8 SUB-CULTURING

After the incubation period of 24 hours, the petri dishes were brought out and were

observed for growth of bacteria. With the aid of sterile wire loop, a small portion was picked

and streaked in a McCartney bottle containing 10ml of the slant freshly prepared culture

media to get pure culture.

3.9 IDENTIFICATION OF BACTERIAL ISOLATES

3.9.1 CULTURAL CHARACTERISTICS

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 Cultural characteristics considered include; the shape, size, colour, elevation, edge

and optical characteristics of the colonies.

3.9.2 GRAM STAINING REACTION

A thin smear of the cell suspension was made on a clean glass slide, and then fixed by

passing the slide containing smear over blue flame and was flooded with crystal violet for 30-

60 seconds. The dye was drained quickly and washed with Lugol’s iodine for 30 seconds.

The slide was washed under the tap water and then decolorized with 70% ethanol for about 5

seconds and rinse. After rinsing under tap water, Safranin was added to the slide for 30-60

seconds, and rinsed off gently in tap water, then drained and blotted dry and observed under

oil immersion objective lens microscope. Gram positive bacteria stained purple while gram

negative bacteria stained red.

3.9.3 BIOCHEMICAL CHARACTERIZATION

3.9.3.1 CATALASE TEST

This was used to differentiate those bacteria that produce Catalase from those that do not.

METHOD

3ml of hydrogen peroxide solution was poured into a sterile test tube and then a sterile glass

rod was used to collect several colonies of the test organisms and inoculated in the hydrogen

peroxide solution. It was observed for immediate active bubbling for positive test.

3.9.3.2 STARCH HYDROLYSIS TEST

Starch hydrolysis was carried out to determine the ability of organism capable of hydrolysis

starch and to differentiate organisms based on their ability to hydrolyzed starch.

METHOD

A single streak of organism to be tested was inoculated at the center of labeled plates, the

plate was incubated, and the surface of the plates was flood with iodine solution for

30seconds. Then the plates were observed.

21
3.9.3.3 METHYL RED TEST

This was carried out to identify Enterobacteria based on the ability to produce and maintain

stable acid end product from glucose fermentation.

METHOD

Glucose phosphate peptone water was used for inoculation of test organism and incubated for

48 hours at 370C after which few drops of methyl red solution was added to the culture and

read immediately. Formation of red color immediately showed a positive test.

3.9.3.4 VOGES-PROSKAUER(VP) TEST

This was also carried out to identify Enterobacteria based on the ability to produce and

maintain stable acid end product from glucose fermentation.

METHOD

After completion of methyl red test, 0.6ml α-naphthol soluttion and 0.2ml 40% KOH solution

was added, the tube was shaked, sloped and it was examined after 15 minutes and 1 hour. A

positive reaction is indicated by a strong red colour.

3.9.3.5 OXIDASE TEST

This was carried out to identify bacteria species that will produce the cytochomeoxidase

enzyme.

METHOD

A piece of filter paper was placed in a clean petri-dish and two to three drops of fresh or

nascent oxidase reagent was added. A colony of test organism was collected using a glass rod

and smeared on the filter paper and observed. Blue-purple colour within few seconds showed

a positive test.

3.9.3.6 INDOLE TEST

22
This test was carried out for indole production by test organism which is important in

identifying Enterobacteria.

METHOD

A sterile wire loop was used to inoculate a colony of test organism into 2ml of peptone water

containing tryptophan. The tube was stopped and incubated at 37 0C for 24 hours, after which

Kovac’s reagent was added to the medium. Observation of red coloration on the surface layer

within ten minutes showed a positive result.

3.9.3.7 COAGULASE TEST

This specifies the ability of the test organism to cause clotting of blood plasma.

METHOD

A drop of physiological saline was placed on each end of a slide. With the loop, straight wire,

a portion of the isolated colony was emulsified in each drop to make thick suspensions. A

drop of human plasma was added to the suspensions and was mixed gently. Clumping of the

organisms was observed within 10 seconds.

3.9.3.8 PROXIMATE ANALYSIS

The proximate analysis system is both comparative and predictive in nature. Proximate

analysis allows us to make legitimate comparisons to feeds on the basis of specific nutrients.

This makes it possible to know how much better one feed is than another in terms of specific

nutrients.

