Science MCB 2012 Balogun

A FERMENTATION STUDY AND
PHYSICOCHEMICAL ANALYSIS OF Prosopis

africana SEEDS TO PRODUCE OKPEHE, A
LOCAL CONDIMENT
BY
BALOGUN, MUTIAT ADEBANKE
B.Sc. (Zaria), M.Sc. (Ilorin)
Matric No: 98/68ER002
A THESIS SUBMITTED TO THE DEPARTMENT OF

MICROBIOLOGY, FACULTY OF SCIENCE, UNIVERSITY OF ILORIN,
ILORIN, IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR
THE AWARD OF DOCTOR OF PHILOSOPHY (Ph.D.) IN
MICROBIOLOGY
AUGUST, 2012
i
CERTIFICATION
This is to certify that the experiments reported in this thesis were conducted under my
supervision in this Department.
..
DR. G.P. OYEYIOLA
DR. G.P. OYEYIOLA
B.Sc. (Maiduguri); M.Sc. (Ibadan)
B.Sc. (Maiduguri); M.Sc. (Ibadan)
M.Phil. (Ilorin); Ph.D. (Kano)
M.Phil. (Ilorin); Ph.D. (Kano)
Supervisor
Head of Department
Examined this ..day of 2012

External Examiner:
Other Examiners.
ii
DEDICATION
This thesis is dedicated in loving memory of my late grandmother, Alhaja Sifawu Bukoye, and
parents, Mr. M.A. Bukoye and Alhaja Hassanat Bukoye, who made sure I had sound education.
May your gentle souls rest in perfect peace (Amen).
iii
ACKNOWLEDGEMENTS
First and foremost, I am very grateful to the Almighty Allah for sparing my life up to this
moment and the opportunity given to me to successfully complete this higher degree. To the
Most High are the glory, adoration and honour.
This research project would not have been successfully executed and written without the
valuable contributions of a number of persons at different stages. My appreciation goes to my
honourable supervisor, Dr. G.P. Oyeyiola, who is also the Head of Department of
Microbiology. His creative advice, constructive criticisms and useful suggestions had
contributed tremendously to the quality of this work. I will forever be grateful.
I must thank Dr. (Mrs.) P.F. Omojasola of Microbiology Department for her support,
encouragement and valuable contributions towards the success of this research. I also appreciate
the roles played by the following people from Department of Microbiology: Prof. Sani, Mrs.
S.T. Balogun, Messers Olabanji, Osho, Jide and Sule (teacher). God bless you all (Amen).
I will ever be grateful to the Dean of Agriculture, Prof. O.A. Omotesho, Prof. J.K. Joseph (how
far?), Prof. M.A. Belewu (Department of Animal Production), Dr. N.O. Muhammad
(Department of Biochemistry), Mr. Jemiri (Department of Chemistry), Mr. L. Adegboyega
(Institute of Education), my HOD (Dr. Mrs. O.R. Karim), Mrs. F.L. Kolawole, Mrs. Hadiza
Jimoh, Dr. R.M.O. Kayode, Mrs. Arungbemi (for typing some parts of this project), Mrs.
Obalowu and other members of staff of Department of Home Economics and Food Science.
They were all very supportive and encouraged me during my research period. Mention must
also be made of another of my teachers, Dr. A. Muhammad-Lawal, for the roles he played.
iv
I am highly indebted to my uncle, Mr. Rauf Bukoye who has always been there for me since
the demise of my dad. My profound gratitude goes to my sister and brothers especially Luqman
for their words of encouragement. I will be an ingrate if I forget to acknowledge my late friend,
Mrs. S.A.Yahaya. May your gentle soul rest in perfect peace (Amen). I also appreciate my late
mum and Mrs. E.O. Olugbemi for being there for my little daughter when I was busy with this
research work.
Finally, I owe the greatest gratitude to my immediate family members; my husband, Gbolahan
and little daughter, Nimah. They were very supportive and endured with me when I was not
around. Thank you very much. I really appreciate it and I pray that Almighty Allah in His
infinite mercy will support you and take you to levels beyond your imaginations (Amen).
TABLE OF CONTENTS
Title Page
Certification
ii
Dedication
iii
Acknowledgements
iv
Table of Contents
vi
List of Tables
List of Figures
xiii
List of Plates
xv
Abstract
xvi
CHAPTER ONE
1.0
Introduction and Literature Review
1.1
Justification for the Study
28
1.2
Research Objectives
29
CHAPTER TWO
2.0
Material and Methods
30
2.1
Sources of Fruits and Okpehe Samples
30
2.2
Traditional Preparation of Okpehe from Prosopis africana Seeds
30
2.3
Laboratory Preparation Using the Modified Traditional Method
31
vi
2.3.1 Using the Autoclave
31
2.3.2 Using the Hot Plate
34
2.4
Inoculation with Starter Culture
36
2.5
Determination of Physicochemical Parameters
38
2.5.1 Determination of pH
38
2.5.2 Determination of Temperature
38
2.5.3 Determination of Moisture Content
38
2.6
Isolation of Microorganisms
39
2.7
Characterization of Bacterial Isolates
40
2.8
Characterization of Yeast Isolate
40
2.9
Proximate Analysis of Okpehe Samples
40
2.9.1 Determination of Dry Matter Content
40
2.9.2 Determination of Ether Extract (Crude Fat) Content
41
2.9.3 Determination of Crude Fibre
42
2.9.4 Determination of Total Ash Content
42
2.9.5 Determination of Crude Protein
43
2.9.6 Determination of Carbohydrate or Nitrogen Free Extract
44
2.10
Determination of Total Sugar
45
2.11
Determination of Amino Acid Profile Using the Amino Acid Analyzer
45
2.12
Determination of Mineral Content
48
vii
2.13
Determination of Shelf Life of Okpehe
48
2.14
Preservation of Okpehe
48
2.14.1 Freezing
49
2.14.2 Refrigerating
49
2.14.3 Sun Drying
49
2.14.4 Oven Drying
50
2.14.5 Salting Plus Oven Drying
50
2.14.6 Smoking
50
2.14.7 Salting
51
2.15
Sensory Evaluation
51
2.16
Experimental Design and Statistical Analysis
52
CHAPTER THREE
3.0
Results
53
3.1
Physicochemical Parameters of Naturally Fermented Okpehe
53
3.2
Microbial Analysis
62
3.3
Nutritional Characteristics of Naturally Fermented Okpehe Samples
70
3.3.1 Proximate Composition of Okpehe Samples
70
3.3.2 Changes in Total Sugar of Naturally Fermented Okpehe Samples
72
3.3.3 Changes in Amino Acid Composition of Naturally Fermented Okpehe Samples
72
3.3.4 Mineral Composition of Naturally Fermented Okpehe Samples
76
viii
3.4
Physicochemical Parameters of Okpehe Samples Inoculated with Starter Cultures 76
3.5
Proximate Composition of Okpehe Samples Inoculated with Starter Culture
85
3.5.1 Proximate Composition of Okpehe Samples Inoculated with Mono Cultures of

Bacterial Isolates
85
3.5.2 Proximate Composition of Okpehe Samples Inoculated with Mixed Cultures of

Bacterial Isolate
93
3.6
Shelf Life of Okpehe Stored at Ambient Temperature
102
3.7
106
3.8
Sensory Analysis of Okpehe
115
CHAPTER FOUR
4.0
Discussion
120
RECOMMENDATIONS
132
REFERENCES
133
APPENDICES
149
ix
LIST OF TABLES
Table 1: Occurrence of Microorganisms in Locally Produced Okpehe
63
Table 2: Occurrence of Microorganisms in Laboratory Produced Okpehe
64
Table 3: Occurrence of Microorganisms in Laboratory Produced Okpehe
65
Table 4: Microbial Counts of Samples after 0 hr of Fermentation
66
Table 5: Microbial Counts of Samples after 24 hrs of Fermentation
67
68
69
Table 8: Proximate Analysis of Naturally Fermented Okpehe Samples
71
Table 9: Amino Acid Composition of Okpehe Samples
74
Table 10: Changes in Essential Amino Acids of Okpehe Samples
75
Table 11: Mineral Composition of Okpehe Samples
76
Table 12: Crude Protein Content of Prosopis africana Seeds Fermented with
Monocultures of Bacterial Isolates
86
Table 13: Ether Extract Content of Prosopis africana Seeds Fermented with
87
Table 14: Crude Fibre Content of Prosopis africana Seeds Fermented with
88
Table 15: Total Ash Content of Prosopis africana Seeds Fermented with
Monocultures of Bacterial Isolate
89
x
Table 16: Moisture Content of Prosopis africana Seeds Fermented with

90
Table 17: Nitrogen Free Extract Content of Prosopis africana Seeds

Fermented with Monocultures of Bacterial Isolates
91
Table 18: Crude Protein Content of Prosopis africana Seeds Fermented with Mixed
Cultures of Bacterial Isolates
94
Table 19: Ether Extract Content of Prosopis africana Seeds Fermented with Mixed
95
Table 20: Crude Fibre Content of Prosopis africana Seeds Fermented with Mixed
96
Table 21: Total Ash Content of Prosopis africana Seeds Fermented with Mixed
97
Table 22: Moisture Content of Prosopis africana Seeds Fermented with Mixed
98
Table 23: Nitrogen Free Extract Content of Prosopis africana Seeds Fermented with
Mixed Cultures of Bacterial Isolates
99
Table 24: Proximate Composition of Okpehe Stored at Ambient Temperature
105
Table 25: Changes in pH of Okpehe During Storage Period
107
Table 26: Microbial Count of Okpehe During Storage Period
108
Table 27: Crude Protein Content of Okpehe During Storage Period
109
xi
Table 28: Ether Extract Content of Okpehe During Storage Period
110
Table 29: Crude Fibre Content of Okpehe During Storage Period
111
Table 30: Total Ash Content of Okpehe During Storage Period
112
Table 31: Moisture Content of Okpehe During Storage Period
113
Table 32: Nitrogen Free Extract of Okpehe During Storage Period
114
Table 33: Sensory Evaluation of Okpehe During Storage
115
116
117
118
xii
LIST OF FIGURES
Fig. 1: Traditional Method of Production of Okpehe from Prosopis africana Seeds
32
Fig. 2: Laboratory Production of Okpehe from Prosopis africana Seeds Using Autoclave 33
Fig. 3: Laboratory Production of Fermented Prosopis africana Seeds (Okpehe) Using
Hot Plate
35
Fig. 4: Laboratory Production of Okpehe Using Starter Culture
37
Fig. 5: pH Changes during the Natural Fermentation of Prosopis africana Seeds to Produce
Okpehe
59
Fig.6: Temperature Changes during the Natural Fermentation of Prosopis africana

Seeds to Produce Okpehe
60
Fig.7: Changes in Moisture Content during the Natural Fermentation of Prosopis africana
61
Fig. 8: Changes in Total Sugar Content during Natural Fermentation of Prosopis africana
73
Fig. 9: Changes in pH during the Fermentation of Okpehe Inoculated with

Monocultures of Bacteria
79
Fig. 10: Changes in pH during the Fermentation of Okpehe Inoculated with Mixed
Cultures of Bacteria
80
xiii
Fig. 11: Changes in pH during the Fermentation of Okpehe Inoculated with Mixed Cultures of
Bacteria
81
Fig. 12: Temperature Changes during the Fermentation of Okpehe Inoculated with
Monocultures of Bacteria
82
Fig.13: Temperature Changes during the Fermentation of Okpehe Inoculated with Mixed
Cultures Bacteria
83
Fig. 14: Changes in pH during the Fermentation of Okpehe Inoculated with Mixed
Cultures of Bacteria
84
Fig. 15: Changes in Total Sugar of Okpehe Fermented with Monocultures of Bacteria
92
Fig. 16: Changes in Total Sugar of Okpehe Fermented with Mixed Cultures of Bacteria
100
Fig. 17: Changes in Total Sugar of Okpehe Fermented with Mixed Cultures of Bacteria
101
Fig. 18: Changes in pH of Okpehe Stored at Ambient Temperature
103
Fig. 19: Changes in Microbial Count of Okpehe Stored at Ambient Temperature
104
xiv
LIST OF PLATES
Plate 1:
Prosopis africana Tree
Plate 2:
Prosopis africana Fruits
Plate 3:
Prosopis africana Seeds
Plate 4:
Broken Fruits (pods) Ready for Winnowing
54
Plate 5:
Locally Fermenting Okpehe in Jute Bags
55
Plate 6:
Locally Fermented Okpehe
56
Plate 7:
Unfermented Samples A, B and C
57
Plate 8:
Fermented Samples A, B and C
58
xv
ABSTRACT
In many countries in Africa including Nigeria, protein malnutrition is a major problem. The
high cost of animal protein has directed interest towards several leguminous seed proteins as
potential sources of vegetable protein for human food. Prosopis africana (African mesquite)
is one of the lesser known legume seed crops used as a food condiment called Okpehe. In
spite of its importance as a condiment, the bio-modification occurring during the
fermentation of Okpehe is not yet fully documented. This study was therefore designed to
ferment P. africana seeds to produce Okpehe in the laboratory by simulating the traditional
method and monitor the physiochemical changes and microbial succession during the
natural fermentation of Okpehe. The commercial and laboratory produced samples were also
compared. The microorganisms isolated from the laboratory sample were used as starter
culture, first singly and later in consortium to produce Okpehe. The study was also to
determine the best preservation method for Okpehe and carry out sensory evaluation of the
condiment.
The commercial Okpehe was obtained from a local producer (sample A) while the autoclave
(sample B) and hot plate (sample C) were used to produce the laboratory samples.
Physicochemical and microbial analyses were carried out on each of the three samples. The
physicochemical and sensory evaluation were also carried out on the inoculated Okpehe
during preservation. The data obtained were subjected to descriptive statistics and one-way
ANOVA.
xvi
Seven organisms were isolated from the commercial sample of Okpehe (sample A) and these
were Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,
Escherichia coli, Staphylococcus aureus and Saccharomyces cerevisiae. Six organisms
were isolated from Okpehe samples produced in the laboratory using both the autoclave and
hot plate. These organisms were B.subtilis, B.licheniformis, B.megaterium, B.pumilus, E.coli
and S.aureus. The microbial load of sample A (4.0 106cfu/g) was significantly higher (p<
0.05) than that of samples B (3.3 106 cfu/g) and C (3.5 106 cfu/g). The pH (6.3 - 8.2),
temperature (270C - 350C), moisture content (45% - 61%), crude protein (30.90% - 40.05%),
total amino acids (80.26g 82.64g) and crude fibre (2.85% 3.63%) increased significantly
(p< 0.05) while the ether extract (11.49% 8.15%), ash (4.89% 4.80%), total sugar
(0.10mg/g 0.05mg/g) and carbohydrate (52.04% - 41.44%) decreased significantly (p<
0.05) for all the samples as fermentation progressed. The Okpehe samples contained an
appreciable amount of potassium, calcium, iron, copper, zinc and manganese while
cadmium and lead were not detected in all the samples.
For the inoculated samples, the crude protein (35% - 44.61%), crude fibre (3.15% - 3.64%)
and moisture content (6.37% - 8.09%) increased significantly (p < 0.05) while the ether
extract (10.81% - 9.00), total ash (4.93% - 3.32%) and nitrogen free extract (39.67% 31.81%) decreased significantly (p < 0.05) as fermentation progressed. The highest crude
protein content of 44.61% was observed with the combination of B. subtilis and B.
licheniformis (AC) while the least was observed with BD which was a combination of B.
megaterium and B. pumilus.
xvii
The highest microbial count after 14 weeks was with the refrigerated Okpehe which had 2.6
106cfu/g while the least of 1.7 106cfu/g was observed with the salted and oven dried
Okpehe. The crude protein (43.97% - 42.81%), total ash (4.13% - 3.88%), crude fibre
(4.02% - 3.40%), moisture content (12.76% - 6.06%) and nitrogen free extract (32.63% 27.08%) decreased significantly as the storage period increased. At the end of the storage
period, the salted and oven dried Okpehe had the highest protein content of 43.64% whilst
the refrigerated Okpehe had the least (42.81%).
The sensory analysis revealed that the refrigerated samples were most preferred by the
panelists in terms of colour with a score of 8.01 and texture with a score of 7.60. Smoking
was rated highest in terms of aroma with a score of 7.68 while the sample with 5% salt and
oven dried was most preferred in terms of general acceptability with a score of 7.61.
The result of this study shows that using starter culture for the fermentation of Okpehe
would improve the protein content of this condiment which can be used to supplement
protein for the populace.
xviii
CHAPTER ONE
1.0
INTRODUCTION AND LITERATURE REVIEW
Legumes are members of the plant family Leguminosae and constitute one of the largest
plant families in the world with perhaps 18,000 species (Anon, 2002). Most species are
tropical and include trees, woody vines and herbaceous plants. All leguminous plants bear
the same type of fruit, the pod which is diversely modified within the family. The three
major groups of leguminosae differ in the appearance of their flowers. The first group is the
Mimosoids in which the flowers have reduced petals and usually have conspicuous, long
stamens that give colour to the flower clusters. They are almost entirely tropical and include
the so-called sensitive plant and the silk tree. The second group, the Caesalpinoids is
characterized by well developed petals and includes ornamentals such as the redbud and the
honey locust in temperate regions and the orchid trees of the tropics. The third and largest
group is the Paillionoids whose members bear a flower that resembles the sweet pea, with a
big petal at the top, a wing on both sides, and a keel of two fused petals that enclose the
stamens at the bottom. This group includes peanuts, garden beans, soybeans, mesquite
(Prosopis) e.t.c which are used for firewood, animal forage, nitrogen fixation, timber,
medicine, natural fibers and flavourings. At least a dozen tropical species growing in the
wild or primitively cultivated, especially in Africa, are locally important for human
consumption (Anon, 2002).
Prosopis is a genus of about 45 species of leguminous spiny trees and shrubs of varying
sizes, predominantly xerophilous that are found in subtropical and tropical regions of
1
America, Africa and southwest Asia (Anon, 2011a). They often thrive in arid soil and are
resistant to drought, on occasions developing extremely deep root systems. Their wood is
usually hard, dense and durable. Their fruits are pods and may contain large amounts of
sugar (Anon, 2011a). Prosopis trees offer shade and forage for wildlife and domestic
animals and the indehiscent pods are palatable to man and animals (Barminas et al. 1998).
According to Geesing (2011), the genus Prosopis possibly originated in tropical Africa
where only Prosopis africana, the least specialized species, persists. At the end of the
Mesozoic times when the land connections between the continents were easier, the ancestors
of todays species may have migrated widely from the African centre to both east and west
and developed into two sharply differentiated parallel groups: a small group of prickly AfroAsian species with P. africana, whose native range stretches from Senegal in the west to
Sudan and Kenya in the east, and with P.farcta, P. cineraria and P. koelziana that are native
to North Africa and the Middle East; and a big group of American species. The spiny
American group of Prosopis species was probably again divided later and became widely
separated into two centres; the Mexican-Texan centre and the Argentine-ParaguayanChilean one, with the centre of polymorphism in Argentina (where 27 out of 44 known
species of Prosopis are found) (Geesing, 2011). Some species of Prosopis include P.
glandulosa Torr (Honey mesquite), P. laevigata (Smooth mesquite), P. pubescens
(Screwbean mesquite), P. reptans Benth. (Tornillo), P.velutina Wooton (Velvet mesquite),
P. juliflora, P. strombulifera (Creeping mesquite), P. nigra, P. africana (Guill & Perr.)
Taub. (African mesquite) and P. farcta (Sol. Ex Russell) (Syrian mesquite) (Anon, 2011a).
Prosopis species contains 5-hydroxytryptamine, apigenin, isorhamnetin-3-diglucoside, larabinose, quercetin, tannin and tryptamine (Anon, 2011a). According to the same source,
the tannins present in Prosopis species are of the phytogallotannins and pyrocatecollic types
and are mainly found in the bark and wood while their concentration is low in the pods.
Prosopis africana (Guill and Perr) Taub syn P. oblonga Benth belongs to the family
Fabaceae and sub family Mimosoideae (United State Department of Agriculture (USDA),
2011). Though the common name of Prosopis africana is African mesquite, (Schuster,
1969; Burkart, 1976; Anon, 2002), its native Nigerian names are Kiriya (Hausa), Kohi
(Fulani), Sam chi lati (Nupe), Ayan (Yoruba), Kpaye (Tiv), Ubwa
(Ibo) and Okpehe
(Idoma) (Ogunshe et al. 2007). It is a savannah tree, 12.2-18.3metres (40-60 feet) high and
up to 2.1metres (7 feet) in girth (Ogunshe et al., 2007) with drooping foliage resembling
Tamarindus indica (Le Houerou, 2011). It has an open crown and slightly rounded
buttresses; bark is very dark, scaly, slash, orange to red-brown with white streaks (Aremu et
al., 2006). It has a deep, fast growing tap root, probably phreatophyte. It has good ability for
coppicing but fairly slow growth (Le Houerou, 2011). Its branches and twigs are thornless;
the leaves are bipinnate, leaflets in 9-16 pairs, oblong lanceolate 12-30 mm and shortly
pubescent. A typical gland lies between pairs of pinnae and leaflets; rachis is 10-15 cm long.
The flowers of P. africana are green-whitish to yellowish; fragrant in dense 6-8 cm axillary
spikes. Its calyx is pubescent, petals are glabrous; 10 free-standing stamens; anthers with a
small apical gland with an ovary that is villous. Flowering occurs shortly before the rains.
The pods are dark red, cylindrical, hard and shiny up to 15 3 cm compartmented with
wood cells (Le Houerou, 2011). Its seeds mature between February and March containing
3
some 10 loose rattling seeds per pod and 7,500 -8,000 seeds (with a thin intermarginal line
around) per kilogram (Plate 1, 2 and 3) (World AgroForestry Centre, 2011).
P. africana can be found on various textured soils (including lateritic soils) and frequently
found on fallow land (Le Houerou, 2011). It can tolerate most soil types. It is the only
tropical African Prosopis species occurring from Senegal to Ethiopia in the zone between
the Sahel and savannah forests. Due to extensive over-exploitation, it has disappeared from
extensive parts of the southern Sahel and the adjacent Sudan savannahs (World
AgroForestry Centre, 2011).
The distribution of Prosopis species ranges from sea level to altitudes of 3700 meters with a
variation in annual rainfall from 100 to 1400 mm but most species are found in most semiarid and arid regions of the world (Geesing, 2011). According to World AgroForestry
Centre, (2011), for Prosopis africana, its altitude can be up to 1000m; mean annual
temperature is between 30 - 400C while its mean annual rainfall can be up to 500mm.
Prosopis africana is the only Prosopis native to intertropical Africa, occurring from Senegal
to Ethiopia throughout the Sudanian and Guinean ecozones, reaching the border of the
Sahelian ecozone in the north (Le Houerou, 2011). Some of the countries where it can be
found include Benin, Cameroon, Central African Republic, Chad, Cote dIvoire, Egypt,
Ethiopia, Gambia, Ghana, Guinea-Bissau, Liberia, Nigeria, Senegal, Sierra Leone, Sudan,
Tanzania, Togo and Uganda (World AgroForestry Centre, 2011). In Nigeria, the trees of P.
africana can be found growing wild in the Middle belt, some parts of the East and South
(Barminas et al., 1998; Aremu et al., 2006).
4
Plate 1: Prosopis africana Tree
(x 1 / 100)
Plate 2: Prosopis africana Fruits
(x 1/ 8)
Plate 3: Prosopis africana Seeds
(x 1/ 4)
P. africana can be propagated by direct sowing on site and seedlings. The hard pod must be
crushed to get the seeds out. The seeds need to be pretreated by placing them in boiling
water for 15 minutes, allowing them to cool and soaking them overnight. They are sown in
bags; ready for out-planting after 14-18 weeks. In its natural habitat, flowering occurs just
prior to the rains while the seeds mature between February and March. Like several other
arid-zone species, P. africana produces a deep tap root with few lateral shonets therefore,
pruning seedlings in pots is necessary. It can be grown as a plantation tree but should be
pruned while young to get a clean bole (World AgroForestry Centre, 2011; Le Houerou,
2011).
According to Le Houerou (2011), there are two varieties of P. africana; one with narrow
cylindrical pods (2.5 cm diameter) and the other with broader, flattened pods (3 cm in
width). The pods remain on the tree long after maturing.
According to World AgroForestry Centre (2011); Le Houerou (2011), the functional uses of
P. africana include:
(i) Young leaves and shoots are a fodder that is highly sought after towards the end of
the dry season.
(ii) The wood has a high calorific value of about 1720 joules/kg and produces excellent
charcoal and firewood.
(iii) Its wood is hard, medium heavy to heavy, with a fine grain that is resistant to
termites and used as timber. The wood is difficult to saw and plane and blunts the
8
cutting tools. It cannot be nailed without pre-drilling however, it is durable and easy
to carve. The wood is used for making mortars, pestles, farm implements, and the
planks are used for construction.
(iv) P. africana yields a gum and also contains tannin.
(v) The bark and roots contain 14-16% tannin and a colouring matter that gives a reddish
tint to leather.
(vi) Pounded dry fruits are suitable as a fish poison just like those of Parkia.
(vii) Almost all parts of the tree are used in medicine e.g. the leaves are used for the
treatment of headache and toothache as well as various other head ailments; the leaves
and bark are combined to treat rheumatism; bark is used as remedies for skin diseases,
fevers and caries, the roots are diuretic and are used to treat gonorrhea, dysentery and
bronchitis; In Ghana, boiled roots are used for treating sore throat, tooth ache and bark as
a dressing or lotion for wounds or cuts.
(viii) In Ghana, the pod ashes are of P. africana are a source of potash for soap making.
(ix) Erosion control: Soil conservation is enhanced by planting P. africana.
(x)
Trees of P. africana are suitable for shade or shelter in homesteads in dry areas.
(xi)
It has the potential to improve soil fertility as it can fix atmospheric nitrogen.
(xii) It improves the soil by providing useful mulch for the soil.
(xiii)
Trees of P. africana are suitable for use as ornamental trees.
(xiv)
P. africana has great potential for parkland agroforestry systems and for
improved
agroforestry technologies in the Sahel, where it grows well in valleys
and rocky soils.

