CHAPTER ONE

1.0 INTRODUCTION
The African oil bean  (Pentaclethra macrophylla) is a native of tropical Africa and
belongs to the family Leguminosea. It is popular in Nigeria where it is known by
several names such as Apara in Yoruba for the seed as well as the fermented product
and the most prominent being Ugba (Igbo), as it is a popular condiment and meat
analogue among consuming populations (Enujiugha and Akanbi, 2005).
The African oil bean plant, Pentaclethra macrophylla Benth has been recognized as a food tree
species for outlaying farms in the forest zone. According to Asoegwu et al (2006) they grow
either wild or semi-wild with no organized cultivation in plantations or orchards in Nigeria.
There are various plant seeds that are fermented and used as food in some rural and urban parts
of Nigeria, among which is ‘ugba’ from African oil bean (Pentaclethra macrophylla). African oil
bean seed also known as “ugba” and “ukpaka” in Igbo language is a popular food delicacy in
Nigeria especially among Igbo ethnic group. It is a fermented product rich in protein and is
obtained by a solid state fermentation of the boiled, shredded seed of African oil bean tree
(Pentaclethra macrophylla Benth). It is an essential food item for various traditional delicacies
where it is mixed with slices of boiled stock fish (ugba and okporoko). Ugba as a source of
protein in developing countries of the world and Africa in particular is of primary importance
because it’s cheap and available. Fermented seeds are not just palatable but serve as a delicacy
amongst consuming regions where it is consumed and garnished with other vegetables or staples.
Consumption of fermented ugba seeds could bridge the prevailing protein energy malnutrition
(PEM) – marasmus in developing countries (Enujiugha and Akanbi, 2008).
It also contains the twenty (20) essential amino acids and essential fatty acids that make up over
10% of the fatty acids in the oil (Enujiugha and Agbede, 2000; Ogbo, 2007). The oil content and
carbohydrate contained in raw African oil bean seeds are 53.98 0.99% and 19.16 0.76% dry wt,
respectively (Enujiugha and Ayodele-Oni, 2003; Enujiugha and Akanbi, 2005). The seed when
boiled, processed and fermented for about 3-4 days (72 hours) is called Ugba ukpkala in Igbo
language of Eastern Nigeria, in Fulani language of Northern Nigeria, it is called Ataa or Atawa,
in Zaire it is known as Twa Bobala, in Wolof language of Senegal, Gambia and Mauritania, it is
called Ataa or Atawa, in the Fula language of West Africa (Mauritania, Senegal, Mali, Guinea,
Burkina Faso, Niger, Nigeria, Cameroon, Gambia, Chad, Sierra Leone, Benin, Guinea-Bissau,
Sudan, Central African Republic, Ivory Coast, Ghana, Togo, Liberia and Gabon), it is also
known as Ataa or Atawa. The fermented seeds are used traditionally as condiments and
seasonings to add flavour to food (Enujiugha and Akanbi, 2002; Ogbo, 2007; Enujiugha and
Akanbi, 2008) and used for the preparation of many delicious African delicacies and snacks
including, African salad, soups and sausages for eating with different staples. It is rich in
vitamins and mineral and in high demand for both local consumption and for export. It is a low
acid food which could be prepared into flour and explored in food fortification and
confectionaries. The seed is a source of edible oil and used for candle making, cooking and soap.
The seed shells are decorative and are often used to make beads which are worn as necklace,
rosaries and sometimes as local dancing apparels. The seeds of African oil bean which are rich in
nutrients (protein, fatty acids and minerals have been found to be a highly nutritive animal feed
when fortified (Egonu and Njoku, 2006).
Processing of these seeds involves boiling, removal from pod, shredding/ cutting into slices,
further boiling, sieving, wrapping in banana/plantain leaves and fermentation. Thermal treatment
induces a resultant rise in nutrient bioavailability and seed digestibility. Processing ugba seeds
drastically reduces the levels of the anti-nutritional compounds mentioned while increasing iron,
calcium, potassium, thiamine and riboflavin levels (Enujiugha, and Ayodele-Oni, 2003).
Some parts of the plant have medicinal values, in Cameroon, the seeds are used to treat infertility
while the pods are used to treat convulsion. In Nigeria comparative study of cancer risks and
cancer levels have been carried out between the Easterners who ate fermented oil bean and those
who did not. An improvement index was also measured between cancer patients who ate ugba as
a meal supplement and those who did not, the result indicated that the fermented form of the
African oil bean seed as a food supplement has greatly reduced the risks of cancer and some
tobacco related diseases. It was discovered that cancer patients who regularly ate fermented oil
bean seed as food supplement showed marked improvements in regaining quality health
(Chidozie, 2006).
1.1 Aims and Objectives
To produce oil from fermented pentaclethra macrophylla Benth seeds.
To determine the mineral composition of the ukpaka oil extract.
 CHAPTER TWO
LITERATURE REVIEW
2.1 Fermented Pentaclethra macrophylla Benth seed (Ukpaka)