METHOD

DETERMINATION OF TITRATABLE ACIDITY

The total titratable acidity (TTA) of ogi was determined for the sample to quantify the acid

produced during sample fermentation. 1ml of ogi liquor was reconstituted in 10 ml of its

fermented water (Omidun). Three drops of phenolphthalein was added as indicator; then

titrated against 0.1M NaOH while gently swirling the content in the conical flask until pink

23
colour appeared. Each ml of 0.1N NaOH used was equivalent to 90.08mg of lactic acid.

Titration reading was taken in triplicate and mean values of the reading was calculated. Total

titratable acidity of lactic acid (ml/M) was calculated.

DETERMINATION OF MOISTURE CONTENT

In the pre-weighed moisture content cans, 5g of the sample was weighed in triplicate. The

sample was dried for 3 h at 105 0c in the Gallenkamp hot-air oven (Gallenkamp, UK) and the

weight was taken. The drying continued until the measure was constant. The sample was

cooled to room temperature in a desiccator and measured. The final weight of the sample was

determined. The moisture content was calculated from weight loss equation below:

Moisture content = (%)

w1= measurement of sample before drying (g)

w2= measurement of sample after drying (g)

DETERMINATION OF TOTAL ASH

The total ash (inorganic residue from the incineration of organic matter) was determined by

dry ashing procedure. The sample (2g) was measured into a pre weighed dry porcelain

crucible. The sample was incinerated in a Gallenkamp muffle furnace (Gallenkamp, UK) at

5500c for 6 h. After ashing, the remaining was removed from the furnace, cooled to room

temperature in a desiccator and weighed. The porcelain crucible was weighed and the % total

ash weight was obtained by using the equation below:

Total ash = (%)

DETERMINATION OF CRUDE FIBRE

The crude fibre was determined using the weighed samples resulting from fat extraction.

Each sample was transferred into conical flask and 100 ml boiling 1.25% H 2SO4 added. Each

beaker was heated for 30 min with periodical rotation to prevent adherence of solids to the

sides of the beakers. The solution was filtered using Whatman No.1 filter paper (28413923)

24
and rinsed with 50ml portions boiling water; repeated trice then dried. Boiling 1.25% (w/v)

NaOH solution (200 ml) was added and the mixture was boiled for 30 min after which the

contents of each beaker was removed and filtered; washed with 25 ml boiling 1% sulphuric

acid, three portions of 50 ml boiling water and 25 ml ethanol. The residue was dried at 100

°C to a constant weight followed by cooling in a desiccator at room temperature and

weighed. The weighed residue was ignited at 600 °C in a Gallenkamp muffle furnace

(Gallenkamp, UK) for 30 min, cooled in a desiccator and reweighed.

The percentage crude fibre in the sample was calculated as:

Crude fibre = (%)

W1 = Weight of sample (g)

W2 = Weight of crucible + sample (g)

W3 = Weight of crucible + Ash (g)

The experiment was carried out in triplicate for the sample and the average calculated for the

sample.

DETERMINATION OF CRUDE PROTEIN

2g of ogi sample was weighed into a digestion flask. Kjeltec catalyst 31835-2501AE (0.8g)

and 15ml of concentrated sulphuric acid was added to each flask. Each flask was heated on

pre heated digester set (K12, Behr LaborTechnik, Germany) at 420 0c in a fume cupboard, and

digested until a clear homogenous mixture was obtained. After digestion, the flask was

removed from the heater, cooled, and the content was diluted with 50ml of distilled water.

The flask was then placed in micro-kjedahl analyser (Kjelmaster K-375, Buchi, Switzerland)

where it received 50ml of NaOH automatically. The mixture was subsequently heated up to

release ammonia which was distilled into a conical flask containing 25ml of 2% (w/v) boric

acid as an indicator for 4 min, the ammonia reacted with boric acid to form ammonium borate

which was titrated against 0.1M hydrochloric (HCl) acid until the purplish – grey end point

25
was attained. The percentage nitrogen content of the samples was calculated using the

equation below:

Nitrogen = (% g)

where A= 0.1 HCl (ml)

DETERMINATION OF CRUDE FAT CONTENT

Fat content of all the ogi samples was determined by a continuous extraction liquid – solid

method using soxhlet extractor with a reflux condenser and a distillation flask (E914, Buchi,