(xv)
Prosopis africana is one of the lesser-known legume seed crops used as food
condiment in Nigeria by the Idoma and Igala people of Middle Belt region and
some parts of the Eastern and Southern Nigeria. It adds variety and pleasure to
the otherwise monotonous traditional diet. It serves not only as a seasoning agent
but also as a low-cost source of protein in the diet (Steinkraus, 1991;
Oguntoyinbo et al., 2010).
Solid State Fermentation (SSF) has existed for several centuries. Aerobic microbial
transformation of solid materials or solid substrate fermentation (SSF) can be defined as the
application of living organisms and their components to industrial products and the process
not an industrial in itself, but an improvement technology that will have a large impact on
many different sectors (Hamlyn, 1998). According to Aderolu (2000), SSF is a process in
which solid substrates are decomposed by mono or mixed cultures of microorganisms under
controlled environmental conditions, with the ultimate aim of producing high quality
products. SSF is characterized by the complete or almost complete absence of free water.
Water, which is essential for microbial activities, is present in an absorbed or in complex
form within the solid matrix and the substrate (Adebiyi, 2007). These cultivation conditions
are especially suitable for the growth of fungi which is known to grow at relatively low
10
water activities. As the microorganisms in SSF grow under conditions closer to their natural
habitats, they are more capable of producing enzymes and metabolites which will not be
produced or may be produced only in low yield in submerged conditions (Jecu, 2000). SSF
has been considered superior in several aspects to submerged fermentation (SmF) due to the
various advantages it renders. According to Pandey (2008), the advantages of SSF include:
(i) Cost effective, due to the use of simple growth and production media.
(ii) Need for little amount of water, which consequently releases negligible or considerably
less quantity of effluent, thus reducing pollution concerns.
(iii) Simplicity, due to the use of low volume equipment (lower cost), which are yet effective
by providing high product titres (concentrated products).
(iv) Ease of aeration of the process, this is due to the availability of atmospheric oxygen to
the substrate since oxygen limitation does not occur as there is an increased diffusion rate of
oxygen into moistened solid substrate, supporting the growth of aerial mycelium.
(v) Potential for effective use at smaller levels also, which makes them suitable for rural
areas too.
Current trends on SSF have focused on its application for the development of bioprocess
such as bioremediation and biodegradation of hazardous compounds, biological
detoxification of agro-industrial residues, biotransformation of crops and crop-residues for
nutritional enrichment, biopulping, and production of value-added products such as
11
biologically active secondary metabolites, including antibiotics, alkaloids, plant growth

factors, enzymes, organic acids, biopesticides, including mycopesticides and bioherbicides,
biosurfactants, biofuel, aroma compounds, etc (Pandey, 2008).
Fermentations are enzyme-induced chemical alterations in food or substrate. The enzyme
involved may be produced by microorganisms or they may be indigenous to the food or
substrate (Ihekoronye and Ngoddy, 1985). Generally, fermentation results in the breakdown
of complex organic substances into smaller ones through the action of catalysis.
Fermentation results in increased nutritional value and wholesomeness over the starting
material (Van Veen and Steinkraus, 1970). Fermentation also contributes to the masking of
undesirable odours and flavours while imparting desirable flavour to the finished product
(Beauchat, 1976). More importantly, fermentation has been reported to enhance digestibility
of starting materials by breaking down complex protein structures to peptides and free
amino acids (Hesseltine and Wang, 1979). It helps in the reduction/elimination of antinutritional factors in foods (Achi, 2005b). It remains an effective, inexpensive method for
extending the shelf life of foods (Tamang, 2009). It reduces the toxicity of some foods e.g.
gari, while others may become extremely toxic during fermentation e.g. bongkrek (Jay et
al., 2005). In African countries, it is a method of making food storable and by this way
attempting to have sufficient food in the lean seasons. Fermentation helps to reduce the
cooking time of foods and hence a reduction in fuel requirements (Ouoba et al., 2007).
During fermentation, the activity of microorganisms resulting from the existence of

favourable environment leads to the breakdown of complex components of food molecules
12
into simpler ones (Ibe and Orabuike, 2009). These substances get metabolized into end
products and such are released to the environment. The major components in food which are
carbohydrates, proteins and fats are metabolized by microbes (Odunfa and Oyewole, 1998).
When microorganisms attack carbohydrates or carbohydrate-like compounds, products such
as alcohols, acids, aldehydes and ketones are released. Since these compounds are only
slightly more oxidisable than the original compound, they can therefore yield energy upon
further oxidation (Ibe and Orabuike, 2009). For normal growth, microorganisms need a
source of nitrogen. This can be met by proteins in food thus microbes attack proteins in
foods through proteolysis by breaking it down to release amino acids with an accompanying
foul smelling odour. Also, microbial growth encourages the synthesis of several vitamins
and amino acids so that fermented products could have an increased nutritive value than the
parent substrate (Odunfa and Oyewole, 1998). Fats are broken down by microbes into free
fatty acids although extensive hydrolysis could diminish nutritional quality (Achi, 2005a).
Achi (2005a) reported that the pulp of African locust bean had more protein and ash while
the oil bean seeds had more lipids and non protein nitrogen (NPN). He also reported that the
4 days fermentation period had varied effects on mineral element levels in African locust
beans seeds.
Parkouda et al. (2009), also reported a reduction of antinutrients like phytate (0.18 0.16)
mg/g and tannins (0.25 0.18) mg/g during the fermentation of baobab seeds. A reduction
in carbohydrate causing flatulence was also reported by them.
13
Fermented foods form an integral and significant part of the diet of many people. Most
fermented products continue to remain of interest since they do not require refrigeration
during distribution and storage (Ibe and Orabuike, 2009). Many fermented foods serve as
main meals, others as beverages while others are highly prized food condiments (Odunfa
and Oyewole, 1998). Those that serve as main meals and beverages are usually products of
carbohydrate-rich materials e.g gari from cassava, ogi from maize and burukutu from
sorghum while those that serve as food condiments are usually made from the fermentation
of protein rich seeds e.g. iru from locust bean. According to Odunfa and Oyewole (1998),
African fermented foods include: fermented non-alcoholic starchy foods such as ogi. koko,
kenkey and mahewu; fermented animal proteins such as fermented milk and fish; and
fermented vegetable proteins such as iru, ugba and Okpehe.
Protein rich seeds are often fermented to make food condiments which enhance the flavor of
foods. The use of hydrolyzed vegetable proteins in seasoning has long been recognized
(Odunfa and Oyewole, 1998). The high cost of animal protein has directed interest towards
several leguminous seeds as potential sources of vegetable protein for human food and
livestock feed (Ezenwah et al., 2008). Most of the fermented vegetable proteins reported are
from leguminous seeds (Ogunshe et al., 2007). Quite often, seeds that are used for
fermentation are inedible in their raw unfermented or cooked state. Seeds of legumes may
account for up to 80% dietary protein and be the only source of protein for some groups of
people. Although fermented food condiments have constituted a significant proportion of the
diet of many people, Nigerians exhibit an ambivalent attitude in terms of consumer tastes
and preferences for such foods (Achi, 2005b). The introduction of foreign high technology
14
products especially processed ones because of globalization and liberalization of the

economy radically changed the Nigerian food culture into a mixed grill of both foreign and
local dishes (Ojo, 1991).
Traditional diets in West Africa often lack variety and consist of large quantities of staple
foods like cassava, yam and maize with supplements of plantain, cocoyam, rice and beans
depending on availability and season (Achi, 2005b). The food diet of Nigerians is mostly
from roots, tubers and cereals. Their low proteins (Karim and Adekunle, 2010) contribute to
low nutrition security of the people. Investigations carried out on methods of improving the
protein and mineral contents of food from plant sources have been reported to be successful.
Soups eaten with the staples are an essential component of the diet and may contain a
variety of seeds, nuts, pulses and leaves (Campbell-Platt, 1980). The staple foods provide
the calories but are poor in other nutrients like proteins and minerals. Soups are the main
sources of proteins and minerals and one of the ways to improve the diet has been to
improve the nutrient content of soups (Achi, 2005b).
According to Websters Reference Library (2009) dictionary, a condiment is a seasoning
that one adds to food in order to increase its flavour e.g. salt, pepper e.t.c. Indigenous
fermented condiments have recently assumed a greater importance in Nigeria since the
majority of the populace can no longer afford the high cost of edible plant and animal
proteins (Ogbadu et al., 1990). Traditional condiments have not attained worldwide
commercial status due to the short shelf life, objectionable packaging materials, stickiness
and the characteristic putrid odour (Arogba et al., 1995). Fermented condiments often have a
15
stigma attached to them as they are considered as food for the poor. They are prepared by
traditional methods of uncontrolled solid substrate fermentation resulting in extensive
hydrolysis of the protein and carbohydrate components (Fetuga et al., 1973; Eka, 1980).
Production of these condiments is by spontaneous fermentation carried out in peoples
homes using rudimentary utensils under varying hygienic conditions (Oguntoyinbo et al.,
2010). Apart from increasing the shelf life and a reduction in the anti-nutritional factors
(Odunfa, 1985; Achi, 2005b), fermentation markedly improves the digestibility, nutritive
value and flavours of the raw seeds. The raw seeds are inedible in the natural state. There
are over thirty different fermented foods in Nigeria (Odunfa, 1985). The methods employed
in the manufacture of fermented condiments differ from one region to another depending on
the prevailing socio-economic circumstances. Plant sources notably legume crops are
fermented and used as traditional condiments. Those in common use include peanuts,
soyabeans, locust beans, oil bean, cowpeas, lentils, alfalfa (Lucerne), bambara nuts e.t.c.
Available information from literature indicates that over nine different fermented products
are condiments. Some of the most important food condiments are iru/dawadawa produced
from locust bean seeds (Parkia biglobosa), ogiri produced from melon seeds (Citrullus
vulgaris) or fluted pumpkin (Telferia occidentale), ogiri-igbo produced from castor oil seed
(Ricinus communis), ugba produced from oil-bean seeds (Pentaclethra macrophylla), owoh
produced from cotton seeds (Gossypium hirsutum) and African yam bean (Sphenostylis
stenocarpa), bambara-daddawa produced from soybean (Glycine max) and bambara
groundnut (Vigna subterranea), mwanza ntuza produced from Hibiscus sabdariffa, oso
produced from seeds of Cathormion altissimum e.t.c. (Campbell-Platt, 1980; Odunfa, 1985;
16
Odunfa and Oyeyiola, 1985; Ogbadu and Okagbue, 1988; Sanni, 1993; Omafuvbe et al.,
2003; Achi, 2005b; Popoola et al., 2006; Ogunshe et al., 2007; Ayodele and Mudsa, 2008).
Fermentation starters (simply starters) are preparations to assist the beginning of the
fermentation process in the preparation of various foods and fermented drinks. A starter
culture is a microbiological culture which actually performs fermentation (Anon, 2012).
These starters usually consist of a cultivation medium, such as grains, seeds, or nutrient
liquids that have been well colonized by the microorganisms used for the fermentation
(Frazier and Westhoff, 2006).
The fermentation of condiments is still being carried out by the traditional village-art
method. There is need to apply modern biotechnological techniques like the use of starter
cultures in improving traditional food processing technologies (Achi, 2005b). It has been
suggested that fermentation processes in developing countries could be improved by using
starter cultures, and back slopping, which entails application of brine from prior
fermentation cycles (Holzapfel, 2002; Naiba, 2003). A pure starter culture is essential for
controlled fermentation. Most starter cultures help to reduce fermentation time as well as
guarantee consistency, product safety and quality (Achi, 2005a; Oguntoyinbo et al., 2010).
Bacillus species, Staphylococcus and Streptococcus enterococcus have been used as pure
culture starters in controlled experimental fermentation (Suberu and Akinyanju, 1996;
Omafuvbe et al., 2003; Ouoba et al., 2003a, b). Controlled fermentation of soybeans was
achieved by using pure single cultures of Bacillus subtilis, B. licheniformis or in
17
combinations (Sarkar et al., 1993; Suberu and Akinyanju, 1996) to obtain soyiru. Mixed
cultures of B subtilis and B. licheniformis were recommended by the same authors while
fermentation was achieved in 72 h. On the other hand, Omafuvbe et al., (2003) used B.
subtilis, B. licheniformis and B. pumilus singly and in combinations to check their abilities to
ferment soybean for the production of daddawa. B. subtilis as single or member of a mixed
starter produced soy-daddawa, which was considered most suitable as it gave acceptable
pure culture condiment supposedly due to its proteolytic enzyme activity and high level of
free amino acids. Report by Gberikon et al. (2010) also showed that fermenting seeds of
Parkia biglobosa, Glycin max and Prosopis africana inoculated with a consortium of B.
subtilis and B. pumilus gave higher values of protein and lipids than when the seeds were
fermented naturally. According to Nwagu et al. (2011), the use of mixed starter culture of B.
megaterium and Corynebacterium sp and B. megaterium and Alkaligenes viscolatis gave a
98% and 97% overall quality respectively in the controlled fermentation of ugba as
compared to using single organism (B. megaterium) that gave a 87% quality. The
fermentation time of 3-4 days was shortened to 2 days in the production of ugba using
mixed starter culture of B. subtilis and B. megaterium (Mbata and Orji, 2008).
According to Parkouda et al. (2009), the following starter cultures have been developed for
the production of the following condiments- B. subtilis var natto (natto), B. subtilis B7 and
B15 for soumbala; B. subtilis MM-4:B12 for ugba; and B. subtilis 24BP2 and B. subtilis
FpdP2 for soydawadawa. The use of a single strain would seem too restrictive for the
production of a foodstuff with a generous range of organoleptic characteristics. The use of a
18
mixture of microorganisms with complementary physiological and metabolic properties