Ukpaka is the Igbo name for the fermented African Oilbean seeds ( Pentaclethra
macrophylla, Benth). It is called Ukana by the Efiks in Southern Nigeria. It is
consumed by an estimated 15 million people in Eastern Nigeria, majority of whom are
Igbos (Odunfa and Oyeyiola, 2005). It is a traditional food generally prepared in homes
as a small family business. The method of production varies from one producer to
another resulting in a non-uniform product ( Njoku and Okemadu, 2009). The beans that
have been fermented for more than three days are taken as a delicacy. Well fermented
beans are added to soup as flavouring ( Odunfa and Oyeyiola, 2005). It is widely
consumed in eastern states of Nigeria with tapioca, stock fish and garden eggs and
leaves. It can also be eaten with bitter kola (Garcinia kola) or kola nuts (Cola
acuminate and C. nitida) and when prepared with gardenegg leaves is used to eat yam
and cocoyam (Okafor et al., 2001; Mbajunwa et al., 2008). It is an important and cheap
source of protein for people whose staple foods are deficient in proteins ( Obeta, 2003).
The quantity of ugba produced annually is not known, since the seeds are collected by
individuals and sold in the market to ugba producers.
2.1.1 Nature of the plant and the seeds 
Oilbean seeds for ugba production are obtained from a perennial legume
tree, Pentaclethra macrophylla, Bentham, commonly called the oilbean tree. The trees
are often planted along the sides of roads as shade trees and around communities as
cash crops. The fruit is a black, hard and woody pod measuring about 35-36 cm long
and 5-10 cm broad. When mature it splits open explosively to release about eight flat,
glossy brown seeds measuring about 5-7 cm in diameter and weighing between 15-20 g
(Keay et al., 2004; Odunfa, 2006a).
2.1.2 Chemical composition of seeds 

The oilbean seeds contain 4-17% carbohydrate, 44-47% oil which has been found to be
rich in oleic acid (Nwokedi, 2005; Odoemelam, 2005) and linoleic acid (Onwuliri et
al., 2004). Onwuliri et al. (2004) also found out that the saturated fatty acid,
lignoceric acid, occurred in high amounts constituting about 10% of the total  fatty
acid concentration. Some workers said that the oil content could be as low as 38% ( Kar
and Okechukwu, 2008). They also reported that the oil contains about 75%
saturated fatty acids and 25% unsaturated fatty acids. Table 1 (Achinewhu, 2003)
shows the fatty acid content of the seeds. Both saturated and unsaturated  fatty acids
are found in the seeds. For the saturated fatty acids, lignoceric acid appears to be
present in the largest amount constituting about 12% while palmitic acid is the least
with 3.4%. Behemic acid is also present with 5.2%. The major unsaturated  fatty
acid in the seeds is linoleic acid constituting 42.8%. Oleic acid is also present in
appreciable amounts (29.0%). Linolenic and gadoleic acids are present in very small
amounts (3.2 and 0.28%, respectively). The presence of appreciable amounts of
behenic and lignoceric acids is not desirable for edible oils (Odunfa, 2006a).

However, Odoemelam (2005) believes that the high degree of unsaturation makes it

suitable for cooking purposes and for use as a drying oil for cosmetics, paints and
varnishes.

Also they have been found to contain 36.2-43.89% crude protein which contains the
20 essential amino acids. However, the sulphur containing amino acid content is much
lower than those found in other plant proteins ( Mbadiwe, 2008; Mba et al.,
2004; Odoemelam, 2005). The high content of other essential  amino acids makes the
seeds a potential source of protein (Achinewhu, 2002). Table 2 shows the amino
acid profile of the seeds. Glutamic acid appears to be the largest  amino acid contained
in the seeds. This may be responsible for its use as a flavouring for soups in south
eastern Nigeria. Aspartic acid, lysine and phenylalanine are also present in appreciable
amounts in the seeds.

Table 1: Fatty acid composition of African oilbean seeds

As percentage of total oil. Achinewhu (2003)

 Table Amino acid content
2: (g/100 g protein) of
African oilbean seeds b,c

Mba et al. (2004), Achinewhu

(2002)

2.1.3 Preparation of Ukpaka 

Methods for ugba preparation vary from one community to the other. In this method
described by Obeta (2003), the seeds are boiled in water for 16-18 h to remove the
tough testa. The cotyledons are then sliced, boiled again for 30 min and left overnight
in water at room temperature. The sliced cotyledons are then washed in water and
packaged in leaves of banana.

Another method described by Odunfa and Oyeyiola (2005) and Odunfa (2006a) shows

that the seeds are boiled in water over an open fire for 4-5 h or even up to 12 h. The
cotyledons are then removed from the seed coats and washed. The cotyledons are again
boiled overnight over a low flame, allowed to cool, drained and washed several times
to remove bitter components in the cotyledons and soaked for a period of 6 h. The
cotyledons are then cut into long thin slices which are mixed with salt, put in a clean
pot, covered and fermented for up to 5 days at room temperature. Usually after 2-3
days of fermentation the sliced cotyledons are wrapped in banana leaves and tied
tightly.

Njoku and Okemadu (2009) also described another production method. The seeds are
boiled for 5-8 h, after which the hard shells are removed. The cotyledons are cooled,
washed and sliced into 4-5x0.1-0.2 cm slices. These are washed again and boiled for
another 1-2 h, cooled and soaked in water for about 10-12 h. They are washed and
allowed to drain for ½-1 h. in a basket lined with banana leaves ( Musa sapientum linn).
They are then wrapped about 40-50 g of slices using another leaf ( Mallotus
oppositifolius) and incubated for 72 h at room temperature.

Another method has been described by Sokari and Wachukwu (2007). These workers
said toasting the bean seeds in hot (100°C) sand and holding for a further 30 min at
100°C significantly improved dehulling. They also said that slicing to 1 mm, boiling
for 30 min and soaking for 2 h removed the bitter taste associated with the seeds. They
claimed that the technique reduced the general production time by 2 days and the
quality of ugba produced from this process was the same as that produced from the
rather more cumbersome and time-consuming traditional technique.

The differences in the various processing methods are responsible for the variations in
the products from one community to the other. The wrapped ugba at different stages of
fermentation are sold to consumers and they are often told the length of fermentation at
the time of purchase.