Switzerland). Each sample (2g) was weighed into a fat free thimble plugged with cotton wool

and placed in the appropriate chamber of the extractor. The distillation flask was filled to two

third capacities with n-hexane (60–80 boiling points); the flask was boiled on a heating

mantle; the distillate was collected. Thereafter, n-hexane was recovered into a clean container

until almost all had been distilled. The remaining solvent in the mixture was evaporated in a

Gallenkamp hot-air oven (Gallenkamp, UK) set at 70 0c. The flask was allowed to cool

subsequently in a desiccator (PYREX, Corning, Inc USA after which the final weight of the

flask was determined. The difference in the final and initial weight of the distillation flask

represented the oil extracted from the sample. The percentage of crude fat was obtained using

the equation below:

Fat = (%)

The experiment was carried out in triplicate for the sample.

DETERMINATION OF CARBOHYDRATE

The carbohydrate content was determined by difference. The sum of the moisture, ash, crude

fiber, fat and protein of the sample was subtracted from 100 to obtain percentage

carbohydrate.

26
 CHAPTER FOUR

RESULT AND DISCUSSION

4.0 RESULTS

4.1 PHYSIOCHEMICAL PROPERTY OF CORN-SLURRY

Table 1: pH of Corn-slurry during fermentation.

Duration pH

0 hrs 4.23

24 hrs 3.66

48 hrs 3.64

72 hrs 3.71

96 hrs 3.84

27
Table 2: Microbial growth during fermentation of corn-slurry

Time P1 P2 P3 Average

0 hrs 1.2×103 1.3×103 1.1×103 1.2×103

24 hrs 1.7×103 1.9×103 2.1×103 1.9×103

48 hrs 3.4×103 3.8×103 3.6×103 3.6×103

72 hrs 5.8×103 6.0×103 5.9×103 5.9×103

96 hrs 8.0×103 8.2×103 8.1×103 8.1×103

28
Table 3: Cultural characteristics of isolates from (Corn-slurry) on De Man Rogosa Sharpe

(MRS)

Colour Optical Elevation Shape Size Edge

IsolateA Creamy Translucent Raised Filamentous Small Lobate

IsolateB Creamy Translucent Raised Filamentous Small Curled

IsolateC Creamy Opaque Raised Rhizoid Large Even

IsolateD Whitish Opaque Raised Rhizoid Large Wavy

IsolateE Whitish Translucent Flat Punctiform Tiny Even

IsolateF Creamy Translucent Flat Round Tiny Wavy

IsolateG Creamy Translucent Flat Round Tiny Lobate

IsolateH Creamy Translucent Flat Round Tiny Even

IsolateI Creamy Transparent Flat Round Tiny Wavy

IsolateJ Creamy Translucent Flat Circular Small Lobate

IsolateK Creamy Translucent Flat Circular Small Curled

IsolateL Creamy Translucent Flat Rhizoid Small Wavy

IsolateM Creamy Translucent Flat Filamentous Small Wavy

IsolateN Creamy Translucent Flat Filamentous Small Even

IsolateO Creamy Translucent Raised Irregular Small Lobate

IsolateP Creamy Translucent Raised Irregular Small Lobate

29
Table 4: Biochemical Analysis of Isolate from corn-slurry (Ogi)
Test Catalase Starch Methyl- Voges- Oxidase Indole Coagulase