seems to be the best approach for obtaining a product with the desired nutritional and
sensory properties (Achi, 2005b).
In most African countries including Nigeria, the problem of food security is not just that of
inadequate food but it is also a problem of loss of food due to spoilage. Lack of adequate
food preservation methods is a major problem contributing to food insecurity in Africa. The
high costs and infrastructural requirement of many advanced food preservation methods
such as refrigeration, freezing, canning and irradiation have greatly reduced their application
in the developing world (Ibe and Orabuike, 2009). This implies that promoting fermentation
and fermentation technology in Africa, is helping to promote food security in the region.
Food preservation is the process of treating and handling food to stop or slow down spoilage
(loss of quality, edibility or nutritional value) and thus allow for longer storage. According
to Sivasankar (2009), the basic principles of food preservation primarily involve the process
of inhibiting such processes as the growth and activity of microorganisms, activity of
endogenous enzymes, chemical reaction which may deteriorate the quality of food and
invasion and spoilage by insects and rodents. Reasons for preserving food include extension
of the safe storage life of food, its safety, acceptability, nutritive value, availability and
economic viability (Anon, 2012).
19
Many processes designed to preserve food involve a number of food preservation methods.
These methods are drying, refrigerating, freezing, addition of salt or sugar, smoking,
canning, high heat processing (pasteurization), ionizing radiation, chemical preservatives,
pickling e.t.c.
Drying is one of the most ancient food preservation techniques, which reduces water activity
sufficiently to prevent or delay bacterial growth. During the process of drying, both the food
and microorganisms are dehydrated. Fellows (2009) observed that microorganisms contain
80% of moisture. Since most disease-causing microorganisms require a moist environment
in which to survive and multiply, drying is a natural method of preventing spoilage (Jay,
2005). According to Banwart (2004), other reasons for drying are (1) to preserve the
product from physical or chemical changes induced or supported by excess moisture; (2) to
reduce the packaging, storage and transportation costs; (3) to prepare a material for a process
when only dry materials can be used; (4) to remove moisture added in previous operations;
(5) to bring the product to a moisture level at which it is normally graded, bought and sold;
(6) to recover waste products; and (7) to provide convenience and a saving of time to users.
Drying can be achieved by sun drying, spray drying, vacuum drying and freeze drying. The
advantages of drying include (1) production of concentrated form of food, (2) inhibition of
microbial growth and autolytic enzymes, (3) retention of most nutrients while its
disadvantages are: (1) loss of some nutrients, particularly thiamin and vitamin C
20
(2) sulphur dioxide is sometimes added to dried fruits to retain vitamin C, but some
individuals are sensitive to this substance (Anon, 2011b).
Refrigeration means storage at temperatures above freezing of water in the foods usually in
the range of 160C (600F) to -2.20C (280F). Refrigerators usually operate at 4 to 70C
(Sivasankar, 2009). According to the same author, it is the simplest method of food
preservation as it has no adverse effects on taste, texture and nutritive value. It preserves
food by slowing down the growth and reproduction of micro-organisms and the action of
enzymes which cause food to spoil. The introduction of commercial and domestic
refrigerators drastically improved the diets of many people in the Western world by allowing
foods such as fresh fruit, salads and dairy products to be stored safely for longer periods,
particularly during warm weather (Anon, 2011b). The advantages of refrigeration are (1)
slows microbial multiplication, (2) slows autolysis by enzymes while its disadvantage is that
there is slow loss of some nutrients with time (Anon, 2011b).
Freezing is also one of the most commonly used processes commercially and domestically
for preserving a very wide range of food including prepared food stuffs which would not
have required freezing in their unprepared state. According to Jay (2005), the two basic
ways of freezing foods are quick or fast and slow freezing. Quick freezing is the process by
which the temperature of foods is lowered to about -200C within 30 minutes and may be
achieved by direct immersion or indirect contact of foods with the refrigerant and the use of
air-blasts of fridge air blown across the foods being frozen while slow freezing refers to the
21
process whereby the desired temperature is achieved within 3 to 72 hours (Jay, 2005).
Freezing has been a major factor in making available a variety of convenience foods to the
consumer apart from preserving foods for prolonged periods. Proper freezing preserves food
without major changes in its size, shape, texture and flavour (Sivasankar, 2009). The
advantages of freezing are (1) it prevents microbial growth by low temperature and
unavailability of water; (2) neither adds nor removes any components (3) imparts no new
flavor nor alters the natural flavor (4) des not diminish the digestibility or cause a significant
loss of nutrient value (Sivasankar, 2009). According to the same author, its disadvantages
include (1) microorganisms maybe reduced in number but not destroyed (2) spore are very
resistant and toxins are not destroyed (3) frozen foods not properly wrapped dehydrate very
rapidly, which cause marked deterioration in the flavor and general appearance of the food
(4) blanching of vegetables prior to freezing causes loss of some B-Group vitamins and
vitamin C (Anon, 2011b).
Smoking is used to lengthen the shelf life of perishable food items. This effect is achieved
by exposing the food to smoke from burning plant materials such as wood. Most commonly
subjected to this method of food preservation are meats and fish that have undergone curing.
Fruits and vegetables like paprika, cheeses, spices, and ingredients for making beverages
such as malt and tea leaves are also smoked, but mainly for cooking or flavoring them. It is
one of the oldest food preservation methods, which probably arose after the development of
cooking with fire (Anon, 2012). The primary purposes of smoking are (1) preservation, (2)
22
development of aroma and flavour, (3) creation of new products, (4) development of colour,
(5) protection from oxidation, (6) formation of protective skin on emulsion-type sausages
(Jay et al., 2005). The chemical composition of wood smoke depends upon factors such as
the type of wood, amount of air, temperature and time (Banwart, 2004). Hickory or other
hardwoods are preferred. Soft woods cause unpleasant flavours in the foods (Banwart,
2004). According to the same author, the preservative effect is a result of the combination of
drying and the deposition of chemicals resulting from the thermal decomposition of wood.
The amount of smoke deposited on a food is a complex function of composition and
concentration of the smoke, the temperature and humidity, and the nature of the food surface
(Banwart, 2004). According to the same author, acids, phenols and carbonyls are chemicals
found in smoke that contribute significantly to the odour and flavour of foods while volatile
phenolic compounds in the vapour phase of the smoke are primarily responsible for the
smoke flavour. The advantage of smoking is that it preserves partly by drying and partly by
incorporation of substances from smoke while its disadvantage is that eating a lot of smoked
foods has been linked with some cancers in some parts of the world (Anon, 2011b).
Treating food with salt was one of the earliest methods of preserving food. Besides its
preservative function, salt is added to foods to enhance flavour, to aid in the development of
colour in processed meats and sauerkraut, to aid in the texture of processed meats, yeastraised baked goods, and certain cheeses, to solubilize myofibrillar proteins that act as
binders in cured meats, and to help in the regulation of the rate of fermentation in pickles,
23
sauerkraut, sausage, cheese and yeast-leavened dough (Banwart, 2004). At high

concentration, salt exerts a drying effect on both food and microorganisms (Jay et al., 2005).
Microorganisms vary in their tolerance to salt. The mesophilic Gram-negative rods and
psychrophiles are inhibited by 4 to 10 percent salt, the lactic-acid producing bacteria vary in
tolerance from 4 to 15 percent salt, least tolerant organisms such as Spirillum can tolerate
only about 1 percent salt, the spore forming bacteria can tolerate from 5 to 16 percent salt
while most non marine bacteria can be inhibited by 20% or less of salt (Banwart, 2004; Jay
et al., 2005). When microbial cells are suspended in saline solution (e.g. 5%), the
concentration of water is greater inside the cells than outside (concentration of water is
highest where solute concentration is lowest), so water passes out of the cells at a greater
rate than it enters. The result to the cells is plasmolysis, which leads to growth inhibition and
possibly death (Jay et al., 2005). This is essentially what is achieved when high
concentrations of salt are added to foods for the purpose of preservation. Both the microbial
cells and the food undergo plasmolysis (shrinkage), resulting in the drying of the food as
well as inhibition or death of microbial cells (Jay, 2005). Enough salt must be used to effect
hypertonic conditions. The higher the concentration, the greater are the preservative and
drying effects. The inhibitory effects of salt are not dependent on pH as are some other
chemical preservatives (Jay et al., 2005). According to Anon (2011b), the advantages of
salting are (1) it makes water unavailable for microbial growth, (2) does not destroy
nutrients while its disadvantage is that it increases the salt content of food thereby affecting
its taste.
24
Preservation of food by the use of heat finds wide application compared to other methods.
The use of high temperature to preserve food is based on their destructive effects on
microorganisms (Jay, 2005). The killing of microorganisms by heat is due to thermal
denaturation of proteins and enzymes of the microorganisms required for their metabolic
activities and growth. The heat treatment necessary to kill the organisms or spores varies
with the kind of organisms, its state and the environment during heating (Sivasankar, 2009).
According to the same author, the use of heat also affects the food adversely hence it is
necessary to use only mild heat treatment that ensures freedom from pathogens and enzyme
activity whilst enhancing the shelf life of the food. With respect to food preservation, any
temperature above ambient is referred to as high. Based on this, there are two temperature
categories in common use in food preservation: pasteurization and sterilization (e.g.
canning). Pasteurization is the heat treatment of foods between 63 -72 oC and it is aimed at
destroying all disease-producing organisms or reduction in number of spoilage organisms
while sterilization is achieved above 100 oC and aimed at destruction of all viable organisms
(Jay et al., 2005). According to Anon (2011b), the advantages of pasteurization are
inactivation of autolytic enzymes and destruction of microorganisms, while its disadvantage
is in the loss of heat-sensitive nutrients. The advantage of canning is that it destroys
microorganisms and autolytic enzymes while its disadvantage is in loss of some watersoluble nutrients into liquid in can (Anon, 2011b).
Sensory analysis is a multi-disciplinary science that uses automated instruments and human
panelists and their senses of sight, smell, taste and touch to measure the sensory
25
characteristics and acceptability of food products (Watts et al., 1990). According to

Sivasankar (2009), sensory evaluation includes quality check on the basis of three basic
factors which are appearance, texture and flavour. These factors determine the organoleptic
properties of food. Sensory test is used for new product development, product/process
improvement, storage stability and consumer preference (Balami et al., 2004). The types of
sensory evaluation methods are paired comparison, duo-trio, triangle, ranking, scalar
difference control, threshold, dilution, attribute rating, flavour profile analysis, texture
profile analysis, quantitative descriptive analysis, hedonic scale rating and food action scale
rating (Iwe, 2007).
The most commonly used scale for preference testing is the nine-point Hedonic scale
(Ihekoronye and Ngoddy, 1985; Iwe, 2007). The term Hedonic means having to do with
pleasure (Iwe, 2007). Hedonic tests are designed to measure the degree of liking for a
product. According to Iwe (2007), the Hedonic scale rating of samples ranged from 1
(dislike extremely), 2 (dislike very much), 3 (dislike moderately), 4 (dislike slightly), 5
(neither like nor dislike), 6 (like slightly), 7 (like moderately), 8 (like very much) and 9 (like
extremely). Panelists usually indicate their degree of liking for each sample by choosing the
appropriate category.
Okpehe fermentation is, like most fermented foods, produced and marketed on a small scale.
Although Okpehe has long been used as a food condiment by the Idoma people of Benue
26
State, the biomodification occurring during the fermentation is yet to be fully documented.
The microorganisms associated with the fermentation may strongly influence the chemical
composition of the commodity. This study is designed to increase the understanding of the
fermentation process involved in the production of Okpehe.
1.1
Justification for the Study
In many countries in Africa including Nigeria, protein malnutrition is a major problem. It

has been reported that the diet of most Nigerians is lacking in substantial protein due to the
high costs of most of the available protein sources such as meat. Therefore food and
nutrition security remains Africas most fundamental challenge for human welfare and
economic growth (Karim and Adekunle, 2010). Nigeria spent approximately $120million on
importation of food flavorings in 1983 with a projected 15% annual future increase (Essien,
1983; Odunfa and Oyewole, 1998). This therefore underscores the importance of fermented
vegetable proteins which have great potential as key protein sources and as basic ingredients
for food supplementation in the diet.
Many people, most especially the rural ones are unable to acquire necessary foods for a
healthy life. This is due to low food availability, profound poverty and lack of nutrition
education (Karim and Adekunle, 2010). The result is that about a third of the Nigerian
populace is malnourished (FAO, 2003). The continuing human costs of inadequate food and
nutrition are enormous. The aggregate costs of food and nutrition security at the national
27
level impose a heavy burden on the government to foster and sustain economic growth and
improve the general welfare of the people. (Benson, 2004; Oladimeji and Karim, 2009).
Due to increasing attention on the Millennium Development Goals (MDGs) and the sad
problems of global economic meltdown, there is the need to urgently find ways to
ameliorate this problem. Among such ways is a deeper research into some unutilized or
underutilized crops. A promising tool for such research is the seeds of Prosopis africana.
Trees of this plant can be found growing wild in many parts of Nigeria (University of Ilorin
campus inclusive) where tonnes of its fruits drop during its season and contribute to
environmental pollution. The raw unfermented seeds of Prosopis africana are inedible but
fermentation improves the nutritional quality and digestibility. Traditionally, the production
process of Okpehe is tedious, wasteful, requires a large volume of water and also takes a
long time to get the end product. The use of microorganisms as starters will shorten the
production time and make the production less tedious while achieving a better quality
product. The use of starter cultures in the right proportion would guarantee consistency,
increase product safety and enhance product quality. Okpehe can serve as a substitute for
meat for low-income earners and can reduce protein-calory malnutrition and essential fatty
acid deficiencies (Oguntoyinbo et al., 2010).
28
1.2
Research Objectives
The objectives of this research were to:

i
ferment Prosopis africana seeds to produce Okpehe in the laboratory simulating the
traditional method;
ii.
monitor the physicochemical changes and microbial succession during the natural
fermentation of Okpehe and a comparism of the laboratory produced and commercial
samples;
iii.
use the microorganisms isolated as starter culture, singly and in consortium for the
fermentation of Okpehe;
iv.
determine the best preservative method for Okpehe using different preservative
regimens; and
v.
carry out sensory evaluation of Okpehe.
29
CHAPTER TWO
2.0
MATERIALS AND METHODS
2.1
Sources of Fruits and Okpehe Samples
The fruits of Prosopis africana were obtained from the Main Campus of the University of
Ilorin, Ilorin, Nigeria and identified at the herbarium of the Plant Biology Department of the
University. Voucher Specimen Number UIH/472 was obtained and sample deposited at the
herbarium.
Okpehe was produced in the laboratory using the autoclave and hot plate
simulating the traditional method. The commercial Okpehe was also collected aseptically
from the home of a local producer and transported to the laboratory in sterile polyethene
bags under ice cubes. All samples were analyzed immediately in the laboratory.
2.2
Traditional Preparation of Okpehe from Prosopis africana Seeds
The seeds of the Prosopis africana were removed by beating the fruits with a club on a
concrete surface to break the tough fruit coat. Two thousand grams of seeds were boiled
overnight in a large earthen-ware pot with wood fire, during which the seed coats became
soft and the seeds swollen. The seeds were allowed to cool. The seed coats were removed
by pressing between fingertips. These coats were later decanted along with the washing
water, leaving the clean seed cotyledons. The clean cotyledons were put in another pot with
small amount of water and cooked for 1-2 hours. The cotyledons were later drained through
sieve, cooled and wrapped with paw-paw leaves. The wrapped cotyledons were put in clean
30
bowls covered with jute bags and then left for 3 days in an incubating unit during which
natural fermentation occurred. After fermentation, the resultant product, which was brown
in colour, was Okpehe, a strong-smelling mass of sticky cotyledons. The Okpehe was made
into balls of 3 -5cm diameter, arranged in trays and dried for 1-2 days in the sun. The
product became black after sun drying. The dried product was ground with mortar and
pestle and it was ready to be sold by the local producers to the consumers. The Okpehe
produced by the local producer was designated as sample A (Fig. 1).
2.3
Laboratory Preparation Using the Modified Traditional Method
2.3.1 Using the autoclave

The method of Ogunshe et al. (2007) was used with some modifications for the fermentation
of Prosopis africana seeds. The seeds were removed by beating the fruits with a club on a
concrete surface to break the tough fruit coat. Two thousand grams of seeds were boiled in
an autoclave at 121 oC for 2 hours. The seeds were allowed to cool. The seed coats were
removed by pressing between fingertips. The cotyledons were separated from the coats and
later rinsed in sterile water, before heating on hot plate set at 60 oC for about 30 minutes to
soften the cotyledons. The cotyledons were later drained through a sterile sieve and cooled
to room temperature before wrapping in paw-paw leaves already disinfected by swabbing
with 70% alcohol, rinsed with sterile water and allowed to dry. The wrapped cotyledons
were covered with layers of sterile aluminium foil and then incubated in an incubating unit
at 30 2 oC for 3 days to produce the fermented mash of Okpehe. The Okpehe produced by
this method was designated as sample B (Fig. 2).
31
Prosopis africana fruits
Remove fruit coats
Prosopis africana seeds
Boiled overnight (with wood fire)
Seeds dehulled and cotyledons washed
Cotyledons cooked for 1-2 hrs
Cotyledons wrapped in pawpaw leaves
Wrapped cotyledons packed in jute bags
Fermentation for 3 days (at 25-30 oC)
Okpehe
Fig.1: Traditional method of production of Okpehe from Prosopis africana seeds
32
Remove fruit coats
Boiled at 121 oC (autoclave)
Cotyledons heated for 30 mins at 60 oC
Cotyledons wrapped in sterile pawpaw leaves
Wrapped cotyledons covered with sterile aluminium foil
Fermentation for 3 days (at 30 2 oC)
Okpehe
Fig. 2: Laboratory production of Okpehe from Prosopis africana seeds using autoclave
33
2.3.2 Using the hot plate

The seeds of the Prosopis africana were removed by beating the fruits with a club on a
concrete surface to break the tough fruit coat. Two thousand grams of seeds were boiled at
1000C for 6 hours in a stainless steel pot on a hot plate, during which the seed coats became
soft and the seeds swollen. The seed coats were removed by pressing between fingertips.
The cotyledons were separated from the seed coats and later rinsed with sterile distilled
water. The clean cotyledons were put in another pot with small amount of water and heated
on the hot plate at 600C for 30 minutes. The cotyledons were later drained through a sterile
sieve and wrapped with paw-paw leaves. The paw-paw leaves used had been swabbed with
70% alcohol, rinsed with sterile and allowed to dry prior to usage. The wrapped cotyledons
were covered with three layers of sterile aluminium foil and then left at 30 20C for 3 days
in an incubating unit during which natural fermentation occurred. The Okpehe produced by
this method was designated as sample C (Fig.3).
For all the three samples, the time of wrapping was regarded as the zero hour of the
fermentation. Samples were taken subsequently after every 24 hours until the end of 72
hours. The samples were stored at 40C in a refrigerator prior to analysis and analyzed within
30 minutes of collection.
34
Remove fruit coats
Boiled at 1000C (on hot plate)
Cotyledons heated for 30mins at 600C
Cotyledons wrapped in pawpaw leaves
Wrapped cotyledons covered with aluminium foil
Fermentation for 3 days (at 30 20C)

35

Okpehe
Fig 3: Laboratory production of fermented Prosopis africana seeds (Okpehe) using hot plate
2.4
Inoculation with Starter Culture
Prosopis africana seeds were processed in the laboratory as previously described. The
cotyledons were drained through a sterile sieve and allowed to cool prior to inoculation of
starter organisms. The method of Oyeyiola (1990) was used with some modifications for the
inoculation with starter organisms. One hundred grams of processed unfermented seeds
were mixed with separate 1 ml broth cultures of the following organisms: B. subtilis, B.
licheniformis, B. megaterium, and B. pumilus. After the single organism inoculation, a
consortium of 2, 3 and 4 organisms was also done into separate one hundred grams of
processed raw seeds in the ratio of 1:1, 1:1:1 and 1:1:1:1 respectively. 1 ml broth cultures of
each of the four organisms were spread on nutrient agar plates, incubated at 370C for 24
hours and counted using the colony counter. All cultures used for inoculation contained
approximately 2 106 cells /ml (Oyeyiola, 1990). Prior to inoculation, a suspension of each
of the starter organisms was made in nutrient broth and incubated at 370C for 24 hours. The
resulting mash after inoculation was first wrapped with pawpaw leaves and then in two
layers of sterile aluminium foil. The pawpaw leaves used had been cleaned and surface
sterilized with 70% alcohol and rinsed with sterile water prior to its usage. The wrapped
cotyledons were left at 30 20C for 72 hours in an incubating unit to ferment (Fig. 4).
Hands were washed thoroughly with an antiseptic detergent solution and rinsed many times
36
with sterile distilled water at every handling stage. Samples were taken at zero time and
subsequently after every 24 hours for physicochemical, total sugar, proximate composition
and microbial analysis.
Remove fruit coats
Boiled at 1000C (on hot plate)
Cotyledons heated for 30mins at 600C

inoculation with starter culture
Cotyledons wrapped in sterile pawpaw leaves
Wrapped cotyledons covered with sterile aluminium foil
Fermentation for 3 days (at 30 20C)

37

Okpehe
Fig 4: Laboratory production of Okpehe using starter culture
2.5
Determination of Physicochemical Parameters
2.5.1 Determination of pH
A pH meter (model Denwer 20) was used for this purpose. It was used to measure the
acidity and alkalinity of the Okpehe samples. It was first standardized using standard buffer
solution of pH 4 and pH 7. The electrode was then rinsed with distilled water and immersed
into the samples. Five grams of the different samples were ground thoroughly in a mortar.
This was then suspended in 100ml of distilled water. The pH of the suspension was
measured using pH meter (model Denwer 20).
2.5.2 Determination of Temperature
The temperature was determined by inserting a sterile thermometer (wiped with alcohol)
into each of the samples on each day of fermentation. The mercury-in-glass thermometer
was used.
2.5.3 Determination of Moisture Content
A dry clean porcelain crucible was weighed (W1) and 2g of each of the different Okpehe
samples was placed in the crucible. The weight of sample and crucible was recorded as W2.
The crucible with sample was then placed in an oven at 60oC for 24 hours. It was then
cooled in a dessicator and reweighed. Further drying, cooling and weighing were carried
38
out until a constant weight W3 was achieved (AOAC, 2002). The percentage moisture
content was given as:
% Moisture
Loss in weight due to drying 100