Anti-Nutritional Content of Ukpaka

The African oil bean seeds are inedible in its unfermented state because it suffers from some drawbacks.
Little is known about anti-nutritional factors in the raw and fermented African oil bean seeds. Although,
Kar and Okechukwu (1978) and Enujiugha and Agbede (2000) reported the presence of a number of
antinutritional and /or toxic factors, recent studies, have revealed the detection of tannins, saponins,
alkaloids, steroids, glycosides, flavonoids, and phytate in the unfermented African oil bean seed (Okorie
and Olasupo, 2014). This study also showed that processing and fermentation drastically reduced the
content of these toxic factors in the fermented product (Okorie and Olasupo, 2014), mainly due to
soaking of the seeds overnight and washing in water before fermentation. This had a significant effect
on all the phytochemicals/anti-nutritional factors identified. Tannin was reduced from 12.58 to 3. 65
mg/100 g, saponin from 52.00 to 22.00 mg/100 g, phytate from 25.63 to 14.47 mg/100 g, glycosides
from 34.76 to 11.33 mg/100 g, alkaloids from 2.52 to 0.14 mg/100 g, flavonoids from 4.66 to 2.49
mg/100 g and steroids from 26.48 to 5.43 mg/100 g. Alkaloids and tannins were completely removed
from the samples after 24 and 48 h of fermentation respectively.

2.2 Microorganisms involved in the fermentation 

Several workers have investigated the microorganisms involved in the fermentation.

Only bacteria are involved in the fermentation ( Obeta, 2003; Odunfa and Oyeyiola,
2005; Ejiofor et al., 2007; Ogueke and Aririatu, 2004). The main fermenting
microorganisms have been identified to be proteolytic  Bacillus  sp. (Obeta, 2003)
which include B. subtilis  (most predominant), B. licheniformis, B. megaterium, B.
macerans and B. circulans.

Their numbers increased tremendously from 10 3  at the start of fermentation to 10 8  at
the end of the fermentation (72 h). Other bacteria identified in the fermenting slices
include coagulase negative Staphylococcus sp., Micococcus sp. (their numbers
decreased after 72h of fermentation), Leuconostoc mesenteroides; Lactobacillus
plantarum, Streptococcus lactis, Proteus sp., Enterobacter sp. and E. coli. Some
workers isolated the yeasts Candida tropicalis and Geotrichum candidum during
fermentation (Ejiofor et al., 2007).

Since protein hydrolysis is the major biochemical change in ugba fermentation

(Oyeyiola, 2001), it can be assumed that the Bacillus sp. are the main fermenting
organisms. They were found to persist until the end of the fermentation and their
numbers increased throughout the period of fermentation while the numbers of others
decreased after 24 h of fermentation (Obeta, 2003). Also Bacillus sp. are important
sources of proteases (Fogarty and Griffin, 2003). The other bacteria only managed to
grow on the little carbohydrate present in the seeds, majority of which may have been
lost due to leaching during the preparatory stages ( Ruiz-Teran and Owens, 2009). The
disappearance of these bacteria could also be due to the activities of  B. subtilis, the
predominant bacterium in the fermentation, which is known to produce the antibiotic
bacitracin. The antibiotic may have inhibited the growth of these bacteria and the
disappearance of Micrococcus sp. especially at 96 h of fermentation is believed to be
due to this (Ogueke and Aririatu, 2004). Micrococcus  sp. are very sensitive to
bacitracin (British Pharmacopoeia Commission, 2003).

Since the bean seeds were boiled for hours before fermentation the microorganisms
involved in the fermentation could not have originated from the beans. The bacteria
involved in the fermentation probably were introduced through the air, water, utensils,
leaves used in wrapping or by handling during the preparatory stages ( Obeta,
2003; Odunfa and Oyeyiola, 2005). Example Staphylococcus sp. are more commonly
associated with the skin and hence are easily disseminated through handling. Also
addition of salt would selectively favour the growth
of Staphylococcus  and Micrococcus sp. which are known to be salt tolerant ( Adam and
Moss, 2009).

2.3 Changes that occur during fermentation 

Various biochemical changes occur during the fermentation.  Obeta (2003) found that
pH increased from 6.5 at 0 h to 9.0 at 48 h and declined to 7.1 at 72 h. The rise in pH
has been attributed to the abundant production of ammonia during the fermentation due
to protein hydrolysis and deaminase activity. The increase in pH would encourage the
growth of Bacillus sp. which have been found to grow well at pH 7.0 to 8.0 ( Odunfa
and Oyeyiola, 2005). The drop in pH to 7.1 at 72 h could be attributed to the fact
that B. subtilis and B. licheniformis use ammonia as nitrogen source (Odunfa, 2006b).
However, Odunfa and Oyeyiola (2005), Njoku and Okemadu (2009) and Ogueke and
Aririatu (2004) found that pH rose throughout the fermentation from 5.0-5.7 at 0 h to
7.9-8.7 after 3-5 days of fermentation.

The temperature of fermentation was observed to increase from about 30.8 to 34.5-
38.5°C within the first 24-36 h of fermentation and decreased gradually afterwards to
30-32.5°C at the end of fermentation (Odunfa and Oyeyiola, 2005; Njoku and
Okemadu, 2009). Thus ugba fermentation is exothermic. This initial increase in
temperature has been attributed to the intense metabolic activities of the
microorganisms (period of maximum microbial activity) and represents the most active
and important period of the fermentation. This is because enzyme studies ( Njoku and
Okemadu, 2009) have revealed that the α-amylase, proteolytic and lipolytic enzyme
activities attained their maximum levels at 24-36 h of fermentation. Thus it could be
the enzymes already produced rather than the presence of the microorganisms that
continued the fermentation later.