hydrolysis red proskauer

IsolateA + + + - + - +

IsolateB + - + - + +

IsolateC + - + - + - +

IsolateD + + - - + - +

IsolateE + - + + + - +

IsolateF + - + - + - -

IsolateG + + - - + - +

IsolateH + - + - + - +

IsolateI + + + - + - +

IsolateJ + + - - + - +

IsolateK + - + - + - +

IsolateL + - - - + - +

IsolateM + + + - + - +

IsolateN + + + + + - +

IsolateO + + - - + - +

IsolateP + - + + + - +

30
Table 5: Morphological Characteristics of Isolate from corn-slurry (Ogi)

Gram staining Cellular shape Cellular Cellular Gram reaction

parameters arrangement pigmentation

IsolateA Rod Cluster Purple +

IsolateB Rod Cluster Purple +

IsolateC Rod Cluster Purple +

IsolateD Rod Cluster Purple +

IsolateE Rod Cluster Purple +

IsolateF Rod Cluster Purple +

IsolateG Cocci Pair Purple +

IsolateH Rod Pair Purple +

IsolateI Rod Cluster Purple +

IsolateJ Rod Cluster Purple +

IsolateK Rod Singly Purple +

IsolateL Rod Pair Purple +

IsolateM Rod Cluster Purple +

IsolateN Rod Cluster Purple +

IsolateO Rod Cluster Purple +

IsolateP Rod Pair Purple +

31
Figure 1: Gram reaction of isolateA

32
Figure 1: Gram reaction of isolateB

33
Figure 3: Gram reaction of isolateC

34
35
Figure 5: Gram reaction of isolateD

36
Figure 5: Gram reaction of isolateE

37
 Figure 6: Gram reaction of isolateF

Figure 7:
Gram
reaction of
isolateG

38
39
40
Figure 10: Gram reaction of isolateJ

41
 Figure

42
Figure 12:

43
Figure 13: Gram reaction of isolateM

44
Figure 14:

45
Figure 15:

46
Figure 16:

47
Table 6: Physiochemical and proximate analysis from corn-slurry (Ogi)

48
 Parameters Percentages (%)

Titratable acid (lactic acid) 0.65

Moisture content 9.00

Ash 1.30

Crude fibre 3.30

Protein 15.71

Fat 4.40

Carbohydrate 67.75

49
4.2 Discussion

According to Bergey’s manual of systematic bacteriology is the main resource for

determining the identification of microorganisms. According to table 1, the pH of the corn-

slurry was checked at 0 hour, 24 hour, 48 hour and it was found decreased from 4.23, 3.66

and 3.64 respectively, that mean the carbohydrate in corn-slurry is converted to lactic acid

and then begin to increase slightly from 3.71 and 3.84 at 72 hour, 96 hour respectively. This

is when the protein content of the corn slurry increased in the fermenting medium.

According to Table 2, triplicate of 10 folds of serial dilution were done and 5 petri

dishes were made for each 10 folds of serial dilution. The odds dilution powers were chosen

throughout (10-1, 10-3, 10-5, 10-7 and 10-9). All the total petri dishes used were 15 and were

labeled according to their power of dilution used. After incubation which is 24 hours, all the

plates were brought out for count and dilution of power 10 -3 were chosen throughout because

they have distinct colonies. The result showed that the microbial population increases with

increase in fermentation period. From Table 2 the microbial load increased from 1.2×10 3 at 0

hour to 8.1×103 at 96 hour.

According to Table 3, which showed the cultural characteristics of the isolates, all the

isolates are creamy colour except isolate D, and isolateE which is whitish in colour. All the

isolates are translucent except isolateC and isolateD which is opaque. All the isolates are flat

except isolatesA,B,C,D,O&P which are raised.

The biochemical characteristics of isolates from corn slurry are shown on Table 4. All

isolates showed positive reaction to catalase test. All the isolates showed no reaction to starch

hydrolysis test except isolatesA,D,G,I,J,M,N,&O. All the isolates showed positive reaction to methyl

red test except isolatesD,G,J,L&O. All the isolates showed no reaction to voges-proskauer test

except isolatesE,N&P. All the isolates showed positive reaction to oxidase test. All the isolates

50
showed no reaction to indole test while all the isolates showed positive reaction to coagulase

test except isolatesF,.

The gram staining reaction of the isolates from corn-slurry (Ogi) is shown on Table 5.

All the isolates showed positive reaction to gram staining. All isolates were Rod in shape

while isolatesG is cocci in shape. Also isolatesG,H,L&P were pair while isolateK is singly and

isolatesA,B,C,D,E,F,I,M,,N&O were in cluster from figure 1 to figure 16 which show the Gram

Staining plates accordingly.

According to Table 6, the proximate analysis carried out on the sample shows the

Titratable acid which is lactic acid to be 0.65%, Moisture content to be 9.00%, Ash to be

1.30%, Crude fibre to be 3.30%, Protein to be 15.71%, Fat to be 4.40% and Carbohydrate to

be 67.75%.

51
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

5.1 CONCLUSION

This shows that the carbohydrate is the major classes of food gotten from corn slurry

produced from guinea corn.

Corn slurry made from guinea corn is rich in nutrient which is needed by the body for

body maintenance.

5.2 RECOMMENDATION

Corn slurry produced from guinea corn is good for winning of baby because of its

nutritional condition.

52