Weight of sample
% Moisture
W1 W3 100
W2 W1
where W1
Weight of Crucible
W2
weight of crucible + sample before drying
W3
weight of crucible + sample after drying
2.6
Isolation of Microorganisms
The serial dilution method was used for the isolation of microorganisms from 1-g samples
before and during fermentation. One gram of sample was added to 9ml of sterile distilled
water and shaken to get 10-1 dilution. Then 1ml of this dilution was transferred to another
9ml of sterile distilled water, and shaken to obtain 10-2 dilution. Nutrient agar was used to
isolate bacteria, while potato dextrose agar and yeast extract agar were used to isolate fungi.
The spread plate method was used for the isolation. Plates were incubated at 37oC for 24 48 hours for bacteria and at room temperature for 2 - 5 days for fungi. Representative
colonies were differentiated on the basis of morphology and colour and then sub-cultured to
obtain pure cultures. Isolation of microorganisms was done within 30 minutes of sample
39
collection. Microorganisms were isolated at zero hour and subsequently after every 24
hours. Colonies were counted using the colony counter from the different mixed culture
plates and representative colonies were sub-cultured.
2.7
Characterization of Bacterial Isolates
This was done by carrying out colonial morphology (which includes shape of colony,
elevation of colony, edge of colony, optical characteristics and pigmentation) and cellular
characteristics which includes gram staining, spore staining and motility. Relevant
biochemical tests were also carried out including catalase, indole, urease, citrate, oxidase,
starch hydrolysis, oxygen relationship, methyl red-voges proskauer, hydrogen sulphide,
nitrate and fermentation of sugars. After determining colonial, cellular and biochemical
characteristics of the bacterial isolates, reference was made to standard texts such as
Buchanan and Gibbons (1974) and Cheesebrough (2004). Detail for characterization and
identification is presented in Appendix II.
2.8
Characterization of Yeast Isolate
The yeast isolate was examined macroscopically (this includes colour, shape and presence or
absence of mycelium) and microscopically (shape of organism, colour, whether budding or
not). Onions et al. (1981) and Fawole and Oso (2004) were consulted for the identification
of the yeast isolate.
2.9
Proximate Analysis of Okpehe Samples
2.9.1 Determination of Dry Matter Content

40
Two grams of samples were weighed into a clean porcelain boat of predetermined weight.
The samples were dried to a constant weight in an oven at 600C for 24 hours. The porcelain
boat was placed in the dessicator and allowed to cool for 1 hour before it was reweighed.
Dry matter was calculated and expressed in percentage (AOAC, 2002).
Dry matter (%) = (weight left after drying/initial weight of sample) 100
2.9.2 Determination of Ether Extract (Crude Fat) Content

Extraction of the fat was carried out by Soxhlet method (AOAC, 2002) using petroleum
ether (Bp 40 60oC) as solvent. The fat was extracted from the dried residue obtained after
the determination of the moisture content. Previously dried thimble was weighed (W1) and
2g of each sample was added into the thimble and reweighed (W2). A 500ml round bottom
flask was filled up to 2/3 of the mark with petroleum ether. The Soxhlet extractor was fixed
up with a reflux condenser and the heat source adjusted between 40 600C boiling range so
that the solvent heated gently. Extraction of the fat continued for 4 hours after which the
thimble was removed. The thimble was placed in a beaker in an oven at 500C and dried to
constant weight with the sample. It was then cooled in the desiccator and reweighed (W3).
The crude fat was then calculated as:
% ether extract
W2 W3 100
W2 W1
where W1
Weight of thimble
41
W2
Weight of the thimble and sample
W3
Weight of the defatted sample and thimble
2.9.3 Determination of Crude Fibre

Two grams of the defatted sample was weighed into a conical flask and reweighed (W0).
Two hundred millimeters (200ml) of 1.25% H2SO4 was added into the flask and heated
gently for 30 minutes. This was then filtered using muslin cloth on a fluted funnel and rinsed
well with boiling water until the washings were no longer acidic. It was then scrapped back
into the flask with spatula. 200ml of 1.25% NaOH was added into the flask and heated
gently for 30 minutes. It was then filtered using muslin cloth and the residue was washed
thoroughly with boiling distilled water. The residue was then transferred into a dry crucible,
dried at 600C for 3 hours, cooled and reweighed (W1). It was then ashed at 5500C for 3
hours in a muffle furnace, cooled in a desiccator and reweighed (W2) (AOAC, 2002)
Crude fibre (%)
W1 W2 100
Wo
where W0
weight of sample and crucible
W1
weight of sample and crucible after oven treatment
W2
weight of sample and crucible after furnace treatment
2.9.4 Determination of Total Ash Content
42
Clean dried crucible was weighed (W1) and two gram (2g) of a sample was placed inside the
crucible and reweighed (W2). The crucible was then heated on a Bunsen burner in order to
burn the volatile organic matter off. The crucible was then transferred to the muffle furnace
and heated at 550oC for 3 hours or more until a light-grey or white ash was obtained. The
crucible was then removed from the furnace and transferred into a desiccator to cool before
reweighing (W3) (AOAC, 2002).
Percentage (%) ash content
W3 W1 100
W2 W1
Where W1
Weight of empty crucible
W2
Weight of crucible and sample
W3
Weight of crucible and ash
2.9.5 Determination of Crude Protein

The Micro-Kjeldhal method (AOAC, 2002) was used for the determination of crude protein
content. The methods involved three stages: Digestion, Distillation and Titration.
Digestion process involves the conversion of nitrogen of the sample into ammonium
sulphate by boiling with concentrated H2SO4. Five gram (5g) each of the dry samples was
weighed into 50ml Kjeldhal digestion flask. Five millilitres of concentrated H2SO4 was
added into each of the flasks and a tablet of selenium catalyst was added. The mixture was
then heated at 100 + 50C on the block digester in a fume cupboard until digested by forming
a clear solution. The flasks were rotated at intervals until the digest was clear. The digests
43
were allowed to cool and transferred into 100ml standard flasks making it up with distilled
water. The digests were transferred into the distillation set up after dilution with water. A
100ml volumetric flask containing 5ml of 2% boric acid and 5 drops of mixed indicator
(0.016g methyl red + 0.083g bromocresol green in 100ml alcohol) was placed under the
condenser for each of the samples. 15ml of 40% NaOH was added to each of the digests in
the reaction vessel via the funnel and made sure it was alkaline by forming a cloudy
solution. Steam from the steam generator was passed into the reaction mixture with all
outlets closed to prevent suck back. The tip of the condensing unit was dipped into the
receiving flask. Distillation continued until 50ml of the distillate had condensed into the
receiving flask. This was done for all the samples. The distillates were then titrated against
0.01M HCl to a pink end point. The total nitrogen content of the samples was calculated
using the formula;
% Nitrogen (N)
A 0.0014 100
W
where A
Volume of acid used in titration
Weight of sample used
% Crude Protein
%N 6.25
Where N is the nitrogen value
2.9.6 Determination of Carbohydrate or Nitrogen Free Extract
44
The carbohydrate or nitrogen free extract content of the samples was determined by the
difference obtained by subtracting the sum of percentage ash, crude protein, crude fibre and
ether extract from 100% on dry matter basis (AOAC, 2002).
% Carbohydrate =
2.10
100 (% ash + % Crude Protein + % Ether Extract + % Crude fibre)
Determination of Total Sugars
The method of Dubois et al. (1956) was used 5.0g of the ground sample was put in a 100ml
conical flask. 50ml of 80% ethanol was added to it and then heated on a boiling water bath
for 1hr 30 minutes to extract the sugars present (Faparusi, 1970). The crude extract was
purified by adding 1% (w/v) basic lead acetate in the ratio of 4ml of extract to 0.5ml of 1%
basic lead acetate. Excess lead was removed with methanol in the ratio of 4ml of extract to
0.25ml of methanol. The extract was centrifuged and filtered. 0.1ml, 0.5mland 1.0ml of the
extract was put into three different test tubes. 1ml of 5% phenol and 5ml of conc. H2SO4 was
added in that order to each of the tubes. The concentrated H2SO4 was added rapidly, the
stream of acid being directed against the liquid surface rather than against the side of the test
tube in order to obtain good mixing. The test tubes were allowed to stand for 10 minutes,
then shaken and placed for 20 minutes in a water bath, at 25oC to 30oC. The optical density
of each solution was read using the spectrophotometer at 490nm. The blank was prepared
by substituting 1ml of distilled water for the sugar extract. The amount of sugar was
estimated from a standard curve of glucose (Appendix III) (Dubois et al. 1956).
45
2.11
Determination of Amino Acid Profile Using the Amino Acid Analyzer
The amino acid profiles of the different samples were determined using the method
described by Speckman et al. (1958). Two grams of dried Okpehe samples were defatted,
hydrolyzed, evaporated in a rotary evaporator and loaded into the Technicon Sequential
Multi-Sample Amino Acid Analyzer (TSM) Model DNA 0209.
Two grams of each Okpehe sample was weighed into extraction thimble; the fat was
extracted with chloroform/ethanol mixture using soxhlet extraction apparatus as described
by AOAC (2002). The extraction lasted for 15 hours.
The fat free sample was placed in a glass ampoule, 7ml of 6M HCL was added and oxygen
was expelled by passing nitrogen into the ampoule (this is to avoid possible oxidation of
some amino acids during hydrolysis e.g. methionine and cystine). The glass ampoule was
then sealed with Bunsen burner flame and put in an oven preset at 105 + 5 oC for 22 hours.
The ampoule was allowed to cool before broken opened at the tips and the contents were
filtered to remove the humins. The filtrate was then evaporated to dryness at 40 oC under
vacuum in a rotary evaporator. The residue obtained was dissolved with 5ml of acetate
buffer (pH 2.0) and stored in plastic specimen bottles, which were kept in the freezer for
further analysis.
Ten microlitres of the hydrolysate was loaded into the catridge of the analyzer. The TSM
analyzer is designed to separate and analyze for free acidic, neutral and basic amino acids in
the hydrolysate. The period of analysis lasted for 76 minutes.
46
The net height of each peak produced by the chart recorder of TSM (each representing an
amino acid) was measured. The half-height of the peak on the chart was found and width of
the peak on the half-height was accurately measured and recorded. Approximate area of
each peak was then obtained by multiplying the height with the width at half-height.
The norleucine equivalent (NE) for each amino acid in the standard mixture was calculated
using the formula,
NE
Area of Norleucine Peak
Area of each amino acid

A constant was calculated for each amino acid in the mixture:
Sstd
NEstd Mol.Weight AAstd
Finally, the amount of each amino acid present in the sample was calculated in g/100g
protein using the following formula;
Concentration (g/100g protein)
where C
Dilution 16
NH W NH/2 Sstd C
NH W (nleu)
Sample wt (g) N% 10 vol loaded

where NH
Net height
Width at half height
nleu
Norleucine
NE
Norleucine equivalent
47
AAstd
2.12
Amino Acid standard
Determination of Mineral Content
The mineral contents of samples were determined using the X-Ray Fluorescence (XRF)
spectroscopy method.
The type of technique that was used was the portable Energy
Dispersive X-ray Fluorescence (EDXRF) technique.
The first stage was the sample
preparation and this was done by pulverizing the samples to fine powdery form using an
agate mortar. Pellets of the samples were formed using a carver model manual pelletizing
machine at a pressure of 10-12 torr. The pelletized sample was inserted into the sample
holder of the XRF system and was bombarded by the X-ray fluorescence produced from the
X-ray tube source at a voltage of 25KeV and current of 50A. The sample characteristic Xray was detected by the solid state Si-Li detector and spectrum acquisition was done using
ADMCA software. The spectrum analysis was done using the AXIL software which relates
the peak areas into concentration values (Funtua, 1999).
2.13
Determination of Shelf Life of Okpehe
The shelf life of Okpehe was determined at room temperature. The samples were stored in
ten clean labeled containers at room temperature for 120 hours. One container was taken for
48
analysis at 12 hours interval. The analyses carried out on the samples are pH, microbial
count and proximate composition.
2.14
One thousand grams of freshly prepared Okpehe was produced using a mixed culture of
Bacillus subtilis and Bacillus licheniformis (as starter) in the ratio 1:1. The Okpehe produced
was divided into ten equal portions and then preserved using the following preservation
treatments: freezing, refrigerating, sun drying, oven drying, salting (5%) plus oven drying,
smoking and salting using the following concentrations; 5%, 10% 15% and 20%. Analyses
carried out on the preserved samples were pH determination, microbial count and proximate
composition.
2.14.1 Freezing
A portion of Okpehe sample (100g) was preserved in ten clean containers using the freezer
at -18 20C. Five grams of sample was taken from a container for analysis on the first day
(0 week), then at 2 weeks interval for fourteen weeks. A separate container was taken every
two weeks interval.
2.14.2 Refrigerating
Another portion of Okpehe (100g) was refrigerated at 4 10C in the refrigerator in ten clean
containers and 5 grams of Okpehe was analysed on the first day (0 week), then every two
weeks for fourteen weeks. A separate container was taken every two weeks interval.
49
2.14.3 Sun Drying

A portion of Okpehe (100g) was spread on a surface sterilized tray and covered with a clean
muslin cloth to prevent dust and contamination. It was then sun dried (3220C). At an hour
interval for eleven hours, the sun dried Okpehe was removed from sun and weighed to check
for the moisture level. A constant moisture level was reached in 2 days. The Okpehe was
ground with a Marlex Excella mixer grinder and stored in ten clean containers at room
temperature. A separate container was taken every two weeks interval. Samples were taken
for analysis on the first day (0 week), then at 2 weeks interval for fourteen weeks.
2.14.4 Oven Drying
One hundred grams of Okpehe was subjected to oven drying at 60C. Moisture level was
checked at an hour interval and constant moisture was reached on the second day.
The Okpehe was ground with a Marlex Excella mixer grinder and stored in ten clean
containers at room temperature. A separate container was taken every two weeks interval.
Samples were taken for analysis on the first day (0 week), then at 2 weeks interval for
fourteen weeks.
2.14.5 Salting Plus Oven Drying
Another portion of the condiment (100g) was mixed with 5% of salt and then oven dried at
60C. Moisture level was checked at an hour interval and constant moisture was reached on
the second day. The Okpehe was ground with a Marlex Excella mixer grinder and stored in
ten clean containers at room temperature. A separate container was taken every two weeks
50
interval. Samples were taken for analysis on the first day (0 week), then at 2 weeks interval
for fourteen weeks.
2.14.6 Smoking
One hundred grams of Okpehe was smoked at 55 20C using a wire mesh. The moisture
level was checked at an hour interval and constant moisture was reached on the second day.
The Okpehe was ground with a Marlex Excella mixer grinder and stored in ten clean
containers at room temperature. A separate container was taken every two weeks interval.
Samples were taken for analysis on the first day (0 week), then at 2 weeks interval for
fourteen weeks.
2.14.7 Salting
For the remaining four portions of Okpehe (100g each), 5%, 10%, 15% and 20% of salt were
added separately and labeled accordingly. They were stored in clean containers at room
temperature. A separate container was taken every two weeks interval. Samples were taken
for analysis on the first day (0 week), then at 2 weeks interval for fourteen weeks.
2.15
Sensory Evaluation
Sensory evaluation of Okpehe samples was determined using the method of Larmond
(1977). It was conducted on all the Okpehe samples alongside other analyses specified
above. Ten trained panelists (consisting of students and workers from the University of
51
Ilorin, Nigeria) conversant with the condiment were selected and briefed about the aim of
the experiment. The panelists were instructed to rate the samples for colour, aroma, texture
and general acceptability in their questionnaires. Detail of the content of the questionnaire is
presented in Appendix IV. The ratings were presented on a nine-point Hedonic Scale
ranging from like very much (9 points) to dislike very much (1 point). Samples were taken
weekly for sensory analysis for fourteen weeks.
2.16
Experimental Design and Statistical Analysis
The experimental design used was completely randomized design (CRD). Data obtained
were subjected to one way analysis of variance (ANOVA) while means were separated by
Duncans Multiple Range Test (DMRT) (Duncan, 1955).
52
CHAPTER THREE
3.0
RESULTS
Plates 4, 5 and 6 show the processing of Okpehe by the local producer. The unfermented and
fermented Okpehe obtained from the local producer and that produced in the laboratory are
shown in plates 7 and 8.
3.1
Physicochemical Parameters of Naturally Fermented Okpehe
The naturally fermented Okpehe samples were samples A, B and C. The natural
fermentation of Prosopis africana seeds to produce Okpehe was accompanied by an increase
in pH, temperature and moisture content from the beginning to the end of fermentation. At
the beginning of fermentation (zero hour), sample B had the highest pH of 6.5 while sample
A had the lowest pH of 7.7 after 72 hours of fermentation (Fig. 5). The highest temperature
of 35oC was recorded for sample B at the end of 72 hours of fermentation (Fig. 6). The
moisture content increased from 45% at zero hour of fermentation to 61% after 72 hours of
fermentation for sample B (Fig. 7).
53
54
Plate 4: Broken Fruits (pods) Ready for Winnowing
55
(x 1/4)
Plate 5: Locally Fermenting Okpehe in Jute Bags After 48 hours
56
( x1/5)
Plate 6: Locally Fermented Okpehe
( x1/5)
57
Plate7: Unfermented Samples A, B and C
Sample A - Okpehe produced by a local producer

Sample B - Okpehe produced using the autoclave
Sample C - Okpehe produced using the hot plate
58
(x1/5)
Plate 8: Fermented Samples A, B and C

59
( x1/5)
10
9
a
8
7
pH
6
5
4
3
2
1
0
0
24
48
Period of fermentation(Hrs)
Sample A
Sample B
72
Sample C
Fig.5: pH changes during the natural fermentation of Prosopis africana seeds to produce
Okpehe
60
40
c
35
b
b
30
a
Temp. (oC)
25
20
15
10
0
0
24
48
Period of fermentation (Hrs)
Sample A
Sample B
72
Sample C
Fig.6: Changes in Temperature during the natural fermentation of Prosopis africana seeds to
produce Okpehe
61
70
a
60
a
a
Moisture content ( %w/w)
50
a
40
30
20
10
0
0
24
48
72
Sample A
Sample B
Sample C
Fig.7: Changes in moisture content during the natural fermentation of Prosopis africana
seeds to produce Okpehe
62
3.2
Microbial Analysis
During the natural fermentation of Prosopis africana seeds to produce Okpehe, different
groups of microorganisms were isolated, characterized and counted. The occurrences of
these isolates in samples A, B and C at the different periods of fermentation are presented in
Tables 1, 2 and 3 respectively. The variety of microorganisms present during the
fermentation produced a whitish mucilaginous substance that covered and linked the
individual light brown to dark brown coloured cotyledons. Seven organisms were isolated
from the commercial sample of Okpehe (sample A) and these were Bacillus subtilis,
Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Staphylococcus aureus,
Escherichia coli and Saccharomyces cerevisiae. Six organisms were isolated from Okpehe
samples produced in the laboratory using both the autoclave (sample B) and hot plate
(sample C). These organisms were Bacillus subtilis, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Staphylococcus aureus and Escherichia coli. The microbial
counts of samples are shown in Tables 4, 5, 6 and 7. The isolated organisms increased in
number as fermentation progressed. The microbial load of sample A (4.0 106cfu/g) was
significantly higher at (p < 0.05) than that of samples B (3.3 106cfu/g) and C (3.5
106cfu/g) at the end of the fermentation period. Five organisms each of the three samples
were able to survive the fermentation conditions and were recovered at the end of the
fermentation. For sample A, the organisms were Bacillus subtilis, Bacillus licheniformis,
Bacillus megaterium, Staphylococcus aureus and Saccharomyces cerevisiae. The organisms
63
recovered for sample B and C after fermentation were Bacillus subtilis, Bacillus
licheniformis, Bacillus megaterium, Bacillus pumilus, and Staphylococcus aureus.
Table 1: Occurrence of Microorganisms in Locally Produced Okpehe (Sample A)

Microorganisms Isolated
`
0hr
24hr
48hr
72hr
Bacillus subtilis
Bacillus licheniformis
Bacillus megaterium
Bacillus pumilus
Escherichia coli
Staphylococcus aureus
Saccharomyces cerevisiae
+ present
- absent
64
Table 2: Occurrence of Microorganisms in Laboratory Produced Okpehe (Sample B)

Period of Fermentation (hr)

0hr
24hr
48hr
72hr
Bacillus subtilis
Bacillus pumilus
Bacillus megaterium
Escherichia coli
+ present
- absent
65
Table 3: Occurrence of Microorganisms in Laboratory Produced Okpehe (Sample C)


0hr
24hr
48hr
72hr
Bacillus subtilis
Bacillus megaterium
Escherichia coli
Bacillus pumilus
+ present
- absent
66
Table 4: Microbial Count of Samples after 0 Hour of Fermentation