Moisture content was also found to increase throughout the period of fermentation (52-
56.90% to 71.20-73%) (Odunfa and Oyeyiola, 2005; Njoku and Okemadu,
2009; Ogueke and Aririatu, 2004). The increase in moisture is believed to be due to the
hydrolytic activities of the microorganisms. However, the high moisture level has been
suggested to predispose the product to rapid spoilage ( Odunfa and Oyeyiola,
2005; Ogueke and Aririatu, 2004). Njoku and Okemadu (2009) detected α-amylase,
proteolytic and lipolytic enzymes from the start of ugba fermentation. These enzymes
attained their maximum levels at 24-36 h. They suggested that this could be assumed to
be the period of maximum microbial activity. The initial enzyme activity detected
could be due to the activity of the natural microflora of the oilbean which developed
particularly during the soaking of the cooked beans. Njoku and Okemadu
(2009) therefore suggested that it could well be that fermentation began much earlier
during the soaking of the sliced beans. Some workers (Enujiugha et al., 2002, 2004)
have demonstrated that the raw seeds contain both α-amylase and lipase. They
observed that the specific activity of the purified α-amylase from the raw and
fermented seeds were 0.037 mL -1  min -1  and 0.88 mL -1  min -1 , respectively. They also
claimed that these enzymes complement the bacterial enzymes during fermentation.
However, since the seeds were boiled for several hours before fermentation they could
not have contributed to the fermentation as the boiling must have inactivated them. The
proteinase enzyme is considered the most important enzyme in ugba
fermentation. Njoku and Okemadu (2009) detected a sevenfold increase in the level of
amino nitrogen while Enujiugha (2003) also observed a steady increase in the level of
amino nitrogen from 1.23 to 13.68 mg Ng -1  DM after 72 h of fermentation. Only a two
fold increase in reducing sugar was found, while the activity of lipase was minimal
compared to the other two enzymes (Njoku and Okemadu, 2009). This agrees with the
report of Achinewhu (2006) that fermentation has no appreciable effect on the  fatty
acid content of P. macrophylla. The minimal activity of the lipase could be attributed
to the effect of NaCl that is usually added during fermentation.  Enujiugha et al.
(2004) in their study of the lipase activity in dormant seeds of African oilbean seeds
observed that the activity of lipase isolated from the seeds were inhibited up to 36% by
NaCl. However, they found that presence of Ca 2+  increased the activity of the enzyme
by 64%.

Microbiological Safety of Fermented African Oil Bean Seeds

Most works on African fermented foods (ugba inclusive) have centered on the isolation and
characterizations of organisms involved in the fermentation processes. Not much effort seems to
have been made toward the occurrence and growth of possible pathogens in the product.
However, Adewunmi et al. (2014) used a combination of genome-based culture dependent and
independent techniques to examine iru microbiota and reported bacterial species with both
spoilage and pathogenic history. In addition, genome typing of Bacillus species isolated from
okpehe and soumbala identified species of Bacillus cereus with enterotoxin production potential
(Ouaba et al., 2008; Oguntoyinbo et al., 2010). It is therefore very important to use genotypic
method in combination with phenotypic data to assess microbial quality of fermenting ugba, in
order to guarantee its microbial safety. Furthermore, because of the stress associated with the
food processing, it would be important to use culture dependent and independent methods in
order to find/detect non-culturable or not yet cultured microorganisms. Available information in
literature shows that organisms such as E. coli, Staphylococcus aureus and other members of the
Enterobacteriaceae have been isolated from condiments in West Africa (Isu and Njoku, 1997;
Okorie and Olasupo, 2013a).
Nutritional Importance of Fermentation of Oil bean

Fermentation has been generally observed to improve the nutritional quality of the products
obtained. The protein content, essential amino acids, vitamins and mineral contents of most
fermented foods have been shown to increase during fermentation. Fermented foods and
beverages harbor diverse microorganisms from the environment, including mycelia molds,
yeasts, and bacteria, mostly lactic acid bacteria and micrococci. These microorganisms transform
the chemical constituents of raw materials during fermentation and enhance the nutritional value
of the products. The activities of these microorganisms are noted to enrich bland diets with
improved flavor and texture; preserve perishable foods; fortify products with essential amino
acids, bioactive compounds, vitamins, and minerals for healthy living. They also bring about
degradation of undesirable compounds and anti- nutritive factors; imparts antioxidant and
antimicrobial properties; improve digestibility; and stimulate probiotic functions. While
fermentation results in a lower proportion of dry matter in the food product, the concentration of
the vitamins, minerals, and protein appear to increase when measured on dry weight basis
(Adams, 1990; Chung et al., 2010; Shil et al., 2010).