Microorganisms
Sample A (cfu/g)
Sample B (cfu/g)
Sample C (cfu/g)
3.1 103
2.3 103
2.5 103
Bacillus licheniformis 2.3 103
3.2 103
3.3 103
Isolated
Bacillus subtilis
Bacillus pumilus
1.0 101
Escherichia coli
5.2 101
3.3 101
3.4 101
Staphylococcus
3.1 101
2.1 101
aureus
Saccharomyces
cerevisiae

67
Table 5: Microbial Counts of Samples after 24 Hours of Fermentation

Microorganisms
Sample A (cfu/g)
Sample B (cfu/g)
Sample C (cfu/g)
5.1 104
4.2 104
4.3 104
4.2 104
4.2 104
Bacillus megaterium
2.0 101
1.6 101
2.1 101
Bacillus pumilus
1.2 101
2.1 101
2.1 101
Escherichia coli
3.4 102
2.8 102
2.9 102
Staphylococcus
3.0 102
2.1 101
2.0 101
1.0 102
Isolated
Bacillus subtilis
aureus
Saccharomyces
cerevisiae

68

Microorganisms
Sample A (cfu/g)
Sample B (cfu/g)
Sample C (cfu/g)
2.4 105
2.0 105
2.0 105
1.4 105
1.5 105
2.4 102
1.3 102
1.3 102
Isolated
Bacillus subtilis
Bacillus megaterium
Bacillus pumilus
1.5 102
2.0 102
Escherichia coli
3.0 103
4.3 102
4.2 102
1.2 103
Staphylococcus
aureus
Saccharomyces
cerevisiae

69

Microorganisms
Sample A (cfu/g)
Sample B (cfu/g)
Sample C (cfu/g)
2.8 106
2.3 106
2.4 106
1.0 106
1.1 106
2.5 103
1.3 103
1.5 103
2.4 103
2.4 103
Isolated
Bacillus subtilis
Bacillus megaterium
Bacillus pumilus
Escherichia coli
Staphylococcus
3.2 104
3.2 103
3.3 103
3.3 103
aureus
Saccharomyces
cerevisiae

70
3.3
Nutritional Characteristics of Naturally Fermented Okpehe Samples
3.3.1 Proximate Composition of Okpehe Samples

Result of the proximate composition of Okpehe samples from 0 - 72 hours are shown in
Table 8. The mean percentage crude protein increased significantly (p < 0.05) throughout
the period of fermentation (0 - 72 hours) for all the samples (Table 8). The increase was
between 30.90% to 36.88% for sample A; 32.68% to 38.44% for sample B and 34.54% to
40.05% for sample C. The crude protein of sample C was highest (40.05%) at the end of
seventy- two hours of fermentation compared to samples B (38.44%) and A (36.88%). The
ether extract content of the Okpehe samples decreased significantly (p < 0.05) as the
fermentation progressed. The mean ether extract content decreased from 9.32% at zero hour
to 8.15% at 72 hours for sample A; 11.41% to 11.35% for sample B and 11.49% to 10.04%
for sample C. The mean crude fibre content increased significantly (p < 0.05) from 2.85% to
2.99% after fermentation for 72 hours for sample A; 3.06% to 3.57% for sample B and 3.16% to
3.63% for sample C. Sample A gave mean values of 4.89% at zero hour and 4.80% after 72
hours; sample B had 4.84% at the initial stage and decreased to 4.82% after 72 hours while
sample C had 4.86% at the initial stage and decreased slightly to 4.84% after 72 hours. The
carbohydrate content decreased significantly (p < 0.05) from 52.04% to 47.18% for sample
A; 48.03% to 41.80% for sample B and 45.95% to 41.44% for sample C after 72 hours. The
dry matter values ranged between 93.03% at zero time and 90.54% after 72 hours of
fermentation for sample A; sample B, it was between 92.42% and 91.88% while that for
sample C, ranged between 92.59% and 91.13% at the end of fermentation.
71
Table 8: Proximate Analysis of Naturally Fermented Okpehe Samples

Proximate
composition
0hr
A
24hr
48hr
72hr
35.98
34.68
36.33
38.01
36.88
38.44
40.05
11.12
8.62a
11.38
10.81
8.15a
11.35
10.04
(%)
Crude Protein
Ether Extract
30.90
32.68a
34.54
32.42
9.32a
11.41b
11.49
9.01a
34.12b
11.40b
Crude Fibre
2.85a
3.06b
3.16c
2.89a
3.25b
3.30b
2.92a
3.40b
3.52c
2.99a
3.57b
3.63c
Ash
4.89c
4.84a
4.86b
4.86c
4.83a
4.85b
4.82a
4.83b
4.84c
4.80a
4.82b
4.84c
Moisture
6.97a
7.41b
7.58c
7.34a
7.83b
7.92c
8.01a
8.21b
8.36c
8.12a
8.87b
9.46c
Nitrogen Free
52.04
48.03b
45.95
50.82
46.40b
44.75
48.96
44.06
42.82
47.18
41.80
41.44
Extract
92.42
92.66
92.17a
92.18
91.79
91.99
91.64
90.54
91.13
9188c
Content
(Carbohydrat
e)
Dry Matter
93.03
a
92.59b
Values are means of triplicate determinations on dry weight basis; means within rows
having different superscripts differ significantly (p < 0.05)
72
3.3.2 Changes in Total Sugar of Naturally Fermented Okpehe Samples

The mean values of the total sugar present in the samples ranged between 0.10 mg/g and
0.05 mg/g for sample A; 0.08mg/g for sample B and 0.10 mg/g and 0.06 mg/ml for sample
C after 72 hours of fermentation (Fig.8). The fermentation of the samples resulted in
decrease in its total sugars.
3.3.3 Changes in Amino Acid Composition of Naturally Fermented Okpehe Samples
Result of the amino acid profile determination reveals that seventeen amino acids were
present in the three Okpehe samples (Table 9). There was an increase in the total amino
acids of the Okpehe samples from 80.26 g at zero hour to 82.64 g after 72 hours of
fermentation. Sample C had the highest total amino acid (82.64 g) while the least was
obtained from sample B (81.87g) at the end of the fermentation. The fermentation resulted
in an increase in the non essential amino acids (50.45 55.56 g) while a decrease was
observed for the essential amino acids (29.35 26.71 g) (Table 10). The essential amino
acids are lysine, tryptophan, phenylalanine, leucine, isoleucine, threonine, methionine,
valine and histidine while the non essential amino acids include glycin, alanine, serine
cysteine, tyrosine, aspartic acid, glutamic acid, proline and hydroxyproline.
73
74
0.14
0.12
Total sugar(mg/g)
0.1
0.08
0.06
0.04
0.02
0
0
24
48
72
Sample A
Sample B
Sample C
Fig. 8: Changes in total sugar contents during the natural fermentation of Prosopis africana
seeds to produce Okpehe
Sample A - Okpehe produced from a local producer
Table 9: Amino Acid Composition of Okpehe Samples

Composition (g/100g Protein)
Amino Acid
0hr
A
Lysine
5.10b
B
5.34c
24hrs
C
5.02a
A
5.05b
48hrs
5.12c
5.00a
75
A
5.02a
72hrs
4.92a
4.89a
A
4.98b
B
4.80a
C
4.80a
Histidine
1.69a
1.76a
1.70a
1.63b
1.52a
1.61b
1.60c
1.51a
1.57b
1.50a
1.49a
1.50a
Arginine
4.34a
4.43b
4.30a
4.32b
4.21a
4.30 b
4.34b
4.00a
4.00a
3.89b
3.83a
3.92b
Aspartic acid
19.59
18.28
18.81
19.40
18.00
18.60
19.35
17.82
18.42
19.20
17.51
18.21
Threonine
2.61a
3.00c
2.72b
2.49a
2.78c
2.62b
2.47a
2.68c
2.57b
2.38a
2.61c
2.54b
Serine
3.87b
3.59a
3.91c
3.82b
3.20a
3.85b
3.75b
3.59a
3.75b
3.51b
3.15a
3.60c
Glutamic
acid
12.20
12.48
13.04
14.00
15.52
15.32
16.21
17.26
17.89
18.64
19.52
19.34
Proline
3.40a
3.68b
3.42a
3.38b
3.68c
3.30a
3.40a
3.70b
3.40a
3.40b
3.70c
3.34b
Glycine
3.55b
3.38a
3.62c
3.42b
3.28a
3.52c
3.26a
3.38b
3.43c
3.16a
3.36b
3.37b
Alanine
4.20a
5.17b
4.18a
4.15b
4.42c
4.06a
4.02a
3.36b
4.01a
3.98b
4.47c
3.92a
Cystine
1.52b
1.39a
1.55b
1.30a
1.33a
1.30a
1.06a
1.26b
1.22b
1.32c
1.16a
1.22b
Valine
4.03b
3.98a
4.04b
4.01b
3.91a
4.00b
3.89a
3.86a
3.90a
3.78b
3.64a
3.82b
Methionine
0.76a
0.83b
0.78a
0.60a
0.69b
0.71b
0.55a
0.65c
0.60b
0.60b
0.55a
0.60b
Isoleucine
3.02b
2.95a
3.04b
2.86b
2.92b
2.98c
2.79a
2.89b
2.92c
2.64a
2.87b
2.85b
Leucine
5.16b
5.00a
5.16b
4.92a
4.89a
5.04b
4.89a
4.89a
4.91a
4.64a
4.62a
4.84b
Tyrosine
2.58b
2.48a
2.60b
2.48b
2.41a
2.51b
2.42b
2.36a
2.42b
2.35b
2.19a
2.38b
Phenylalanin
e
2.64a
2.64a
2.62a
2.56ab
2.58b
2.52a
2.46a
2.56b
2.46a
2.30a
2.40b
2.39b
Total
80.26
80.38
80.51
80.31
80.46
81.24
81.48
80.69
82.36
82.27
81.87
82.64
Values are means of triplicate determinations on dry weight basis; means within rows
having different superscripts differ significantly (p < 0.05).
Sample A - Okpehe obtained from local producer
76
Table 10: Changes in Essential Amino Acids of Okpehe Samples

Amino Acids
Composition (g/100g)
0hr
A
24hrs
C
48hrs
72hrs
C
29.35
29.93
29.38
28.44
28.62
28.78
28.01
27.96
27.82
26.71
26.81
27.26
Percentage
36.57
37.24
36.49
35.51
35.57
35.43
34.38
34.65
33.78
32.47
32.75
32.99
Non
50.91
50.45
51.13
51.87
51.84
52.46
53.47
52.73
54.54
55.56
55.06
55.38
63.43
62.76
63.51
64.59
64.43
64.57
65.62
65.35
66.22
67.53
67.25
67.01
Essential
(g/100g)
Essential
(g/100g)
Percentage

77
3.3.4 Mineral Composition of Naturally Fermented Okpehe Samples

The mineral compositions of the three Okpehe samples are shown in Table 11. Potassium
was found to be the most abundant mineral and decreased significantly (p < 0.05) during the
period of fermentation for all the samples. It ranged between 224.9 mg/100g at zero time
and 183.1mg/100g after 72 hours for sample A; 210.1mg/100g at zero time and
164.2mg/100g after 72 hours for sample B and 312.9mg/100g at zero time and
229.5mg/100g after 72 hours for sample C. Calcium was found to be the second highest
mineral with a range of 35.8mg/100g at zero time and 45.3mg/100g after72 hours sample A;
39.5mg/g at time zero and 44.4mg/100g after 72 hours for sample B; and 44.9mg/100g at
zero time and 55.6mg/100g after 72 hours. The amount of calcium increased significantly (p
< 0.05) as fermentation progressed. Zinc was also found in an appreciable amount. The
amount was constant 14.2mg/100g throughout the period of fermentation for all the samples.
The concentration of iron was in the range of 10.1mg/100g at zero time and 10.2mg/100g
after 72 hours for sample A; 10.2mg/100g at zero time and 10.7mg/100g after 72 hours for
sample B and 10.1mg/100g at zero time and 10.5mg/100g after 72 hours for sample C. The
amount of manganese detected in all the three samples was moderate. The values ranged
between 4.8mg/100g at zero time and 4.2mg/100g for sample A; 6.1mg/100g at zero time
and 5.6mg/100g after 72 hours of fermentation for sample B and 15.2mg/100g at zero time
and 6.1mg/100g after 72 hours for sample C. The values decreased significantly (p < 0.05)
as the period of fermentation increased. Copper was also detected in all the samples and it
increased significantly (p < 0.05) as fermentation continued. The values at zero time are
78
8.7mg/100g, 9.1mg/100g and 8.9mg/100g for samples A, B and C respectively while after
72 hours of fermentation, the values are 9.3mg/100g, 9.2mg/100g and 9.2mg/100g for
samples A, B and C respectively. Lead and cadmium were not detected in all the samples.
3.4
Physicochemical Parameters of Okpehe Samples Inoculated with Starter

Cultures
There was a significant increase in pH of the Okpehe samples inoculated with both mono
and mixed starter cultures of bacteria (Figs. 9, 10 and 11) as the period of fermentation
increased. The range of pH for the mono culture inoculated Okpehe was between 6.80 and
8.92 while that of the mixed culture inoculated Okpehe was between 6.98 and 8.92. As
fermentation progressed, there was a rise in temperature (28.5 35oC) in both the mono and
mixed culture inoculated Okpehe samples (Figs. 12, 13 and 14).
Table 11: Mineral Composition of Okpehe Samples

79
Mineral
Composition (mg/100g)
0hr
A
Potassium
24hrs
48hrs
72hrs
224.9
210.1
312.9
210.2
190.2
311.1
200.1
182.3
295.1
183.1
164.2
229.5
Calcium
35.8a
39.5b
44.9c
39.1a
42.8b
48.0c
41.2a
43.2b
50.4c
45.3b
44.4a
55.6c
Manganes
4.8a
6.1b
15.2c
4.7a
6.0b
10.2c
4.4a
5.8b
7.8c
4.2b
5.6b
6.1c
Iron
10.1a
10.2b
10.1a
10.1a
10.3c
10.2b
10.2a
10.3b
10.3b
10.2a
10.7c
10.5b
Copper
8.7a
9.1c
8.9b
8.8a
9.1c
8.9b
9.1a
9.2b
9.1a
9.3b
9.2a
9.2a
Zinc
14.2a
14.1b
14.2a
14.2a
14.1b
14.2a
14.2a
14.1b
14.2a
14.2a
14.1b
14.2a
Lead
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Cadmium
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Values are means of triplicate determinations on dry weight basis; means within rows with
different superscripts differ significantly (p < 0.05).
ND- Not Detected
80
10
b
9
e
8
7
d
b
a a a a a
b c
c
a
a a a a a
pH
6
5
4
3
2
1
0
0
24
48
72
Period of fermentation (hr)

B.subtilis
B. megaterium
B. licheniformis
B. pumilus
Control
Fig.9: Changes in pH during the fermentation of Okpehe inoculated with monocultures of

bacteria
81
10
c a
c a a
b
9
8
7
b b c a ab c
c b d a d d
d b c
bc c
pH
6
5
4
3
2
1
0
0
Sample DE
Sample DF
24
48
Sample DG
Sample EF
Sample EG
72
Sample FG
Fig. 10: Changes in pH during the fermentation of Okpehe inoculated with mixed cultures of
bacteria
D B. subtilis
E B. megaterium
F B. licheniformis
G B. pumilus
82
10
9
8
a
c
c
b
a
pH
6
5
4
3
2
1
0
0
Sample DEF
24
48
Sample DFG
Sample EFG
72
Sample DEFG
Fig. 11: Changes in pH during the fermentation of Okpehe inoculated with mixed cultures of
bacteria
D B. subtilis
E B. megaterium
F B. licheniformis
G B. pumilus
83
45
40
c
35
Temperature (0C)
30
c b b
b c a b
d
a
a a a a a
25
20
15
10
5
0
0
B. subtilis
24
48
72
B. megaterium
B. licheniformis
B. pumilus
Control
Fig. 12: Temperature changes during the fermentation of Okpehe inoculated with
Monocultures of bacteria
84
40
35
e
a b b a a b
Temperature (0C)
30
d
b
b c
a a
25
20
15
10
5
0
0
Sample DE
24
48
Sample DF
Sample DG
Sample EF
Sample EG
72
Sample FG
Fig. 13: Temperature changes during the fermentation of Okpehe inoculated with mixed
cultures of bacteria
D B. subtilis
E B. megaterium
F B. licheniformis
G B. pumilus
85
10
9
8
d
a
c
c
b ab
b
a
pH
6
5
4
3
2
1
0
0
Sample DEF
24
48
Sample DFG
Sample EFG
72
Sample DEFG
Fig.14: Changes in pH during the fermentation of Okpehe inoculated with mixed cultures of
bacteria
D B. subtilis
E B. megaterium
F B. licheniformis
G B. pumilus
86
3.5
Proximate Composition of Okpehe Samples Inoculated with Starter Cultures
3.5.1 Proximate Composition of Okpehe Samples Inoculated with Mono Cultures of

Bacterial Isolates
The result of the proximate composition of Okpehe inoculated with mono cultures of
bacterial isolates are shown in Tables 12, 13, 14, 15, 16 and 17. As the fermentation period
increased from 0 72 hours, the crude protein, crude fibre and moisture content increased
significantly (p < 0.05) while the ether extract, total ash and nitrogen free extract decreased
significantly (p < 0.05). The Okpehe inoculated with the monoculture of B. subtilis had the
highest crude protein of 41.25% while B. pumilus had the least of 38.45% after 72 hours of
fermentation (Table 12). The range of 9.28% - 10.25% was obtained for the ether extract
content at the end of fermentation (Table 13). The crude fibre increased from 3.16% at zero
hour to 3.53% after 72 hours of fermentation (Table 14). The total ash decreased from
4.79% to 4.72% (Table 15) while the nitrogen free extract decreased from 39.95% - 31.96%
after the fermentation period (Table 17). The range of the moisture content was between
6.66% - 8.19% at the end f the 72 hours of fermentation (Table 16).
The changes in total sugar content of Okpehe samples inoculated with mono cultures of
bacterial isolates are presented in Fig. 15. The total sugar decreased from 10.2mg/g to
7.5mg/g after the fermentation period. The least total sugar of 7.5mg/g was obtained with
the control sample at the end of 72 hours of fermentation.
87
Table 12: Crude Protein Content of Prosopis africana Seeds Fermented with
Bacterial Isolate
Crude Protein (% w/w)

0hr
24hr
48hr
72hr
34.62a 0.04
36.32d 0.07
38.89d 0.03
41.25d 0.08
B. megaterium
34.62a 0.04
35.02a 0.06
35.61a 0.03
39.85b 0.06
B. licheniformis
34.62a 0.04
36.01c 0.07
38.28c 0.05
41.20c 0.06
B. pumilus
34.62a 0.04
35.42b 0.06
35.63b 0.05
38.45a 0.05
Control
34.62a 0.04
36.38e 0.06
38.92e 0.05
41.91e 0.04
B subtilis
Values are means of triplicate determinations SD on dry weight basis; means within rows
with different superscripts differ significantly (p < 0.05).
SD Standard Deviation
Table 13: Ether Extract Content of Prosopis africana Seeds Fermented with
Bacterial Isolate
Ether Extract (% w/w)

88
0hr
24hr
48hr
72hr
B subtilis
10.85 0.02 a
10.42 0.01 a
10.23 0.03 a
10.13 0.02 b
B. megaterium
10.85a 0.02
10.51cd 0.02
10.30b 0.02
10.25c 0.01
B. licheniformis
10.85a 0.02
10.44b 0.01
10.24a 0.01
10.22c 0.01
B. pumilus
10.85a 0.02
10.53d 0.01
10.40d 0.03
9.28a 0.02
Control
10.85a 0.02
10.50c 0.03
10.35c 0.02
10.25c 0.04
Table 14: Crude Fibre Content of Prosopis africana Seeds Fermented with
Bacterial Isolate
Crude Fibre (% w/w)

0hr
B subtilis
3.16a 0.01
24hr
48hr
72hr
3.21b 0.01
3.52d 0.03
3.52d 0.02
89
B. megaterium
3.16a 0.01
3.19a 0.02
3.19a 0.02
3.20a 0.01
B. licheniformis
3.16a 0.01
3.19a 0.02
3.52d 0.01
3.53d 0.04
B. pumilus
3.16a 0.01
3.22bc 0.01
3.29c 0.03
3.29c 0.02
Control
3.16a 0.01
3.23c 0.02
3.23b 0.01
3.24b 0.03
Table 15: Total Ash Content of Prosopis africana Seeds Fermented with Monocultures
of Bacterial Isolates
Bacterial Isolate
Total Ash (% w/w)