African oil bean seeds support diet and improve nutritional availability. Proximate analysis of
raw oil bean seed reveals that it is mainly composed of proteins (36–42%), lipids (43–47%) and
carbohydrates (4–17%; Odunfa and Oyeyiola, 1985; Njoku and Okemadu, 1989; Ogueke and
Aririatu, 2004). Slight increases in the crude protein and ash contents of the fermented beans
have been reported. Enujiugha (2003) reported a steady increase in the level of amino nitrogen
from 1.23 mg/Ng1 DM at the start of fermentation to 13.68 mg/Ng-1 DM after 72 h. The amino
acid component of the fermented seed has been shown to contain the 20 essential amino acids.
The high content of the essential amino acids makes the seed a potential source of protein
(Achinewhu, 1982). Glutamic acid appears to be the largest amino acid contained in the seed and
its fermented product. This may be responsible for its use as a flavoring agent for soups in south
eastern Nigeria. Aspartic acid, lysine and phenylalanine are also present in appreciable amounts
in the fermented seeds. In their study of compositional changes in oil bean seeds observed during
thermal treatment, Enujiugha and Akanbi (2005) reported a reduction of the protein content from
22.32% dry wt. in the raw seeds to 19.00% dry wt. in the canned product. Each processing step
brought about a decrease in levels of anti-nutritional factors analyzed. Oxalates, tannins and
phytic acid were reduced from 2.79 mg/g, 0.38 g/100 g, and 2.11 g/100 g in the raw seeds to 0.81
mg/g, 0.22 g/100 g, and 1.16 g/100 g in the canned product, respectively. The oil component of
the seed contains about 75% of saturated fatty acids and 25% of unsaturated fatty acids (Kar and
Okechukwu, 1978). For the saturated fatty acids, lignoceric acid appears to be present in the
largest amount constituting about 12% of the total fatty acid concentration, while palmitic acid is
the least with 3.4%. The major unsaturated fatty acid in the seeds is linoleic acid constituting
42.8%. Oleic acid is also present in appreciable amounts (29.0%). Linolenic and gadoleic acids
are present in very small amounts (3.2 and 0.28%, respectively). The presence of appreciable
amounts of behenic and lignoceric acids is not desirable for edible oils (Odunfa, 1986).
However, Odoemelam (2005) noted that the high degree of unsaturation makes it suitable for
cooking purposes and for use as a drying oil for cosmetics, paints and varnishes. Fermentation
has been found to have minimal effect on the fatty acid content of the oil bean seed. (Onwuliri et
al., 2004) reported that fatty acid concentrations did not change appreciably with processing and
fermentation. Enujiugha and Akanbi (2005) however observed an increase in the oil content from
53.98 to 60.11%. Information available shows that fatty acid content of the oil bean seeds is not
qualitatively affected by fermentation. The principal fatty acid linoleic acid however has been
shown to increase from 60.68 to 67.57% of the total fatty acids while oleic acid decreased from
26.95 to 22.59% during fermentation. Palmitic acid and other saturated fatty acids in the seed oil
are also slightly affected by fermentation. Available information shows that the vitamin content
of the seeds is low while they are a poor source of calcium and phosphorus (Duke, 1981). The
mineral and vitamin contents are observed to decrease during fermentation. The niacin and
riboflavin of the seeds have been found to decrease during fermentation. Enujiugha and Akanbi
(2005) noted that fermentation and canning significantly (P < 0.05) reduced the phosphorus and
iron contents of the seeds while processing generally raised the calcium and magnesium
contents.

Utilization of Ukpaka

Oil bean seed as a delicacy, is consumed in the fermented form and is known as ugba or ukpaka.
This fermented product is obtained by boiling the oil bean seed overnight, after which it is
dehulled by hand and then the cotyledon is sliced. The sliced cotyledons are boiled again for
about 2 hours, washed in water and put in a covered basket and left in a warm environment to
ferment for at least 12 hours. Fermentation can be prolonged depending on the temperature of the
environment (Elizabeth et al., 2006). The fermented slices can be eaten alone or in combination
with other foods. The delicacy prepared with this food is ugba agworoagwu – ugba mixed with
palm oil, pepper, salt and akanwu (trona). Variations to the food were noted as crayfish, fish,
cowskin (kpomo or canda), cassava slices, solanum leaf or various fruits being added. Ugba is
used for several social activities such as marriage, naming ceremonies and union meetings. They
are sold and served in hotels and other eating houses popularly known as “Ugba joints”. It is a
tree plant and the seeds are picked or harvested between August and November. The seeds are
stored in containers until ready for use (Elizabeth et al., 2006)

Health benefits of Fermented food

Fermented food, Fermented food, enjoyed across the globe, conveys health benefits through
lactic acid fermentation. The fermentation process can transform the flavour of food from the
plain and mundane to a mouth-puckering sourness enlivened by colonies of beneficial bacteria
and enhanced micronutrients.

 Studies have revealed that Lactobacillus rhamnosus and L. reuteri which are common
organisms in Nigerian fermented foods like ogi and kunun- zaki could colonize the vagina, kill
viruses, and reduce the risk of infections, including bacterial vaginosis (Reid et al., 2001a;
Cadieux et al., 2002). The potential therapeutic effects of Lactic Acid Bacteria (LAB) and ogi,
including their immunostimulatory effect, are due primarily to changes in the gastrointestinal
(GI) microflora to suppress the growth of pathogens. Increase in population of LAB in the
intestinal or vagina reduces the cause of bacterial vaginosis, which is a major risk factor for the
contraction of HIV (Reid, 2002a). It also reduces the occurrence of gonorrhoea, chlamydia, and
other sexually transmitted diseases (Reid et al., 2001b) and diarrhoea (Adebolu et al., 2007).

 All lactic acid producing bacteria (E.g Lactobacillus acidophilus, L.bulgaricus, L. plantarum,
L. caret, L. pentoaceticus, L. brevis and L. themophilus) produces high acidity during
fermentation. The lactic acid they produce is effective in inhibiting the growth of other bacteria
that may decompose or spoil the food. Despite their complexity, the whole basis of lactic acid
fermentation centres on the ability of lactic acid bacteria to produce acid, which then inhibits the
growth of other non-desirable organisms. Other compounds are important as they improve
particular testes and aromas to the final products. The L. mesenteroides initiates growth in
vegetables more rapidly over a range of temperatures and salt concentrations than any other
lactic acid bacteria. It produces carbon dioxide and acids which rapidly lower the pH and inhibit
the development of undesirable microorganism.

 Over 200 species of bacteria live in gut of humans. These microbes help break down food in
the intestines, aid in the digestion process, help fight off disease, and boost the immune system.
A good balance of intestinal flora is very important to the overall health. If we eat nothing but
overly processed and hard to digest foods, then the fermentation process occurs within the GIT
resulting into gas, bloating, diarrhoea, and constipation might possibly lead to other diseases like
cancer. However, providing the body with predigested foods such as fermented foods will help
the existing microbes within to do the job they need to do.

 Fermentation is not only a way to preserve certain foods, in some cases it actually adds to the
nutrient value of it. Fermented vegetables contain more vitamin C and fermented milk products
have ample amounts of B vitamins. The bioavailability of these vitamins also increases with
fermentation. Probiotics, or "good bacteria" are also formed through the process of fermentation.
Fermented soy products contain more vitamin B12 (Chung et al, 2010).
The desirable bacteria cause less deterioration of the food by inhibiting the growth of the
spoiling types of bacteria. Some fermenting processes lower the pH of foods preventing harmful
microorganisms to live with too acidic an environment. Controlled fermentation processes
encourage the growth of good bacteria which starves, or fights off, the bad microbes.

 The fermentation process can be stopped by other means of preserving, such as, canning
(heating), drying, or freezing. Heat (pasteurization, 63°C), and low temperatures (freezing, 0°C
or below) stops the fermenting process by slowing, or killing, the preferred microorganisms, and
other bacteria. A few undesirable bacteria are not killed by either means, and continue to grow.
When the beneficial bacteria are gone, the unfavorable bacteria take over, growing
exponentially! This causes rotting, disease, illness, and inedible foods. When the good guys are
present and happy, the food remains edible.

 Phytates (phytic acid) are the storage form of phosphorus [a mineral] bound to inositol [a B
vitamin] in foods high in fiber (all plant foods), and particularly the fiber of raw whole grains,
legumes, seeds, and nuts. Although these foods have high phosphorus content, the phosphates in
phytates are not released by human digestion. Phytates, particularly in such raw foods as bran,
are a concern because they can bind a portion of the iron, zinc, and calcium in foods, making the
minerals unavailable for absorption. When bread is leavened (fermented) by yeast, enzymes
degrade phytic acid, and phytates pose no problem. Enzymes, called phytases, destroy phytates
during fermentation processes such as: the yeast-raising of dough, Even a small amount of
phytates in food can reduce iron absorption by half (by 50%), but the effect is less marked if a
meal is supplemented with ascorbic acid (Vitamin C) which also helps the absorption of zinc and
calcium.

 Fermented food, enjoyed across the globe, conveys health benefits through lactic acid
fermentation. The fermentation process can transform the flavor of food from the plain and
mundane to a mouth-puckering sourness enlivened by colonies of beneficial bacteria and
enhanced micronutrients. While fermented food like yogurt, sauerkraut and kefir are well-known
many other lesser-known foods also benefit from the lactic acid fermentation process. Indeed,
virtually every food with a complex or simple sugar content can be successfully fermented.

 Born of both necessity and practicality, lactic acid fermentation proved to be not only an
efficient method of preserving food for our ancestors, but also a critical one. Indeed, fermented
food like sauerkraut, cheese, wine, kvass, soured grain porridge and breads often sustained tribes
and villages during harsh winters when fresh food simply wasn’t available let alone plentiful.

 In many societies including our own where yogurt has been heralded as a health food since the
19th century, fermented food has gained a reputation for its beneficial effects on immunity,
intestinal health and general well-being. Modern researchers are just beginning to understand
what the sages of old were tuned in to: fermented food conveys clear and calculable health
benefits to the human diet. Lactic acid fermentation in and of itself enhances the micronutrient
profile of several foods.

Extraction of Oil from Oilseeds

The vast majority of plants, especially the agricultural stock, contain extractable oil that may be
of some commercial value. Since the beginning of human civilization, rural communities from
around the globe have used various traditional methods to extract mainly edible oil from
materials of plant origin. Many edible vegetable oils such as palm, corn, soybean, peanut,
coconut etc (CODEX-STAN 210-1999) are used as table oils because of their high nutritive
value. For instance, fats and oils are the most concentrated form of energy, providing
approximately 9 kcal of energy per gram compared to only 4 kcal per gram for proteins and
carbohydrates (Ali et al., 2005). This is in addition to their industrial application as raw materials
for the synthesis of polyols, polymers, resins, biodiesel, pharmaceuticals etc.

Oilseed Pretreatment

Irrespective of the extraction method to be used, oilseed pretreatment is necessary. Basic steps in
this process are dehulling, pod or seed coat removal, winnowing, sorting, cleaning, grinding or
milling and preheating (Ogunniyi, 2006; Yusuf et al., 2015). Grinding or crushing of oilseeds
prior to extraction is to ensure that oil-bearing minute cells embedded in fibrous structures are
broken or ruptured to release the oil (Akpan et al., 2006; Tayde et al., 2011). Heat treatment
further facilitates the oil release process by reducing moisture content and hardening the interior
of the oilseed (Patel et al., 2016). In recent times, preheating of oilseed done conventionally by
hot air oven, is being replaced by microwave-assisted heat treatment, the latter offering some
advantages (Mgudu et al., 2012). Additionally, grinding or size reduction prior to solvent
extraction increases the surface area for solvent penetration to bring out the oil by leaching.

Old Traditional Methods

In terms of oil recovery and oil yield, the old traditional or informal wet extraction methods used
by rural communities around the globe is regarded as inefficient, often yielding below the range
of plant oil content found in literature (Alonge and Olaniyan, 2006; Olaniyan, 2010). Olaniyan
(2010) has outlined three major means of recovering oil from oleaginous materials of plant origin
as wet extraction (hot water or steam extraction), solvent extraction and mechanical expression.
With regard to the wet extraction method, Oluwole et al. (2012) proffered nine major operations
that are involved in the extraction of castor oil by the old traditional method namely, collection
of seed pods, shelling of the pods/winnowing, boiling the seeds to reduce moisture, grinding of
seeds to form paste, mixing the paste with water/boiling to extract oil, scooping of oil and drying
the oil by heating.
Olaniyan and Yusuf (2012) described the old traditional method of extraction of seed oils as
involving the roasting of seed kernels by mortar and pestle or between two stones, mixing the
crushed mass with water, cooking of the mixed paste in order to obtain the oil by floating and
skimming, and then drying of the oil by further heating. They further described this method as
tedious, time consuming, energy sapping, drudgery prone, inefficient and low in yield and
quality. In other words, the old traditional methods are crude, largely unscientific, inefficient,
and yielding poor quality extracted oil.