0hr
24hr
B subtilis
4.79a 0.02
4.76c 0.01
4.75bc 0.02
4.74b 0.01
B. megaterium
4.79a 0.02
4.73a 0.01
4.72a 0.01
4.72a 0.01
90
48hr
72hr
B. licheniformis
4.79a 0.02
4.77d 0.02
4.77d 0.01
4.75c 0.01
B. pumilus
4.79a 0.02
4.74b 0.03
4.74b 0.02
4.72a 0.02
Control
4.79a 0.02
4.77d 0.02
4.76c 0.01
4.72a 0.02
Table 16: Moisture Content of Prosopis africana Seeds Fermented with Monocultures
Bacterial Isolate
Moisture Content (% w/w)

0hr
24hr
48hr
72hr
B subtilis
6.66b 0.06
7.21a 0.03
7.94a 0.05
8.40d 0.03
B. megaterium
6.67b 0.06
7.24b 0.04
8.10c 0.05
8.25b 0.04
B. licheniformis
6.63a 0.05
7.39c 0.04
8.01b 0.03
8.12a 0.06
B. pumilus
6.65ab 0.06
7.45e 0.05
7.99b 0.04
8.31c 0.05
91
6.70c 0.06
Control
7.42d 0.05
8.01b 0.04
8.49e 0.06
Bacterial Isolate
Nitrogen Free Extract (% w/w)

0hr
24hr
48hr
72hr
B subtilis
39.92b 0.05
38.08b 0.06
34.67a 0.04
31.96a 0.05
B .megaterium
39.91b 0.05
39.31e 0.06
38.08e 0.05
33.73d 0.05
B. licheniformis
39.95c 0.04
38.20c 0.07
35.02c 0.04
32.18b 0.07
B. pumilus
39.93bc 0.05
38.64d 0.05
37.95d 0.07
35.95e 0.05
Control
39.88a 0.04
37.30a 0.07
34.73b 0.06
32.39c 0.04
92
93
12
Total Sugar (mg/g)
10
0
0
B. subtilis
24
48
B. megaterium
B. licheniformis
72
B. pumilus
Control
Fig. 15: Changes in total sugar of Okpehe inoculated with monocultures of bacteria
3.5.2 Proximate Composition of Okpehe Samples Inoculated with Mixed Cultures of

Bacterial Isolates
94
For the mixed culture inoculated Okpehe, the result of the proximate composition are
presented in Tables 18 23. The crude protein (35% - 44.61%) (Table 18), crude fibre
(3.15% - 3.64%) (Table 20) and moisture content (6.37% - 8.09%) (Table 22) increased
significantly (p < 0.05) while the ether extract (10.81% - 9.00) (Table 19), total ash (4.93% 3.32%) (Table 21) and nitrogen free extract (39.67% - 31.81%) (Table 23) decreased
significantly (p < 0.05) as fermentation progressed. The highest crude protein content of
44.61% was observed with the combination of B. subtilis and B. licheniformis (EG) while
the least was observed with FH which was a combination of B. megaterium and B. pumilus.
The total sugar of Okpehe samples inoculated with mixed starter cultures of bacteria are
shown in Figures 16 and 17. There was a decrease in total sugar as the period of
fermentation increased. The highest total sugar at zero hour was recorded in Sample EGH
(B. subtilis, B. licheniformis and B. pumilus) while sample EG (B. subtilis and B.
licheniformis) had the least after 72 hours of fermentation.
Table 18: Crude Protein Content of Prosopis africana Seeds Fermented with Mixed
Bacterial Isolate
Crude Protein (%)

95
0hr
24hrs
48hrs
72hrs
EF
35.02a 0.06
36.82f 0.09
37.24d 0.11
43.50i 0.15
EG
35.02a 0.06
36.98g 0.12
39.58j 0.11
44.61j 0.07
EH
35.02a 0.06
36.65e 0.09
37.42f 0.09
43.42h 0.13
FG
35.02a 0.06
37.01g 0.12
37.84h 0.09
43.35g 0.13
FH
35.02a 0.06
36.26c 0.12
36.31a 0.12
42.24a 0.11
GH
35.02a 0.06
36.16a 0.09
36.92b 0.08
43.20f 0.10
EFG
35.02a 0.06
36.42d 0.11
38.05i 0.08
43.09e 0.11
EGH
35.02a 0.06
36.61e 0.08
37.51g 0.08
42.77d 0.10
FGH
35.02a 0.06
36.19ab 0.09
36.97c 0.10
42.33b 0.10
EFGH
35.02a 0.04
36.23bc 0.11
37.32e 0.10
42.60c 0.09
Values are means of triplicate determinations SD on dry weight basis; means within
columns with different superscripts differ significantly (p < 0.05).
E B. subtilis
F B. megaterium
G B. licheniformis
H B. pumilus
Table 19: Ether Extract Content of Prosopis africana Seeds Fermented with Mixed
Bacterial Isolate
Ether Extract (%)

96
0hr
24hrs
48hrs
72hrs
EF
10.81a 0.03
9.90d 0.05
9.50e 0.01
9.12b 0.03
EG
10.81a 0.03
9.43b 0.01
9.09b 0.04
9.03a 0.02
EH
10.81a 0.03
10.17f 0.04
9.27c 0.05
9.03a 0.02
FG
10.81a 0.03
9.41b 0.05
9.53f 0.05
9.36d 0.04
FH
10.81a 0.03
10.10e 0.01
9.48e 0.02
9.40d 0.05
GH
10.81a 0.03
9.92d 0.04
9.31d 0.02
9.23c 0.05
EFG
10.81a 0.03
10.25g 0.04
10.19h 0.03
10.03f 0.01
EGH
10.81a 0.03
9.31a 0.03
9.06a 0.04
9.00a 0.03
FGH
10.81a 0.03
10.08e 0.05
10.05g 0.06
9.53e 0.02
EFGH
10.81a 0.03
9.61c 0.04
9.05a 0.06
9.05a 0.02
E B. subtilis
F B. megaterium
G B. licheniformis
H B. pumilus
Table 20: Crude Fibre Content of Prosopis africana Seeds Fermented with Mixed
Bacterial Isolate
Crude Fibre (%)
97
0 hr
24 hrs
48 hrs
72 hrs
EF
3.15a 0.01
3.15a 0.02
3.16a 0.01
3.17a 0.03
EG
3.15a 0.01
3.15a 0.02
3.16a 0.02
3.22cd 0.03
EH
3.15a 0.01
3.15a 0.02
3.19b 0.01
3.20bc 0.01
FG
3.15a 0.01
3.15a 0.02
3.16a 0.01
3.18ab 0.03
FH
3.15a 0.01
3.15a 0.02
3.16a 0.02
3.17a 0.01
GH
3.15a 0.01
3.15a 0.02
3.24c 0.02
3.64f 0.03
EFG
3.15a 0.01
3.15a 0.02
3.17a 0.01
3.23d 0.01
EGH
3.15a 0.01
3.15a 0.02
3.16a 0.01
3.16a 0.01
FGH
3.15a 0.01
3.16a 0.01
3.25c 0.02
3.29e 0.03
EFGH
3.15a 0.01
3.15a 0.02
3.16a 0.01
3.18ab 0.01
E B. subtilis
F B. megaterium
G B. licheniformis
H B. pumilus
Table 21: Total Ash Content of Prosopis africana Seeds Fermented with Mixed
Bacterial Isolate
Total Ash (%)

0hr
24hrs
48hrs
72hrs
EF
4.93a 0.01
4.20b 0.01
4.10cd 0.01
3.69c 0.01
EG
4.93a 0.01
4.64g 0.02
4.33g 0.01
4.02g 0.02
98
EH
4.92a 0.01
4.63fg 0.02
4.27ef 0.01
4.03g 0.01
FG
4.93a 0.01
4.61f 0.01
4.23e 0.03
3.64b 0.02
FH
4.93a 0.01
4.63fg 0.02
4.09c 0.02
3.32a 0.01
GH
4.93a 0.01
4.62fg 0.01
4.28f 0.02
3.93e 0.01
EFG
4.92a 0.01
4.02a 0.02
3.93a 0.01
3.90d 0.02
EGH
4.93a 0.01
4.23c 0.01
4.14d 0.01
4.02g 0.01
FGH
4.93a 0.01
4.28d 0.02
4.00b 0.02
3.97f 0.01
EFGH
4.93a 0.01
4.46e 0.01
4.33g 0.02
4.26h 0.03
E B. subtilis
F B. megaterium
G B. licheniformis
G B. pumilus
Table 22: Moisture Content of Prosopis africana Seeds Fermented with Mixed Cultures
Bacterial Isolate
Moisture Content (%)

0hr
24hrs
48hrs
72hrs
EF
6.39a 0.09
6.85de 0.08
6.98b 0.08
7.18b 0.07
EG
6.39a 0.10
6.53a 0.08
6.88a 0.07
7.31c 0.08
EH
6.37a 0.09
6.99g 0.09
7.01b 0.09
7.30c 0.08
99
FG
6.39a 0.09
6.63b 0.10
7.01b 0.09
7.43d 0.10
FH
6.39a 0.10
6.88ef 0.10
7.73e 0.08
8.09f 0.07
GH
6.38a 0.09
6.60b 0.08
6.84a 0.10
7.05aa 0.09
EFG
6.38a 0.08
6.72c 0.08
7.32cd 0.09
7.41d 0.10
EGH
6.39a 0.09
6.82d a 0.09
7.28c 0.09
7.48e 0.08
FGH
6.38a 0.09
6.81d 0.10
7.33d 0.11
7.48ea 0.07
EFGH
6.38a 0.09
6.92f 0.09
7.33d 0.11
7.51e 0.07
E B. subtilis
F B. megaterium
G B. licheniformis
H B. pumilus
Mixed Cultures of Bacterial Isolates
Bacterial Isolate
Nitrogen Free Extract (%)

0hr
24hrs
48hrs
72hrs
EF
39.64a 0.26
39.60cd 0.19
39.50f 0.19
33.76e 0.21
EG
39.64a 0.26
39.29a 0.21
36.96a 0.21
31.81a 0.19
100
EH
39.67b 0.25
39.65d 0.20
38.84d 0.21
33.02c 0.19
FG
39.64a 0.26
39.49b 0.21
38.31b 0.18
33.04c 0.24
FH
39.64a 0.26
39.63d 0.21
39.61g 0.21
36.18g 0.18
GH
39.65a 0.25
39.63d 0.20
39.41e 0.20
32.95c 0.21
EFG
39.66a 0.25
39.46b 0.20
38.34b 0.18
32.34b 0.19
EGH
39.64a 0.26
39.88e 0.24
38.85d 0.19
34.57f 0.22
FGH
39.65a 0.25
39.57c 0.24
38.40c 0.17
33.40d 0.20
EFGH
39.65a 0.25
39.63d 0.27
38.81d 0.21
33.40d 0.20
E B. subtilis
F B. megaterium
G B. licheniformis
101
H B. pumilus
10
9
8
Total Sugar (mg/g)
7
6
5
4
3
2
1
0
0
24
48
72

Sample EF
Sample EG
Sample EH
Sample FG
Sample FH
Sample GH
Fig. 16: Changes in total sugar of Okpehe fermented with mixed cultures of bacteria
E B. subtilis
F B. megaterium
G B. licheniformis
H B. pumilus
102
9.6
9.4
9.2
Total Sugar (mg/g)
9
8.8
8.6
8.4
8.2
8
7.8
7.6
0
24
48
72

Sample EFG
Sample EGH
Sample FGH
Sample EFGH
Fig. 17: Changes in total sugar of Okpehe fermented with mixed cultures of bacteria
E B. subtilis
F B. megaterium
G B. licheniformis
H B. pumilus
3.6
Shelf Life of Okpehe Stored at Ambient Temperature
103
The pH, microbial count and nutritional characteristics of Okpehe were observed when it
was stored at ambient temperature for 120 hours. There was a rise in pH of Okpehe as the
time of storage increased (Fig. 18). An increase in microbial count was observed in Okpehe
from 0 72 hours while there was a decline in microbial population after this time till the
end of 120 hours (Fig. 19). The result of the proximate composition of Okpehe stored at
ambient temperature is shown in Table 24. A significant decrease (p < 0.05) was observed in
crude protein, ether extract and nitrogen free extract at the end of the period of storage.
104
9
8
7
6
pH
5
4
3
2
1
0
0
12
24
36
48
60
72
84
96
108
120
Time (hr)
Fig. 18: Changes in pH of Okpehe Stored at Ambient Temperature (30 20C)
105
14
Microbial Count ( 105cfu/g)
12
10
0
0
12
24
36
48
60
72 84 96
Time (hrs)
108 120
Fig. 19: Changes in Microbial Count of Okpehe Stored at Ambient Temperature (30 20C)
Table 24: Proximate Composition of Okpehe Stored at Ambient Temperature (30

20C)
Period of Crude
Storage
Protein
Ether
Extract
Crude
Fibre (%)
106
Total Ash Moisture

(%)
Content
Nitrogen
Free
(hrs)
(%)
(%)
(%)
Extract
(%)
42.62h 0.08
9.41h 0.03
3.43a 0.01
4.03a 0.01
7.47a 0.09
33.04i 0.05
12
42.62h 0.08
9.37g 0.02
3.43a 0.01
4.03a 0.01
7.53b 0.08
33.02j 0.04
24
42.62h 0.08
9.31f 0.02
3.43a 0.01
4.03a 0.01
7.64c 0.08
32.97g 0.06
36
42.58g 0.06
9.31f 0.01
3.44ab 0.01
4.05b 0.01
7.68d 0.07
32.94f 0.05
48
42.50f 0.10
9.27e 0.02
3.46bc 0.02
4.05b 0.02
8.00e 0.08
32.72e 0.05
60
42.50f 0.10
9.26d 0.03
3.47cd 0.02
4.08c 0.02
8.03f 0.09
32.70d 0.04
72
42.36e 0.11
9.23d 0.03
3.47cd 0.02
4.09cd 0.01
8.15g 0.10
32.70d 0.04
84
42.34d 0.09
9.16c 0.02
3.49d 0.01
4.09cd 0.02
8.20h 0.08
32.70d 0.04
96
42.20c 0.07
9.14c 0.02
3.52e 0.01
4.10d 0.02
8.27i 0.09
31.77c 0.06
108
41.98b 0.07
9.08b 0.01
3.52e 0.03
4.11ef 0.01
10.01j 0.10
31.28b 0.08
120
41.23a 0.06
9.01a 0.01
3.58f 0.02
4.12f 0.02
10.96k 0.07
31.10a 0.05
3.7
107
Table 25 shows the result of the pH changes observed in Okpehe preserved for 14 weeks
using different preservation treatments. The pH increased significantly (6.53 7.46) as the
storage period increased for all the treatments (Table 25). There was a reduction in microbial
count (3.7 106 1.7 106cfu/g) of all samples after the 14 weeks storage period. The
highest microbial count after 14 weeks was with the refrigerated Okpehe which had 2.6
106cfu/g while the least of 1.7 106cfu/g was observed with the salted and oven dried
Okpehe (Table 26). The crude protein (43.97% - 42.81%) (Table 27), total ash (4.13% 3.88%) (Table 30), crude fibre (4.02% - 3.40%) (Table 29), moisture content (12.76% 6.06%) (Table 31) and nitrogen free extract (32.63% - 27.08%) (Table 32) decreased
significantly as the storage period increased. At the end of the storage period, the salted and
oven dried Okpehe had the highest crude protein content of 43.64% whilst the refrigerated
Okpehe had the least (42.81%) (Table 27). The trend of the crude protein content for all the
treatments after 14 weeks is shown thus salt + oven drying > oven drying > sun drying >
salting > smoking > freezing > refrigerating
108
Table 25: Changes in pH of Okpehe During Storage Period