Conventional Methods

The conventional methods are the well-known and widely practiced methods of oil extraction
namely, solvent extraction and mechanical expression. Many seed oils are extracted by either of
the two methods, or a combination of the two. This classification may also include rendering (Ali
et al., 2005), though perhaps not as widely used. Regardless of extraction method, extracted and
refined oil must be evaluated for its physico-chemical properties to determine its application or
usage.

Solvent extraction

The solvent extraction method is a conventional extraction method commonly applied to oilseeds
with low oil content (< 20%), like soybean. This method is considered as one of the most
efficient methods in vegetable oil extraction, with less residual oil left in the cake or meal
(Buenrostro and Lopez-Munguia, 1986; Tayde et al., 2011). The choice of solvent is based
mainly on the maximum leaching characteristics of the desired solute substrate (Dutta et al.,
2015). Solvents commonly used are hexane, diethyl ether, petroleum ether and ethanol. Other
considerations are high solvent-solute ratio, relative volatility of solvent to oil, oil viscosity and
polarity, as well as cost and market availability (Muzenda et al., 2012; Takadas and Doker,
2017). The solvent extraction method offers a number of advantages. Bhuiya et al. (2015) who
researched on the optimization of oil extraction process from Australian Native Beauty Leaf
Seed (Calophyllum innophyllum) reported that the solvent extraction process is a very effective
method, with high yield and consistent performance, though cost of production was relatively
higher than mechanical press methods due to high cost of solvent. According to Muzenda et al.
(2012) in their work on the optimization of process parameters for castor oil production, they
observed that oil extraction ability of solvent during solvent extraction is enhanced with increase
in extraction time; with solvent-solute ratio in favour of the solvent preferably by a factor of 6:1.
Ikya et al. (2013) studied the effects of extraction methods on the yield and quality
characteristics of oils from shea nut. They compared results of physical, chemical and sensory
properties of oil extracted by solvent extraction and old traditional extraction methods. They
reported a higher oil yield of 47.5% for the solvent extraction method compared to 34.1% for the
old traditional method, and better keeping quality for the solvent-extracted oil (lower moisture
content and lower flash and fire point values). In their work on the extraction, characterization
and modification of castor seed oil, Akpan et al. (2006) employed the solvent extraction method
to extract castor seed oil from castor bean paste using Soxhlet extractor. They obtained 33.2% oil
yield, which was within the expected range for castor beans found in literature. They concluded
that mode of extraction and seed variety are very important parameters affecting oil yield.

Mechanical expression

Mechanical expression Mechanical expression involves the application of pressure (using

hydraulic or screw presses) to force oil out of an oilbearing material (Arisanu, 2013). By this
method, oil yield is enhanced by increased mechanical pressure on the oilbearing material. In a
study of the yield characteristics of ground soybean sample at various operating pressures,
pressing durations and product bulk temperatures, Mwithiga and Moriasi (2007) found that oil
yields increased linearly with compression pressure (40-80 kgf/m2 ), duration of pressing (6-12
mins) and increase in the bulk temperature of preheated oilseeds, reaching a peak yield at about
750C. With regard to oil yield, screw presses have an advantage over hydraulic presses for
churning out slightly higher yields, in addition to their continuous mode of operation (Arisanu,
2013). Mechanical presses (manual or powered) meant for small (laboratory) scale oil extraction
are simple, safer and containing fewer steps compared to solvent extraction of vegetable oils
(Oyinlola et al., 2004). However, in developing countries even simpler devices are in use to
achieve similar results (Mwithiga and Moriasi, 2007). On the industrial scale, industrial
machines or expellers are used for the purpose of extracting vegetable oils mechanically.

Mechanical press methods are often used to extract vegetable oil from oilseeds having oil content
higher than 20% (Sinha et al., 2015). Generally, these methods have the advantage of low
operation cost, and of producing high quality light coloured oil with low concentration of free
fatty acids (FFAs) (Carr, 1976; Kirk-othmer, 1979). However, it has a relatively low yield
compared to solvent extraction and is therefore comparatively inefficient, often with a large
portion of oil left in the cake or meal after extraction (Buenrostro and Lopez-Munguia, 1986;
Anderson, 1996). In addition, it is time consuming and labour intensive (Bhuiya et al., 2015). In
castor oil extraction for instance, mechanical pressing removes only about 45% of the oil, with
remaining oil in meal extractable by solvent extraction method (Ogunniyi, 2006).

There are two types of mechanical press methods namely, cold-press and hot-press methods.
Cold-press or scarification method is carried out at low temperature (below 500C) and pressure,
whereas the hot-press method is carried out at elevated temperature and pressure. Cold-pressed
seed oils are safer than hot-pressed seed oils as adverse effects caused by high temperatures are
avoided in the former. Some of the likely adverse effects are decreased oxidative stability,
degradation of valuable oil components and reduced oil keeping quality. In cold-pressed oils, the
purity and natural properties of seed oils are preserved (AzadmardDamirchi et al., 2011; Bhatol,
2013). This includes the retention of valuable nutraceuticals like phytosterols and tocopherols in
the extracted oil (Kittiphoom et al., 2015). Because of these attractive qualities, there is growing
global demand for cold-pressed oil. In contrast, hot-press methods give higher oil yield due
largely to decreased seed oil viscosity at high temperatures. This enhances oil flow during
extraction. Thus high temperature increases the efficiency of the extraction process and yields of
up to 80% of available oil in seed are possible (Patel et al., 2016), but they may also engender oil
degradation, with attendant deterioration of oil quality.

CHAPTER THREE
3.0 COLLECTION OF SAMPLES
Fermented African oil bean seed (Pentaclethra Macrophylla) used for this study was purchased
from Eke market Oko Orumba North LGA Anambra state.
3.2 METHODS
3.2.1 PROCESSING METHODS

Collection and Preparation of sample

The purchased sample was thereafter reduced to particle sizes ranging from 0.75mm to 2.00mm
and later it was dried at room temperature for five days to eliminate moisture. The dried sample
was later grounded at Eke-Oko market to a fine powered form for a better extraction and analysis
to take place.

3.2 METHODS
Extraction of Oil
The oils were extracted by solvent extraction method using petroleum spirit for castor oil seeds.
The oils were separated from the solvent by distillation after which they were collected as the
residues.
Mineral Content Analysis

Determination of minerals like Ca, P, Fe, Mg and K, were carried out according to AOAC
(2005). Sample weighing 10g will be transferred into a crucible, and heated over flame to
volatilize as much of the organic matter as possible, before transferring into a muffle furnace
to burn at 450°C for 5-7hours. To the ash will be added 10ml of dilute HCl, boiled for a few
minutes, and made up to 100ml with distilled water.
This will be used for the mineral analysis as described below.

Determination of Phosphorous

Determination of phosphorous will be done according to the method of AOAC (2005).

Twenty five grams (25g) of ammonium molybdate and 1.25g of ammonium metavanadate
were added to 300ml of distilled water, warmed to dissolve, cooled and made up to 500ml
with water. Concentrated HCl (215ml) will be diluted to 500ml with water and mixed with
ammonium molybdate-ammonium metavanadate reagent.
Phosphorous stock will be prepared by dissolving 0.879g of dried phosphorous dihydrogen
orthophosphate (dried at 105oC for one hour) with water and 1ml of conc. HCl added. It will
be diluted to 200ml with the first reagent, and 2ml of toluene will be added to give 1mg/ml.
The working standard will be prepared by measuring 2ml of phosphorous to 0, 2, 4, 6, 8, and
10ml of standard phosphorous solution into six 200-ml volumetric flasks and diluted to mark
with water.
Each phosphorous standard solution (5ml) will be pipetted into a 500-ml graduated flask.
Molybdate mixture (10ml) will be added and diluted to the mark with water. It will be
allowed to stand for 15 minutes for colour development, and the absorbance measured at
400nm against blank. A calibration curve (see appendix 1) relating absorbance to mg of
phosphorous will be used to read the phosphorus content of the sample solution in mg/ml,
and the number of phosphorous equivalent to the absorbance of the sample blank determined
will be calculated.
Iron Determination
Phenanthroline method as described in AOAC (2005), will be used. Phenanthroline solution
will be prepared by dissolving 100mg I,10-phenanthroline molybdate in 100ml distilled
water by stirring and heating to 80oC. Hydroxylamine solution will be prepared by dissolving
10g in 100ml of distilled water, while ammonium acetate buffer solution will be prepared by
dissolving 250g in 150ml distilled water. 5ml of the digested sample will be added in a test-
tube. Then, 3ml of phenanthroline solution and 2ml of HCl will be added. Hydroxylamine
solution (1ml) will be added to the mixture and boiled in a steam bath at
600oC for 2 minutes. Then, 9ml of ammonium acetate buffer solution will be added and
diluted to 50ml with water. The absorbance will be taken at 510nm. Calibration curve (see
appendix 2) will be prepared by pipetting 2, 4, 6, 8, and 10ml standard iron solution into
100ml volumetric flasks to prepare a solution of known concentrations. The curve obtained
will be used to read off the value of iron.

Determination of Calcium

Titrimetric method will be used for calcium determination as described in AOAC (2005).
The test solution (10ml) will be added into 250-ml conical flask. Potassium hydroxide
(25ml), water (25ml) and a pinch of calcium indicator were added, and titrated against
Ethylene Diamine Tetra Acetate (EDTA) dissolution salt solution to an end point. The
volume of EDTA is the volume equivalent of calcium in the solution. The value of calcium
will be calculated as shown below:
% Ca = volumeofEDTA×mol.EDTA× At.wt.ofcalcium× DF ×100 .

1000×wt.ofsampleused ×10

Where EDTA- Ethylene Diamine Tetra Acetate

DF- Dilution factor

Determination of Potassium

Determination of potassium will be done according to the method of AOAC (2005). The
ashed sample (2ml) will be pipetted and transferred into 3 test tubes and 3ml of water added;
2ml of sodium cobalt nitrite reagent will be added, shaken vigorously and allowed to stand
for 45 minutes and centrifuged 15 minutes. The supernatant will be drained off and to the
residue will be added 2ml of ethanol and shaken vigorously, centrifuged and the supernatant
drained off. The residue will be further will be washed twice with ethanol and centrifuged
respectively. 2ml of water will be added to the residue, boiled for 10 minutes with frequent
shaking to dissolve the precipitate, cooled and 1ml of 1% choline hydrochloride, 1ml of 2%
sodium ferricyanide added, and made up to 6ml with water.
The absorbance will be taken at 620nm against a blank.
Determination of Magnesium
Determination of magnesium will be done according to the method of AOAC (2005). Ashed
sample (2ml) sample will be transferred into 3 test tubes and 3ml of water added; 2ml of 10%
sodium tungstate, 2ml of 0.67N sulfuric acid were added, centrifuged for 5 minutes. 5ml of the
supernatant will be taken added 1ml water, 1ml of 0.05% titan yellow, and 1ml of 0.1% gum
ghatti. 2ml of 10% sodium hydroxide will be added and the absorbance taken at 520nm against a
blank.