Preservation period (weeks)
Preservation
method
0
Sun drying
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
10
12
14
7.01a
0.00
7.01a
0.01
7.02a
0.01
7.06ab
0.01
7.10b
0.00
7.10b
0.02
7.13b
0.02
7.19c
0.01
7.05a
0.01
7.10b
0.03
7.19c
0.02
7.20c
0.01
7.23cd
0.01
7.24d
0.01
7.32e
0.02
7.42f
0.03
7.03a
0.01
7.12b
0.02
7.18c
0.01
7.28d
0.01
7.37e
0.03
7.41ef
0.02
7.42f
0.01
7.46g
0.00
6.84a
0.01
7.02b
0.01
7.03b
0.02
7.13c
0.01
7.14c
0.02
7.23d
0.03
7.27e
0.01
7.33f
0.01
6.53a
0.00
6.58b
0.01
6.98c
0.03
7.00c
0.00
7.05d
0.01
7.07d
0.01
7.15e
0.02
7.23f
0.02
6.71a
0.02
6.89b
0.02
6.99c
0.01
7.01c
0.00
7.03cd
0.00
7.04cd
0.02
7.07d
0.01
7.15e
0.02
6.85a
0.01
6.94b
0.02
7.15c
0.00
7.29d
0.01
7.34d
0.02
7.40e
0.02
7.44e
0.03
7.54f
0.03
6.74a
0.01
6.83b
0.01
6.86b
0.03
7.06c
0.01
7.22d
0.02
7.23d
0.01
7.31e
0.00
7.45f
0.02
6.62a
0.00
6.85b
0.01
6.85b
0.01
6.89bc
0.00
7.00c
0.00
7.16d
0.02
7.23e
0.03
7.37f
0.02
6.41a
0.01
6.48b
0.02
6.76c
0.01
6.90d
0.00
6.98e
0.01
7.06f
0.02
7.09fg
0.00
7.13g
0.01
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
Values are means of triplicate determinations SD; means within rows with different
superscripts differ significantly (p < 0.05).
Table 26: Microbial Count (cfu/g) of Okpehe During Storage Period
109
Microbial Count ( 106cfu/g)
Preservation
method
Sun drying
0wk
2wks
4wks
6wks
8wks
10wks
12wks
14wks
3.6h
3.3g
3.0f
2.6e
2.4d
2.3b
2.3b
2.0a
3.7g
3.6f
3.6f
3.2e
3.1d
3.0c
2.6b
2.4a
3.7h
3.6g
3.4f
3.3e
3.2d
2.9c
2.8b
2.6a
3.7h
3.4g
3.2f
2.5e
2.4d
2.2c
2.1b
1.9a
3.6h
3.5g
3.2f
2.7e
2.5d
2.1b
2.2c
1.8a
3.7h
3.6g
3.0f
2.7e
2.4d
2.2c
2.0b
1.7a
3.6h
3.5g
3.3f
2.8e
2.6d
2.5c
2.4b
2.2a
3.6h
3.4g
3.3f
2.7e
2.5d
2.3c
2.2b
2.1a
3.6h
3.4g
3.2f
2.6e
2.4d
2.3c
2.1b
2.0a
3.6g
3.3f
3.2e
2.5d
2.3c
2.3c
2.0b
1.8a
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
Values are means of triplicate determinations; means within rows with different superscripts
differ significantly (p < 0.05).
Table 27: Crude Protein Content (%) of Okpehe During Storage Period
110
Preservation
method
0 wk
Sun drying
43.92e
0.05
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
2 wks
4 wks
6 wks
8 wks
10 wks
12 wks
14 wks
43.85de
0.03
43.81d
0.04
43.75c
0.05
43.68b
0.04
43.58a
0.05
43.90e
0.04
43.90e
0.07
43.16d
0.06
43.16d
0.06
43.15cd
0.03
43.13c
0.05
43.10c
0.04
43.04b
0.04
43.00ab
0.05
42.96a
0.04
43.16e
0.05
43.16e
0.04
43.14de
0.07
43.11d
0.05
43.05c
0.06
43.01c
0.04
42.91b
0.03
42.81a
0.04
43.97f
0.04
43.96e
0.04
43.94e
0.06
43.89de
0.05
43.84d
0.05
43.79c
0.05
43.72b
0.04
43.62a
0.06
43.41d
0.05
43.39d
0.05
43.38d
0.04
43.36cd
0.06
43.31c
0.04
43.23b
0.05
43.23b
0.03
43.14a
0.07
43.95c
0.04
43.93c
0.05
43.93c
0.05
43.90c
0.06
43.86bc
0.08
43.86bc
0.04
43.82b
0.04
43.64a
0.03
43.52e
0.07
43.51e
0.06
43.51e
0.06
43.48de
0.05
43.44d
0.04
43.38c
0.05
43.30b
0.03
43.23a
0.04
43.53e
0.07
43.51e
0.08
43.51e
0.04
43.49de
0.03
43.45d
0.04
43.39c
0.04
43.30b
0.05
43.23a
0.05
43.53f
0.06
43.52f
0.06
43.52f
0.05
43.48e
0.05
43.42d
0.04
43.37c
0.05
43.28b
0.04
43.20a
0.03
43.50e
0.04
43.50e
0.05
43.50e
0.07
43.48de
0.06
43.43d
0.05
43.34c
0.05
43.27b
0.03
43.20a
0.04
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
111
Table 28: Ether Extract Content (%) of Okpehe During Storage Period
Preservation
method
0 wk
Sun drying
9.22a
0.01
9.27b
0.01
9.31b
0.01
9.41c
0.01
9.49d
0.01
9.55e
0.01
9.77f
0.01
9.95g
0.02
9.12a
0.01
9.12a
0.01
9.15a
0.01
9.23b
0.02
9.30c
0.03
9.40d
0.04
9.52e
0.02
9.65f
0.01
9.13a
0.02
9.14a
0.01
9.18ab
0.03
9.23b
0.02
9.30c
0.02
9.40d
0.01
9.58e
0.01
9.81f
0.03
9.44a
0.01
9.48ab
0.02
9.51b
0.01
9.61c
0.03
9.72d
0.02
9.83e
0.01
9.96f
0.03
10.18g
0.02
9.29a
0.03
9.35b
0.04
9.40c
0.01
9.52d
0.03
9.58e
0.02
9.75f
0.02
10.23g
0.02
10.23h
0.01
9.32a
0.02
9.38b
0.01
9.43c
0.02
9.56d
0.04
9.65e
0.04
9.75f
0.02
9.83g
0.01
10.10h
0.02
9.27a
0.03
9.31ab
0.03
9.35b
0.01
9.45c
0.02
9.49c
0.02
9.64d
0.01
9.83e
0.01
10.00e
0.02
9.32a
0.02
9.36ab
0.01
9.37b
0.02
9.46c
0.03
9.53d
0.02
9.67e
0.04
9.84f
0.02
10.01g
0.03
9.30a
0.04
9.35ab
0.01
9.39b
0.03
9.53c
0.02
9.64d
0.02
9.77e
0.01
9.93f
0.03
10.02g
0.02
9.33a
0.01
9.37ab
0.01
9.42b
0.02
9.47c
0.03
9.56d
0.01
9.70e
0.02
9.85f
0.02
10.07g
0.01
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
2 wks
4 wks
6 wks
8 wks
10 wks
12 wks
14 wks
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
112
Table 29: Crude Fibre Content (%) of Okpehe During Storage Period
Preservation
method
0 wk
Sun drying
4.02b
0.03
4.02b
0.01
4.02b
0.02
4.02b
0.01
4.00b
0.01
3.98a
0.02
3.96a
0.01
3.95a
0.01
3.82b
0.02
3.82b
0.03
3.82b
0.01
3.81b
0.01
3.80a
0.02
3.78a
0.02
3.78a
0.01
3.77a
0.02
3.82b
0.02
3.82b
0.02
3.81b
0.03
3.81b
0.01
3.81b
0.03
3.80ab
0.01
3.79a
0.01
3.76a
0.02
4.00c
0.01
4.00c
0.01
3.99c
0.01
3.97c
0.02
3.92b
0.03
3.90ab
0.03
3.87a
0.01
3.85a
0.01
3.75c
0.01
3.74c
0.01
3.74c
0.01
3.72bc
0.03
3.71bc
0.01
3.68b
0.03
3.68b
0.02
3.62a
0.02
3.85b
0.02
3.85b
0.02
3.84b
0.03
3.84b
0.01
3.81ab
0.02
3.77a
0.01
3.76a
0.02
3.75a
0.02
3.54b
0.01
3.53b
0.01
3.52b
0.02
3.51b
0.02
3.51b
0.02
3.50a
0.02
3.48a
0.00
3.46a
0.01
3.49b
0.01
3.49b
0.02
3.49b
0.03
3.47b
0.01
3.47b
0.02
3.45a
0.01
3.43a
0.01
3.41a
0.03
3.49b
0.01
3.48b
0.03
3.48b
0.03
3.47b
0.02
3.46b
0.02
3.45a
0.01
3.42a
0.01
3.41a
0.02
3.45a
0.01
3.45a
0.01
3.44a
0.04
3.44a
0.02
3.43a
0.01
3.43a
0.02
3.42a
0.01
3.40a
0.03
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
2 wks
4 wks
6 wks
8 wks
10 wks
12 wks
14 wks
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
113
Table 30: Total Ash Content (%) of Okpehe During Storage Period
Preservation
method
0 wk
Sun drying
4.02b
0.01
4.01b
0.01
4.00b
0.02
3.99ab
0.01
3.99ab
0.02
3.96a
0.02
3.95a
0.01
3.94a
0.01
3.95a
0.02
3.95a
0.01
3.95a
0.01
3.95a
0.02
3.95a
0.01
3.95a
0.01
3.93a
0.02
3.93a
0.03
3.95b
0.03
3.94b
0.02
3.94b
0.02
3.93ab
0.01
3.92a
0.01
3.91a
0.02
3.89a
0.01
3.88a
0.01
4.02b
0.02
4.02b
0.02
4.02b
0.01
4.00b
0.02
4.00b
0.01
3.98ab
0.01
3.97ab
0.02
3.93a
0.02
4.09c
0.01
4.08c
0.03
4.07c
0.02
4.03b
0.01
4.03b
0.02
4.00b
0.01
4.00b
0.01
3.91a
0.01
4.13c
0.01
4.12c
0.01
4.10c
0.01
4.06bc
0.02
4.05b
0.01
4.01b
0.03
3.99ab
0.02
3.95a
0.01
4.05b
0.01
4.05b
0.01
4.05b
0.01
4.03ab
0.03
4.03ab
0.01
4.02ab
0.01
4.01a
0.02
3.98a
0.01
4.07b
0.01
4.07b
0.01
4.07b
0.02
4.03a
0.01
4.03a
0.01
4.01a
0.01
4.00a
0.01
4.00a
0.02
4.07b
0.01
4.07b
0.01
4.07b
0.02
4.04a
0.02
4.04a
0.01
4.01a
0.01
4.01a
0.02
4.00a
0.01
4.08b
0.02
4.07b
0.01
4.06b
0.01
4.03ab
0.02
4.02a
0.02
4.01a
0.01
4.00a
0.02
3.98a
0.03
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
2 wks
4 wks
6 wks
8 wks
10 wks
12 wks
14 wks
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
114
Table 31: Moisture Content (%) of Okpehe During Storage Period

Preservation
method
0 wk
Sun drying
8.61c
0.14
8.60c
0.10
8.58c
0.10
12.76c
0.09
12.75c
0.09
12.47c
0.11
12.47c
0.07
6.78c
0.13
8.68c
0.07
6.12b
0.07
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
2 wks
4 wks
6 wks
8 wks
10 wks
12 wks
14 wks
8.55b
0.08
8.53b
0.06
8.52b
0.08
8.49ab
0.08
8.44a
0.07
12.73bc
0.08
12.70b
0.10
12.70b
0.06
12.68b
0.08
12.64a
0.07
12.61a
0.10
12.47c
0.08
12.47c
0.08
12.45bc
0.06
12.43b
0.06
12.40b
0.11
12.34a
0.09
6.76bc
0.06
6.76bc
0.05
6.76bc
0.09
6.75b
0.08
6.73b
0.07
6.72ab
0.07
6.68a
0.10
8.67c
0.10
8.66c
0.06
8.65bc
0.07
8.65bc
0.06
8.62b
0.06
8.60b
0.10
8.54a
0.08
6.11b
0.09
6.11b
0.08
6.11b
0.07
6.10ab
0.10
6.09a
0.07
6.08a
0.08
6.06a
0.08
10.70d
0.09
10.69d
0.11
10.66cd
0.08
10.63c
0.06
10.63c
0.10
10.57b
0.07
10.53ab
0.07
10.48a
0.06
10.17c
0.08
10.17c
0.08
10.17c
0.06
10.16c
0.07
10.15bc
0.09
10.12b
0.09
10.10b
0.10
10.05a
0.10
10.62d
0.10
10.61d
0.07
10.58c
0.06
10.54c
0.07
10.50b
0.08
10.46b
0.07
10.41a
0.07
10.40a
0.10
10.23c
0.10
10.22c
0.08
10.20bc
0.07
10.20bc
0.09
10.19b
0.08
10.17b
0.06
10.15ab
0.08
10.09a
0.06
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
115
Table 32: Nitrogen Free Extract (%) of Okpehe During Storage Period
Preservation
method
0 wk
2 wks
Sun drying
30.21b
0.14
30.20b
0.12
27.22c
0.08
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
4 wks
6 wks
30.19ab
0.12
30.18a
0.11
30.18a
0.09
30.16a
0.11
30.15a
0.10
30.14a
0.12
27.20bc
0.10
27.20bc
0.10
27.18b
0.08
27.15b
0.10
27.15b
0.11
27.13ab
0.09
27.08a
0.09
27.47b
0.10
27.47b
0.10
27.46b
0.09
27.45b
0.09
27.47b
0.12
27.45ab
0.11
27.43a
0.08
27.40a
0.08
31.78a
0.09
31.78a
0.09
31.78a
0.08
31.77a
0.10
31.77a
0.10
31.77a
0.08
31.76a
0.07
31.74a
0.11
30.78c
0.11
30.77bc
0.09
30.75b
0.09
30.73b
0.08
30.72b
0.09
30.72b
0.10
30.72b
0.08
30.56a
0.09
32.63b
0.08
32.61b
0.07
32.59b
0.11
32.53a
0.12
32.53a
0.11
32.52a
0.09
32.52a
0.08
32.50a
0.10
28.92b
0.12
28.91b
0.11
28.91b
0.11
28.90b
0.09
28.90ab
0.08
28.89a
0.09
28.85a
0.09
28.85a
0.10
29.42c
0.11
29.40bc
0.09
29.39b
0.13
29.39b
0.08
29.37b
0.07
29.36b
0.10
29.33a
0.08
29.30a
0.11
28.99b
0.10
28.97ab
0.12
28.96ab
0.11
28.94a
0.09
28.94a
0.09
28.94a
0.08
28.95a
0.10
28.92a
0.09
29.41c
0.13
29.39b
0.07
29.38b
0.08
29.38b
0.07
29.37b
0.08
29.35b
0.10
29.31ab
0.09
29.26a
0.10
8 wks
10 wks
12 wks
14 wks
Drying
(600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
116
3.8 Sensory Evaluation of Okpehe

The results of the sensory analysis of Okpehe stored for 14 weeks using different
preservation treatments are shown in Tables 33, 34, 35 and 36. At the end of the storage
period, the refrigerated sample was preferred by the panelists in terms of colour with a score
of 8.01 0.01 while the least preferred was the smoked sample with a score of 6.89 0.02
(Table 33). In terms of texture, the least preferred was the salted (20%) sample with a score
of 5.48 0.02 and the most preferred was the refrigerated sample with a score of 7.60 0.01
(Table 34). The smoked sample was rated highest in terms of aroma (7.68 0.04) over the
others (Table 35). For the general acceptability, panelists most preferred the salted and oven
dried sample (7.61 0.01) while the least preferred were the salted (15% and 20%) samples
with a score of 5.50 0.01 (Table 36).
117

Preservation
method
Period of Storage (weeks) / Colour

0
Sun drying
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt (5%)+Oven
Drying (600C)
Salting
10
12
14
7.10b
0.01
7.18c
0.01
7.13b
0.02
7.06a
0.03
7.42e
0.03
7.25d
0.04
7.40e
0.02
7.51f
0.01
7.08a
0.02
7.47d
0.01
7.07a
0.01
7.41c
0.04
7.51d
0.04
7.37b
0.02
7.67f
0.02
7.60e
0.03
7.13a
0.04
7.56d
0.04
7.08a
0.02
7.41b
0.02
7.53c
0.02
7.49c
0.02
7.68e
0.01
7.81f
0.01
7.11a
0.04
7.17b
0.04
7.17b
0.03
7.53c
0.03
7.50c
0.01
7.52c
0.02
7.64d
0.01
7.62d
0.01
6.82a
0.03
6.83a
0.04
7.21c
0.02
7.34d
0.01
7.44e
0.03
7.42e
0.01
7.29b
0.03
6.89b
0.02
7.07a
0.02
7.22c
0.02
7.16b
0.02
7.37d
0.01
7.45e
0.02
7.22c
0.01
7.43e
0.01
7.47e
0.01
7.08ab
0.02
7.04a
0.01
7.13b
0.01
7.28d
0.02
7.20c
0.03
7.24c
0.03
7.24c
0.04
7.03a
0.04
7.07b
0.01
7.04a
0.01
7.13c
0.02
7.27e
0.13
7.21d
0.03
7.23d
0.02
7.24ab
0.01
7.01a
0.02
7.06b
0.03
7.04a
0.02
7.12c
0.03
7.26e
0.01
7.20d
0.01
7.23d
0.04
7.23d
0.01
7.00a
0.03
7.06b
0.01
7.04a
0.02
7.12c
0.02
7.25d
0.04
7.24d
0.02
7.23d
0.01
7.22d
0.03
7.01a
0.01
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
118

Period of Storage (weeks) /Texture
Preservation
method
0
Sun drying
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt (5%)+Oven
Drying (600C)
Salting
10
12
14
7.23d
0.03
7.32e
0.02
6.80c
0.05
6.62b
0.03
6.41a
0.03
6.38a
0.02
6.43a
0.04
7.19d
0.02
7.02e
0.02
7.17g
0.01
7.00e
0.02
5.80a
0.02
6.80d
0.01
6.21c
0.01
6.00b
0.04
7.10f
0.03
7.32a
0.01
7.32a
0.03
7.28a
0.01
7.30a
0.02
7.30a
0.01
7.34b
0.03
7.44c
0.01
7.60d
0.01
7.34e
0.02
7.41f
0.03
7.12d
0.03
6.53c
0.01
6.47b
0.02
6.50b
0.03
6.21a
0.03
7.32e
0.01
6.64d
0.02
6.54c
0.02
7.02f
0.03
6.79e
0.03
7.05f
0.01
7.13g
0.02
6.24b
0.01
5.93a
0.03
6.73a
0.04
6.79b
0.04
6.90c
0.02
6.73a
0.02
7.01d
0.03
6.90c
0.03
7.00d
0.02
6.77a
0.02
6.00b
0.01
5.81a
0.01
5.83a
0.02
6.00b
0.01
5.80a
0.02
7.25e
0.02
6.24c
0.03
6.62d
0.02
6.00b
0.03
5.84a
0.01
6.03b
0.01
6.07c
0.03
6.25d
0.01
6.39e
0.01
6.42e
0.04
6.61f
0.01
5.96f
0.01
5.24b
0.02
5.42c
0.01
5.62e
0.03
5.44c
0.02
5.19b
0.01
5.07a
0.01
5.51d
0.03
5.96f
0.02
5.13c
0.01
5.22d
0.02
5.25d
0.02
5.00a
0.03
5.00a
0.04
5.07b
0.02
5.48e
0.02
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
119

Period of Storage (weeks) / Aroma
Preservation
method
0
Sun drying
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt (5%)+Oven
Drying (600C)
Salting
10
12
14
6.16a
0.01
7.33e
0.02
6.83c
0.02
6.86c
0.04
7.24d
0.04
6.49b
0.02
7.20d
0.03
7.34e
0.01
6.08a
0.02
7.17f
0.04
7.26g
0.04
6.92d
0.01
6.61c
0.02
6.40b
0.02
7.00e
0.03
7.21f
0.02
6.08a
0.01
7.16d
0.01
7.24e
0.03
6.93c
0.03
6.62b
0.01
6.05a
0.01
6.89c
0.03
7.16d
0.01
6.17a
0.01
7.34f
0.03
6.79b
0.01
7.00d
0.03
7.00d
0.01
6.92c
0.03
7.10e
0.01
7.29f
0.01
6.16a
0.01
7.34e
0.04
7.32e
0.01
7.19d
0.02
7.05b
0.02
7.12c
0.01
7.40f
0.01
7.68g
0.04
6.17a
0.02
7.34e
0.02
7.40f
0.03
6.78c
0.03
6.42b
0.01
7.21d
0.01
7.40f
0.04
7.40f
0.03
6.06a
0.01
7.35f
0.01
7.37f
0.03
6.82e
0.02
6.61d
0.02
6.15b
0.03
6.42c
0.03
6.42c
0.04
6.06a
0.01
7.36e
0.02
7.37e
0.03
6.83d
0.01
6.61c
0.03
6.31b
0.01
6.32b
0.02
6.31b
0.02
6.07a
0.02
7.35e
0.02
7.36e
0.01
6.83d
0.01
6.63c
0.02
6.19b
0.01
6.17b
0.02
6.18b
0.01
6.07a
0.02
7.35d
0.01
7.36d
0.02
6.84c
0.02
6.63b
0.03
6.03a
0.02
6.02a
0.03
6.02a
0.02
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
120

Preservation
method
Period of Storage (weeks) /Acceptability

0
Sun drying
(3220C)
Freezer
(-1820C)
Refrigeration
(410C)
Oven drying
(600C)
Smoking
(5520C)
Salt
(5%)+Oven
10
12
14
6.60c
0.01
6.43b
0.03
6.03a
0.04
6.86d
0.02
7.04e
0.03
7.23g
0.01
7.23g
0.02
7.12f
0.01
6.48b
0.01
6.87d
0.01
6.86cd
0.03
7.02e
0.02
6.81c
0.02
6.20a
0.01
7.00e
0.01
7.24f
0.01
6.08a
0.03
6.16b
0.03
6.43c
0.03
6.43c
0.02
6.52d
0.02
6.52d
0.01
6.48d
0.02
7.16e
0.02
6.47b
0.02
6.84c
0.01
6.82c
0.02
7.05d
0.01
6.84c
0.01
6.25a
0.02
7.01d
0.02
7.39e
0.01
5.86c
0.01
6.25d
0.01
5.82b
0.02
5.47a
0.01
6.01d
0.02
5.81b
0.02
6.02d
0.03
6.01d
0.03
6.37a
0.01
6.86b
0.02
7.00c
0.03
7.07d
0.01
7.40e
0.02
7.42e
0.01
7.60f
0.01
7.61f
0.01
6.01a
0.02
6.65b
0.02
6.63b
0.01
6.81c
0.01
6.86d
0.03
6.85cd
0.02
6.84c
0.02
6.82c
0.01
6.06d
0.03
6.64e
0.04
6.63e
0.01
5.72a
0.01
5.85b
0.02
5.83b
0.03
5.82b
0.01
6.01c
0.02
5.86d
0.02
6.04e
0.02
5.62c
0.02
5.67c
0.02
5.64c
0.03
5.54a
0.02
5.57b
0.01
5.50a
0.02
5.86d
0.01
6.03e
0.01
5.62c
0.01
5.65c
0.02
5.63c
0.03
5.53a
0.01
5.56b
0.02
5.50a
0.01
Drying (600C)
Salting
(5%)
Salting
(10%)
Salting
(15%)
Salting
(20%)
121
CHAPTER FOUR
4.0
DISCUSSION
Okpehe, with its characteristic appearance and aroma was produced from the spontaneous
fermentation of Prosopis africana seeds. The mash became soft and dark with a strong
ammoniacal odour after seventy two hours of fermentation. The organisms growing in the
fermenting Okpehe during the fermentation produced a whitish mucilaginous substance that
covered and linked the individual light brown to dark brown coloured cotyledons. These
organisms increased in number as the fermentation period progressed.
The involvement of a variety of microorganisms in spontaneous food fermentation is normal
and does not render the product unsafe for human consumption, especially when none of the
microorganisms is pathogenic to man (Oyeyiola, 2002). The growth of microorganisms
during the fermentation of Okpehe is likely to have a significant influence on the quality and
flavour of the final product. The organisms involved in the samples fermentation would
have been introduced by chance inoculation from the environment but the initial boiling
would eliminate most of the surface microflora of the Prosopis seeds. Boiling of the seeds
before fermentation has the effect of eliminating the species responsible for an acid
fermentation and encouraging a non-acid fermentation that is dominated by Bacillus species
(Achi, 1992).
Saccharomyces cerevisiae was found in only sample A. These organisms were present
throughout the period of fermentation. Similar result was obtained by Oyeyiola (2002). S.
cerevisiae is known to be able to grow well in the complete absence or presence of very
122
little oxygen as was likely to prevail during the fermentation (Oyeyiola, 2002). However,
Achi (1992) and Ogunshe et al. (2007) did not isolate any yeast in the fermentation of
Prosopis africana seeds.
The higher microbial load obtained in sample A (Okpehe obtained from a local producer)
may be attributed to the production procedure carried out for the sample. Aseptic techniques
were observed during the production of Okpehe in the laboratory while the local producer
(sample A) may not pay too much attention to practising good hygiene.
The presence of Bacillus species in the samples is expected since they have been found to be
associated with fermenting legume seeds for Okpehe (Achi, 1992; Ogunshe et al., 2007),
ugba (Obeta, 1983), dawadawa (Odunfa, 1981; Antai and Ibrahim, 1986), iru (Oyeyiola,
1987) and soumbala (0uoba et al., 2008). The presence of these organisms, which are
proteolytic, may lead to an increased proteinase activity, causing the breakdown of proteins.
The presence of Staphylococcus species in the samples was typical of the microflora of
fermenting beans (Obeta, 1983; Antai and Ibrahim, 1986). Staphylococcus species have
been associated with fermenting foods of plant origin especially vegetable proteins (Odunfa
and Komolafe, 1989; Jideani and Okeke, 1991). The coagulase-negative Staphylococcus
species are non-pathogenic and safe organisms on vegetable proteins (Odunfa, 1981). These
organisms may contribute to the flavour of the fermenting Okpehe because of their lipolytic
activity.
Escherichia coli was also found to be present in the three samples at the beginning of
fermentation but disappeared after 24 hours of fermentation. E. coli though fermentative and
123
found in the air and soil, has been isolated from some fermentation (Ogunshe et al., 2007).
Enterobacter cloacae was isolated by Achi (1992) from Okpehe, although no
enterobacterium was isolated by Oyeyiola (2002) in Okpehe from local producers. Most of
these microorganisms did not survive until the end of fermentation, perhaps because of the
modified environment, which had developed at the later stages of fermentation.
The rise in pH which occurred during fermentation could be due to the high proteinase
activity of the microorganisms involved (Odunfa, 1985), which ultimately resulted in the
liberation of ammonia as was reported for some other fermenting protein foods such as natto
(Hesseltine and Wang, 1967), koji (Yong and Wood, 1977), iru (Odunfa, 1985), ugba
(Oyeyiola, 1989), kawal (Dirar, 1993) and soumbala (0uoba et al., 2007). During the period
of the rise in pH, desirable flavour components of the condiment presumably were
developed. Organic acids which may result from protein decomposition may contribute to
the darkening of colour (Achi, 1992). The pH of Okpehe being slightly alkaline agrees with
earlier reports of Achi (1992), Oguntoyinbo et al. (2001), Omafuvbre et al. (2004) and
Ogunshe et al. (2007), all of who recorded a slightly alkaline pH in fermented food
condiments from vegetable proteins. All fermented condiments are characterized by a very
strong pungent smell. The increase in pH is generally due to the production of ammonia,
which is characterized by the pungent smell of fermented condiments (Ogunshe et al.,
2007). Whitaker (1978) also reported that ammonia production might be due to protease
deaminase enzymes produced by Bacillus isolates.
124
The rise in temperature indicates that the fermentation was exothermic with the changes
being due to the metabolic activities of the microorganisms. This is in line with the work of
Hesseltine and Wang (1967) who reported that during the short period of fermentation of
dawadawa, heat was evolved and there was also an increase in pH due to the abundant
production of ammonia during the later stages of fermentation. The heat generated in the
fermenting mash possibly provided the ideal temperature conditions for the optimal activity
of the proteolytic enzymes (Odunfa, 1985).
The increase in crude protein is in agreement with the work of Gernah et al. (2005) and
Campbell-Platt (1980) where the crude protein of dawadawa increased from 24.8% to
33.5% and 30.0% to 38.5% respectively. They attributed this to the organism Bacillus
subtilis associated with the fermentation. The increase in protein may be due to the
fermenting organisms and also to the fact that fermentation results in increased nutritional
values which are in agreement with the results of Odunfa (1985). Protein in Prosopis has
been reported to constitute about 60% or more of the seed kernels weight (Felker and
Bandurski, 1977). The fresh (unboiled and unfermented) seeds of Prosopis africana contain
30.12% crude protein of Table 18.
The progressive decrease in crude fat during the fermentation for all the three samples is
desirable because it will result in the breakdown of fat into simpler substances which will
enhance the digestibility of the product in human body. The source of lipase activity in this
fermentation may be attributed to the Staphylococcus species, in which lipolytic activities
are well known (Ogunshe et al., 2007). This result of decrease in crude fat content in
125
Okpehe samples is however not in agreement with the work of Gernah et al. (2005), who
reported an increase in fat content of iru during fermentation. The decrease in fat has been
reported by Odunfa (1985) to be desirable, since high amounts of fatty acids in foods can
cause rancidity thereby making the food taste sour.
The slight increase in crude fibre may be due to the utilization of other nutrients by the
fermenting organisms because they may not be able to degrade the cellulose and
hemicellulose present in the samples. Crude fibre consists largely of cellulose and
hemicellulose. Azokpota et al. (2006) also observed a slight increase in crude fibre in the
production of afitin, iru and sonru from Parkia biglobosa.
The ash content observed is an indication that Okpehe samples are rich in minerals. The
slight increase in ash content is in agreement with work of Gernah et al. (2005) and
Azokpota et al. (2006) in the fermentation of some local condiments from Parkia biglobosa
seeds.
The sharp decrease in carbohydrate content of the samples may be due to the hydrolytic
effect of microbial amylase converting the carbohydrate into sugars easily utilizable by the
isolated organisms during fermentation. Similar result was obtained by Gernah et al. (2005)
after fermenting iru for 72 hours. Bacillus species are important sources of amylases
therefore, the high recovery rates of these organisms from the fermentation may account for
their high amylase activity (Ogunshe et al., 2007) thereby leading to a reduction in the
carbohydrate as fermentation progressed.
126
There was a corresponding reduction in the percent dry matter, as it was observed with the
carbohydrate content. This is probably due to carbohydrate utilization by the fermenting
organisms.
The fermentation of the samples resulted in a decrease in its total sugars. The pattern of
change in soluble sugar level have been reported in similar fermented condiments
(Omafuvbre and Oyedapo, 2000; Omafuvbre et al., 2000). From previous works, it has been
found that oligosaccharides are present in the unfermented vegetable beans, but the quantity
decreases during fermentation (Oyenuga, 1968). The decrease in total sugars may be
attributed to its utilization by the fermenting organisms.
There was a gradual increase in the total amino acids in the samples with sample C having
the highest amount. This may be because microorganisms are capable of synthesizing amino
acids themselves. Amino acids produced were due to protein metabolism which is
responsible for the gradual pH increase. The result shows that the samples contained
nutritionally useful quantities of most of the essential amino acids. Similar increases in the
level of amino acids with fermentation have been reported in leguminous vegetable seeds
(Ogunshe et al., 2007; Omafuvbre et al., 1999, 2000). This rapid increase in the total amino
acids may be a reflection of the increased protease activity in the fermenting seeds (Ogunshe
et al., 2007). Soluble low molecular weight peptides and amino acids that contribute to
flavour are produced through the enzymatic breakdown of proteins (Odunfa, 1985; Ogbonna
et al., 2001; Achi, 2005b). Free amino acids increase but longer fermentation results in
losses of lysine or other essential amino acids (Achi, 2005a).
127
There was a significant increase in the non essential amino acids over the essential amino
acids in all the samples. Similar increase in non essential amino acids over essential amino
acids was reported by Muhammad and Oloyede, (2009) in Terminalia catappa seed meal
fermented by Aspergillus niger. This may be an indication that the non essential amino acids
and nucleic acids have been synthesized at the expense of the essential amino acids
(Muhammad and Oloyede, 2009), thus the reduction in the essential amino acids.
Potassium was found to be the most abundant mineral in all the samples although it
decreased as fermentation progressed. The abundance of potassium is in close agreement
with the observation of Aremu et al. (2006) that potassium was the most predominant
mineral in agricultural products. The appreciable high amount of potassium is good because
the element helps in regulation of body fluids and maintenance of normal body pressure. It
helps in controlling kidney failure, heart oddities and respiratory flaw (Anhwange, 2008).
Aremu et al. (2006) also observed that potassium was the most abundant mineral in
Prosopis africana flour. The work of Nda-Umar et al. (2008) gave a contrary result that
calcium is the most abundant mineral in Okpehe.
Calcium, which was found to be the second highest mineral increased with fermentation for
all the samples. Similar result was obtained by Aremu et al. (2006) on the flour of P.
africana. The presence of calcium in this condiment is good because the element is needed
for bone development and strong teeth.
Zinc was also found in an appreciable amount. The amount was constant throughout the
period of fermentation for all the samples. Results obtained by other workers were higher;
128
90.70 0.03 mg/100g by Nda Umar et al. (2008) in Okpehe and 22.4mg/100g by Aremu et
al. (2006) in P. africana flour. Zinc aids digestion and body functions.
The concentration of iron slightly increased as fermentation progressed for all the samples.
Nda-Umar et al. (2008) also detected iron in moderate amounts (79.38mg/100g) in Okpehe.
Lower amounts of iron (15.5 0.4 mg/100g) was obtained by Aremu et al. (2006) in P.
africana flour. Iron carries oxygen to the cells and is necessary for the production of energy,
synthesis of collagen and the proper functioning of the immune system (Anhwange, 2008).
The amount of manganese detected in all the three samples was moderate. Observation by
Aremu et al. (2006) showed that P. africana flour contained 36 0.4mg/100g of
manganese. Manganese is known to aid formation of skeleton and cartilage of the body.
Manganese dearth is scarce but could affect glucose tolerance, normal reproductive, skeletal
and cartilage formation (Anhwange, 2008).
Copper was also detected in all the samples and it increased slightly during fermentation.
Copper (46.2 0.7mg) was among the minerals present in P. africana flour (Aremu et al.,
2006. Copper is a trace element that serves as a co-factor and is required for enzyme
function.
Lead and cadmium were not detected in all the samples. Similar result was also obtained by
Aremu et al. (2006) for P. africana flour. These elements are known to be toxic to the
human body.
129
The Okpehe obtained from the mixed culture had a higher crude protein than either the
monoculture inoculated or naturally fermented Okpehe. The increase in crude protein is in
agreement with the work of Gernah et al. (2005) and Campbell-Platt (1980) where the crude
protein of dawadawa increased from 24.8% to 33.5% and 30.0% to 38.5% respectively. The
higher crude protein obtained is in line with the work of Gberikon et al. (2010), where the
protein and lipid values were higher when a mixed culture of Bacillus pumilus and Bacillus
subtilis were used as starter in the fermentation of Glycin max, Parkia biglobosa and P.
africana seeds as compared to when these seeds were fermented naturally. A better quality
ugba was also produced with a mixed starter culture of B. megaterium and Corynebacterium
sp and B. megaterium and Alkaligenes viscolatis which gave a 98% and 97% overall quality
respectively in the controlled fermentation of ugba as compared to using single organism (B.
megaterium) that gave a 87% quality (Nwagu et al. 2011). Also mixed cultures of B subtilis
and B. licheniformis were recommended (because of the better product obtained) for the
controlled fermentation of soybeans against the use of single organisms (Sarkar et al., 1993;
Suberu and Akinyanju, 1996) This is due to the fact that when organisms responsible for
legume seeds fermentation are used as inoculum or starters in the right percentage; it
guarantees consistency, product safety and quality (Holpzapfel, 2002; Achi, 2005a;
Oguntoyinbo et al., 2010). Inconsistencies do occur in naturally fermented seeds when there
are no appropriate starters to initiate fermentation.
In the shelf life study of Okpehe, after the initial increase in microbial population, a decline
was observed after 72 hours. This was probably because deterioration had started setting in.
The changes in microbial population observed depend on the generation time of each
130
microorganism. Generation time is not uniform for all microorganisms (Frazier and
Westhoff, 2010). The reduced crude protein, ether extract and nitrogen free extract during
the storage period is in agreement with the work of Mbata et al. (2008) that food nutrients
are lost during storage period. The shelf life of Okpehe from this study is 72 hours. Similar
result (3 5 days) was obtained for the shelf life of ugba (Mbata et al., 2008).
For the preservation of Okpehe, the pH increased slightly during the storage period although
the pH of the treated samples was lower than fresh Okpehe. The lower pH obtained was due
to the different preservation treatments carried out on the samples. Evaporation of ammonia
during drying results in decrease in pH (Parkouda et al., 2008). The lowest microbial count
obtained for the salted (5%) and oven dried sample was because, the presence of salt helped
to decrease the water activity and removed water from microorganisms through osmosis
thereby discouraged microbial growth and prevented the spoilage of Okpehe during the
storage period. This is inconsonance with the report of Ademola et al. (2011) that common
salt helps to preserve food condiment. Drying helps to dehydrate both the food and
microorganisms (Fellows, 2009). It also helped to concentrate the soluble ingredients in
Okpehe, and these high concentrates prevented the growth of microorganisms (Ohenhen et
al., 2008) thus the low microbial load recorded in the salted (5%) and oven dried sample.
The significant decrease in total ash, crude fibre, nitrogen free extract and crude protein
observed during storage is consistent with the findings of Ademola et al. (2011) when they
worked on preservation of processed locust bean (iru). They observed a decrease in the
nutrient composition of iru during storage. The decrease in protein may be explained by the
131
presence of proteolytic enzyme present in the fermented Okpehe. This enzyme is responsible
for the breakdown of proteins and the release of ammonia.
In the sensory analysis, the refrigerated sample was rated highest by panelists in terms of
colour probably because it looked fresh and people tend to like such. The process of
refrigeration preserves foods by slowing autolysis by the food enzymes (Anon, 2011b)
which may lead to spoilage. Sivasankar, (2009) observed that refrigeration has no adverse
effects on the colour, texture and nutritive value of foods. The smoked sample was rated
lowest because it looked a little darker in appearance and probably because the people were
not used to it. During smoking, there was the deposition of chemicals resulting from the
thermal decomposition of wood (Banwart, 2004) on the sample and thus its appearance. In
terms of texture, the least preferred was the salted (20%) sample while the most preferred
was the refrigerated sample. This is because the texture of foods is mostly determined by the
moisture and fat contents, and the types and amounts of structural carbohydrates and
proteins that are present in foods (Fellows, 2009). According to Sivasankar (2009),
refrigeration has no adverse effects on the colour, texture and nutritive value of foods. The
smoked sample was preferred most in terms of aroma over the others because the process of
smoking helped in imparting some flavour on the Okpehe. According to Anon, (2011b),
smoking preserves partly by drying and partly by incorporating substances from smoke thus
imparting some aroma in food. On the average, the panelists rated the sample that was salted
(5%) and oven dried highest in terms of general acceptability while the salted (15% and
20%) samples were rated lowest. This may be because the combination of oven drying and
salting (5%) helped to reduce the water activity of Okpehe and thus discouraged microbial
132
growth. It may also be because the panelists were used to drying the condiment after
processing before consumption. This result is consistent with the findings of Ibe and
Orabuike, (2009).
In conclusion, laboratory produced sample using the hot plate (600C) (sample C) was better
in terms of nutrition than the other two samples. A better quality Okpehe can be produced
from Prosopis africana seeds by processing the seeds with the hot plate set at 600C to avoid
loss of some essential nutrients; and using starter culture of B. subtilis and B. licheniformis
prepared in the stated proportion. Okpehe can be best preserved by combining salting (5%)
with oven drying at 600C.
133
RECOMMENDATIONS
(i) It is recommended that the overnight boiling of the Prosopis africana seeds by the
local producers should be discouraged because of the loss of some essential
nutrients, energy and time wastage.
(ii) The local producers should be trained to ferment foods under hygienic conditions.
(iii)Since the trees of Prosopis africana are found in abundance in the Southern Guinea
Savannah zone including Kwara State without being utilized, it is also recommended
that a pilot project be set up in the state to produce the condiment using the available
data from this study as baseline information.
(iv) Okpehe has a high amount of protein and thus is recommended as a supplement to
meat for the populace to reduce protein- calory malnutrition.
(v) Finally, it is recommended that Okpehe should be made into cubes and packaged
well to attract prospective buyers.
134
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APPENDIX I
LIST OF PUBLICATIONS FROM THIS THESIS
1. Balogun, M .A. and Oyeyiola, G. P. (2011). Microbiological and Chemical Changes
During the Production of Okpehe from Prosopis africana Seeds. Journal of Asian
Scientific Research 1 (8): 390 398.
2. Balogun, M. A. and
Oyeyiola, G. P. (2012). Changes in the Nutrient Composition
of Okpehe During Fermentation. Pakistan Journal of Nutrition 11 (3): 270 - 275.
151
APPENDIX II
MICROBIAL ANALYSIS
Table 37: Cellular and Biochemical Characteristics of Bacterial Isolates
Spore
Catalas
e
O2
r/ship
MR
VP
Indole
Urease
Citrate
Starch
Oxidase
Nitrate
re
H2S
prdn
Sucrose
Glucose
Lactose
Maltose
B. subtilis
FA
A A
B. pumilus
FA
A A
B.
licheniformis
FA
A A
B.
megaterium
FA
A A
E. coli
FA
A
G
A
G
A A
G G
A
G
S. aureus
FA
A
G
A A
FA Facultative anaerobe; A Acid production; AG Acid and gas production
152
Fructos
e
Mannit
ol
Identification
Gram
Reactio
nnnnn
Motility
SUGAR FERMENTATION
APPENDIX III
STANDARD CURVE FOR GLUCOSE
14
Total sugar (mg/g)
12
10
8
6
4
2
0
0.04
0.1
0.15
0.2
0.22
0.3
0.35
Absorbance
Fig. 20: Standard curve for glucose
153
0.39
0.42
0.48
0.53
0.57
APPENDIX IV
QUESTIONNAIRE FOR SENSORY EVALUATION
Name:
Date:
Instruction:
You have been presented with ten coded Okpehe produced from Prosopis africana seeds
preserved using different preservation treatments. Please evaluate these samples for colour,
texture, aroma and general acceptability using the scale below;
Like extremely
9
Like very much
8
Like moderately
7
Like slightly
6
Neither like nor dislike 5
Dislike slightly
4
Dislike moderately 3
Dislike very much
2
Dislike extremely
1
Sample
Colour
Texture
305
347
459
561
587
619
667
741
815
945
Thanks
154
Aroma
General
Acceptability

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A FERMENTATION STUDY AND

PHYSICOCHEMICAL ANALYSIS OF Prosopis

A THESIS SUBMITTED TO THE DEPARTMENT OF

DR. G.P. OYEYIOLA

DR. G.P. OYEYIOLA

B.Sc. (Maiduguri); M.Sc. (Ibadan)

B.Sc. (Maiduguri); M.Sc. (Ibadan)

M.Phil. (Ilorin); Ph.D. (Kano)

M.Phil. (Ilorin); Ph.D. (Kano)

Examined this ..day of 2012

Introduction and Literature Review

Justification for the Study

Material and Methods

Sources of Fruits and Okpehe Samples

Traditional Preparation of Okpehe from Prosopis africana Seeds

Laboratory Preparation Using the Modified Traditional Method

2.3.1 Using the Autoclave

2.3.2 Using the Hot Plate

Inoculation with Starter Culture

Determination of Physicochemical Parameters

2.5.2 Determination of Temperature

2.5.3 Determination of Moisture Content

Characterization of Bacterial Isolates

Characterization of Yeast Isolate

Proximate Analysis of Okpehe Samples

2.9.1 Determination of Dry Matter Content

2.9.2 Determination of Ether Extract (Crude Fat) Content

2.9.3 Determination of Crude Fibre

2.9.4 Determination of Total Ash Content

2.9.5 Determination of Crude Protein

2.9.6 Determination of Carbohydrate or Nitrogen Free Extract

Determination of Total Sugar

Determination of Amino Acid Profile Using the Amino Acid Analyzer

Determination of Mineral Content

Determination of Shelf Life of Okpehe

2.14.3 Sun Drying

2.14.4 Oven Drying

2.14.5 Salting Plus Oven Drying

Experimental Design and Statistical Analysis

Physicochemical Parameters of Naturally Fermented Okpehe

Nutritional Characteristics of Naturally Fermented Okpehe Samples

3.3.1 Proximate Composition of Okpehe Samples

3.3.2 Changes in Total Sugar of Naturally Fermented Okpehe Samples

3.3.3 Changes in Amino Acid Composition of Naturally Fermented Okpehe Samples

3.3.4 Mineral Composition of Naturally Fermented Okpehe Samples

Physicochemical Parameters of Okpehe Samples Inoculated with Starter Cultures 76

Proximate Composition of Okpehe Samples Inoculated with Starter Culture

3.5.1 Proximate Composition of Okpehe Samples Inoculated with Mono Cultures of

3.5.2 Proximate Composition of Okpehe Samples Inoculated with Mixed Cultures of

Shelf Life of Okpehe Stored at Ambient Temperature

Sensory Analysis of Okpehe

Table 2: Occurrence of Microorganisms in Laboratory Produced Okpehe

Table 3: Occurrence of Microorganisms in Laboratory Produced Okpehe

Table 4: Microbial Counts of Samples after 0 hr of Fermentation

Table 5: Microbial Counts of Samples after 24 hrs of Fermentation

Table 6: Microbial Counts of Samples after 48 hrs of Fermentation

Table 7: Microbial Counts of Samples after 72 hrs of Fermentation

Table 8: Proximate Analysis of Naturally Fermented Okpehe Samples

Table 9: Amino Acid Composition of Okpehe Samples

Table 10: Changes in Essential Amino Acids of Okpehe Samples

Table 11: Mineral Composition of Okpehe Samples

Table 16: Moisture Content of Prosopis africana Seeds Fermented with